Patent Grant
7999080
The invention relates generally to a variety of moieties and tools for the detection of protein phosphorylation. Moreover, the invention relates to the use of the same for diagnostic and therapeutic purposes.
The activation of proteins by post-translational modification is an important cellular mechanism for regulating most aspects of biological organization and control, including growth, development, homeostasis, and cellular communication. Cellular signal transduction pathways involve protein kinases, protein phosphatases, and phosphoprotein-interacting domain (e.g., SH2, PTB, WW, FHA, 14-3-3) containing cellular proteins to provide multidimensional, dynamic and reversible regulation of many biological activities. See e.g., Sawyer et al., Med. Chem. 1(3): 293-319 (2005).
Protein phosphorylation on a proteome-wide scale is extremely complex as a result of three factors: the large number of modifying proteins, e.g. kinases, encoded in the genome, the much larger number of sites on substrate proteins that are modified by these enzymes, and the dynamic nature of protein expression during growth, development, disease states, and aging. The human genome, for example, encodes over 520 different protein kinases, making them the most abundant class of enzymes known. See Hunter, Nature 411: 355-65 (2001). Most kinases phosphorylate many different substrate proteins, at distinct tyrosine, serine, and/or threonine residues. Indeed, it is estimated that one-third of all proteins encoded by the human genome are phosphorylated, and many are phosphorylated at multiple sites by different kinases. See Graves et al., Pharmacol. Ther. 82: 11.1-21 (1999).
Many of these phosphorylation sites regulate critical biological processes and may prove to be important for diagnostic or therapeutic modalities useful in the treatment and management of many pathological conditions and diseases, including inter alia cancer, developmental disorders, as inflammatory, immune, metabolic and bone diseases.
For example, of the more than 100 dominant oncogenes identified to date, 46 are protein kinases. See Hunter, supra. Understanding which proteins are modified by these kinases will greatly expand our understanding of the molecular mechanisms underlying oncogenic transformation. Therefore, the identification of, and ability to detect, phosphorylation sites on a wide variety of cellular proteins is crucially important to understanding the key signaling proteins and pathways implicated in the progression of many disease states.
Understanding reversible protein phosphorylation and its role in the operation and interrelationship between cellular components and functions provides the opportunity to gain a finer appreciation of cellular regulation. In spite of the importance of protein modification, phosphorylation is not yet well understood due to the extraordinary complexity of signaling pathways, and the slow development of the technology necessary to unravel it.
In many instances, such knowledge is likely to provide valuable tools useful to evaluate, and possibly to manipulate target pathways, ultimately altering the functional status of a given cell for a variety of purposes.
The importance of protein kinase-regulated signal transduction pathways is underscored by a number of drugs designed to treat various cancer types by the inhibition of target protein kinases at the apex or intermediary levels of pathways implicated in cancer development. See Stern et al., Expert Opin. Ther. Targets 9(4):851-60 (2005).
Leukemia, a disease in which a number of underlying signal transduction events have been elucidated, has become a disease model for phosphoproteomic research and development efforts. As such, it represent a paradigm leading the way for many other programs seeking to address many classes of diseases (See, Harrison's Principles of Internal Medicine, McGraw-Hill, New York, N.Y.)
Depending on the cell type involved and the rate by which the disease progresses leukemia can be defined as acute or chronic myelogenous leukemia (AML or CML), or acute and chronic lymphocytic leukemia (ALL or CLL).
Most varieties of leukemia are generally characterized by genetic alterations e.g., chromosomal translocations, deletions or point mutations resulting in the constitutive activation of protein kinase genes, and their products, particularly tyrosine kinases. The most well known alteration is the oncogenic role of the chimeric BCR-Abl gene. See Nowell, Science 132: 1497 (1960)). The resulting BCR-Abl kinase protein is constitutively active and elicits characteristic signaling pathways that have been shown to drive the proliferation and survival of CML cells (see Daley, Science 247: 824-830 (1990); Raitano et al., Biochim. Biophys. Acta. December 9; 1333(3): F201-16 (1997)).
The recent success of Imanitib (also known as ST1571 or Gleevec®), the first molecularly targeted compound designed to specifically inhibit the tyrosine kinase activity of BCR-Abl, provided critical confirmation of the central role of BCR-Abl signaling in the progression of CML (see Schindler et al., Science 289: 1938-1942 (2000); Nardi et al., Curr. Opin. Hematol. 11: 35-43 (2003)).
The success of Gleevec® now serves as a paradigm for the development of targeted drugs designed to block the activity of other tyrosine kinases known to be involved in many diseased including leukemias and other malignancies (see, e.g., Sawyers, Curr. Opin. Genet. Dev. February; 12(1): 111-5 (2002); Druker, Adv. Cancer Res. 91:1-30 (2004)). For example, recent studies have demonstrated that mutations in the FLT3 gene occur in one third of adult patients with AML. FLT3 (Fms-like tyrosine kinase 3) is a member of the class III receptor tyrosine kinase (RTK) family including FMS, platelet-derived growth factor receptor (PDGFR) and c-KIT (see Rosnet et al., Crit. Rev. Oncog. 4: 595-613 (1993). In 20-27% of patients with AML, an internal tandem duplication in the juxta-membrane region of FLT3 can be detected (see Yokota et al., Leukemia 11: 1605-1609 (1997)). Another 7% of patients have mutations within the active loop of the second kinase domain, predominantly substitutions of aspartate residue 835 (D835), while additional mutations have been described (see Yamamoto et al., Blood 97: 2434-2439 (2001); Abu-Duhier et al., Br. J. Haematol. 113: 983-988 (2001)). Expression of mutated FLT3 receptors results in constitutive tyrosine phosphorylation of FLT3, and subsequent phosphorylation and activation of downstream molecules such as STAT5, Akt and MAPK, resulting in factor-independent growth of hematopoietic cell lines.
Altogether, FLT3 is the single most common activated gene in AML known to date. This evidence has triggered an intensive search for FLT3 inhibitors for clinical use leading to at least four compounds in advanced stages of clinical development, including: PKC412 (by Novartis), CEP-701 (by Cephalon), MLN518 (by Millenium Pharmaceuticals), and SU5614 (by Sugen/Pfizer) (see Stone et al., Blood (in press) (2004); Smith et al., Blood 103: 3669-3676 (2004); Clark et al., Blood 104: 2867-2872 (2004); and Spiekerman et al., Blood 101: 1494-1504 (2003)).
There is also evidence indicating that kinases such as FLT3, c-KIT and Abl are implicated in some cases of ALL (see Cools et al., Cancer Res. 64: 6385-6389 (2004); Hu, Nat. Genet. 36: 453-461 (2004); and Graux et al., Nat. Genet. 36: 1084-1089 (2004)). In contrast, very little is know regarding any causative role of protein kinases in CLL, except for a high correlation between high expression of the tyrosine kinase ZAP70 and the more aggressive form of the disease (see Rassenti et al., N. Eng. J. Med. 351: 893-901 (2004)).
Despite the identification of a few key molecules involved in progression of leukemia, the vast majority of signaling protein changes underlying this disease remains unknown. There is, therefore, relatively scarce information about kinase-driven signaling pathways and phosphorylation sites relevant to the different types of leukemia. This has hampered a complete and accurate understanding of how protein activation within signaling pathways is driving these complex cancers. Accordingly, there is a continuing and pressing need to unravel the molecular mechanisms of kinase-driven oncogenesis in leukemia by identifying the downstream signaling proteins mediating cellular transformation in this disease. Identifying particular phosphorylation sites on such signaling proteins and providing new reagents, such as phospho-specific antibodies and AQUA peptides, to detect and quantify them remains particularly important to advancing our understanding of the biology of this disease.
Presently, diagnosis of leukemia is made by tissue biopsy and detection of different cell surface markers. However, misdiagnosis can occur since some leukemia cases can be negative for certain markers, and because these markers may not indicate which genes or protein kinases may be deregulated. Although the genetic translocations and/or mutations characteristic of a particular form of leukemia can be sometimes detected, it is clear that other downstream effectors of constitutively active kinases having potential diagnostic, predictive, or therapeutic value, remain to be elucidated. Accordingly, identification of downstream signaling molecules and phosphorylation sites involved in different types of leukemia and development of new reagents to detect and quantify these sites and proteins may lead to improved diagnostic/prognostic markers, as well as novel drug targets, for the detection and treatment of this disease.
Several novel protein phosphorylation sites have been identified in a variety of cell lines. Such novel phosphorylation sites (tyrosine), and their corresponding parent proteins are reported (see Table 1). The elucidation of these sites at long last provides the elements necessary to attain those much needed proteomics tools and modalities.
The invention discloses novel phosphorylation sites identified in signal transduction proteins and pathways underlying various disease states including for example human leukemias. The invention thus provides new reagents, including phosphorylation-site specific antibodies and AQUA peptides, for the selective detection and quantification of these phosphorylated sites/proteins. Also provided are methods of using the reagents of the invention for the detection and quantification of the disclosed phosphorylation sites.
FIG. 1—Is a diagram broadly depicting the immunoaffinity isolation and mass-spectrometric characterization methodology (IAP) employed to identify the novel phosphorylation sites disclosed herein.
FIG. 2—Is a table (corresponding to Table 1) enumerating the Leukemia signaling protein phosphorylation sites disclosed herein:
Column A=the name of the parent protein; Column B=the SwissProt accession number for the protein (human sequence); Column C=the protein type/classification; Column D=the tyrosine residue (in the parent protein amino acid sequence) at which phosphorylation occurs within the phosphorylation site; Column E=the phosphorylation site sequence encompassing the phosphorylatable residue (residue at which phosphorylation occurs (and corresponding to the respective entry in Column D) appears in lowercase; Column F=the type of leukemia in which the phosphorylation site was discovered; and Column G=the cell type(s), tissue(s) and/or patient(s) in which the phosphorylation site was discovered.
FIG. 3—is an exemplary mass spectrograph depicting the detection of the tyrosine 173 phosphorylation site in PSMC6 (see Row 251, SEQ ID NO: 252 in FIG. 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* indicates the phosphorylated serine (shown as lowercase “y” in
FIG. 4—is an exemplary mass spectrograph depicting the detection of the tyrosine 50 phosphorylation site in NCK2 (see Row 5, SEQ ID NO: 4 in FIG. 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* indicates the phosphorylated tyrosine (shown as lowercase “y” in
Several novel protein phosphorylation sites have been identified in a variety of cell lines. Such novel phosphorylation sites (tyrosine), and their corresponding parent proteins are reported (see Table 1). The elucidation of these sites at long last provides the elements necessary to attain those much needed proteomics tools and modalities.
The disclosure of the phosphorylation sites provides the key to the production of new moieties, compositions and methods to specifically detect and/or to quantify these phosphorylated sites/proteins. Such moieties include for example reagents, such as phosphorylation site-specific antibodies and AQUA peptides (heavy-isotope labeled peptides). Such reagents are highly useful, inter alia, for studying signal transduction events underlying the progression of many diseases known or suspected to involve protein phosphorylation e.g., leukemia in a mammal. Accordingly, the invention provides novel reagents—phospho-specific antibodies and AQUA peptides—for the specific detection and/or quantification of a target signaling protein/polypeptide (e.g., a signaling protein/polypeptide implicated in leukemia) only when phosphorylated (or only when not phosphorylated) at a particular phosphorylation site disclosed herein. The invention also provides methods of detecting and/or quantifying one or more phosphorylated Target signaling protein/polypeptide using the phosphorylation-site specific antibodies and AQUA peptides of the invention.
These phosphorylation sites correspond to numerous different parent proteins (the full sequences (human) of which are all publicly available in SwissProt database and their Accession numbers listed in Column B of Table 1/
In part, the invention provides an isolated phosphorylation site-specific antibody that specifically binds a given Target signaling protein/polypeptide only when phosphorylated (or not phosphorylated, respectively) at a particular tyrosine enumerated in Column D of Table 1/
In one embodiment, the invention provides an isolated phosphorylation site-specific antibody that specifically binds a Target signaling protein/polypeptide selected from Column A of Table 1 (Row 2-492) only when phosphorylated at the tyrosine residue listed in corresponding Column D of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-70, 72-79, 81-465, 468-488, 490-497), wherein said antibody does not bind said signaling protein when not phosphorylated at said tyrosine. In another embodiment, the invention provides an isolated phosphorylation site-specific antibody that specifically binds a Target signaling protein/polypeptide selected from Column A of Table 1 only when not phosphorylated at the tyrosine residue listed in corresponding Column D of Table 1, comprised within the peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-70, 72-79, 81-465, 468-488, 490-497), wherein said antibody does not bind said signaling protein when phosphorylated at said tyrosine. Such reagents enable the specific detection of phosphorylation (or non-phosphorylation) of a novel phosphorylatable site disclosed herein. The invention further provides immortalized cell lines producing such antibodies. In one embodiment, the immortalized cell line is a rabbit or mouse hybridoma.
In another embodiment, the invention provides a heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Target signaling protein/polypeptide selected from Column A of Table 1, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-70, 72-79, 81-465, 468-488, 490-497), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D of Table 1. In certain embodiments, the phosphorylatable tyrosine within the labeled peptide is phosphorylated, while in other embodiments, the phosphorylatable residue within the labeled peptide is not phosphorylated.
Reagents (antibodies and AQUA peptides) provided by the invention may conveniently be grouped by the type of Target signaling protein/polypeptide in which a given phosphorylation site (for which reagents are provided) occurs. The protein types for each respective protein (in which a phosphorylation site has been discovered) are provided in Column C of Table 1/
Subsets of the phosphorylation sites (and their corresponding proteins) disclosed herein are those occurring on the following protein types/groups listed in Column C of Table 1/
The patents, published applications, and scientific literature referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.
In one subset of embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds an adaptor/scaffold protein selected from Column A, Rows 2-28, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 2-28, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 2-28, of Table 1 (SEQ ID NOs: 1-27), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the adaptor/scaffold protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of an adaptor/scaffold protein selected from Column A, Rows 2-28, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 2-28, of Table 1 (SEQ ID NOs: 1-27), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 2-28, of Table 1.
Among this subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following adaptor/scaffold protein phosphorylation sites are: NCK2 (Y50), PAG (Y387) and SAP97 (Y806) (see SEQ ID NOs: 4, 9 and 21).
In a second subset of embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a cell cycle regulation protein selected from Column A, Rows 41-54, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 41-54, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 41-54, of Table 1 (SEQ ID NOs: 40-53), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the cell cycle regulation protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a signaling protein that is a cell cycle regulation protein selected from Column A, Rows 41-54, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 41-54, of Table 1 (SEQ ID NOs: 40-53), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 41-54, of Table 1.
Among this subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following cell cycle regulation protein phosphorylation site is: securin (Y111) (see SEQ ID NO: 51).
In another subset of embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds an enzyme protein selected from Column A, Rows 83-142, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 83-142, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 83-142, of Table 1 (SEQ ID NOs: 82-141), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the enzyme protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a signaling protein that is an enzyme protein selected from Column A, Rows 83-142, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 83-142, of Table 1 (SEQ ID NOs: 82-141), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 83-142, of Table 1.
Among this subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following enzyme protein phosphorylation sites are: p47phox (Y48), PLCG1 (Y379), PLCG1 (Y833) and PLCG2 (Y495) (see SEQ ID NO's: 92, 110, 115 and 118).
In still another subset of embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a G protein or regulator protein selected from Column A, Rows 141-166, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 141-166, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 141-166, of Table 1 (SEQ ID NOs: 142-167), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the G protein or regulator protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a signaling protein that is a G protein or regulator protein selected from Column A, Rows 141-166, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 141-166, of Table 1 (SEQ ID NOs: 142-167), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 141-166, of Table 1.
Among this subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following G protein or regulator protein phosphorylation sites are: Rap1a (Y159) and RAPGEF4 (Y857) (see SEQ ID NOs: 152 and 157).
In still another subset of embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a kinase (non-protein) selected from Column A, Rows 171-193, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 171-193, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 171-193, of Table 1 (SEQ ID NOs: 172-194), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the kinase (non-protein) when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a signaling protein that is a kinase (non-protein) selected from Column A, Rows 171-193, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 171-193, of Table 1 (SEQ ID NOs: 172-194), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 171-193, of Table 1.
Among this subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following kinase (non-protein) phosphorylation sites are: NM23 (Y52), PIK3CA (Y361), PIK3R1 (Y657), PIK3R3 (Y184) and PIK4CA (Y973) (see SEQ ID NOs: 172, 180, 183, 186 and 189).
In still another subset of embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a phosphatase selected from Column A, Rows 209-241, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 209-241, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 209-241 of Table 1 (SEQ ID NOs: 210-242), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds a phosphatase when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a signaling protein that is a phosphatase selected from Column A, Rows 209-241, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 209-241, of Table 1 (SEQ ID NOs: 210-242), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 209-241, of Table 1.
Among this subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following phosphatase phosphorylation sites are: PHPT1 (Y93), PPP6C (Y261), PFEN (Y176), SHP-1 (Y301) and SHP-2 (Y242) (see SEQ ID NOs: 214, 223, 225, 241 and 242).
In yet another subset of embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a protein kinase (Ser/Thr) selected from Column A, Rows 260-283, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 260-283, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 260-283, of Table 1 (SEQ ID NOs: 261-284), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the protein kinase (Ser/Thr) when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a signaling protein that is a protein kinase (Ser/Thr) selected from Column A, Rows 260-283, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 260-283, of Table 1 (SEQ ID NOs: 261-284), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 260-283, of Table 1.
Among this subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following protein kinase (Ser/Thr) phosphorylation sites are: PAK2 (Y252), PKCT (Y545) and PLK1 (Y217) (see SEQ ID NOs: 264, 272 and 275).
In yet another subset of embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a receptor/channel/transporter/cell surface protein selected from Column A, Rows 284-306, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 284-306, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 284-306, of Table 1 (SEQ ID NOs: 285-307), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the receptor/channel/transporter/cell surface protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a signaling protein that is a receptor/channel/transporter/cell surface protein selected from Column A, Rows 284-306, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 284-306, of Table 1 (SEQ ID NOs: 285-307), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 284-306, of Table 1.
Among this subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following receptor/channel/transporter/cell surface protein phosphorylation sites are: NMDAR2B (Y239) and PAR1 (Y397) (see SEQ ID NOs: 286 and 301).
In still another subset of embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a RNA binding protein selected from Column A, Rows 307-364, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 307-364, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 307-364, of Table 1 (SEQ ID NOs: 308-365), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the RNA binding protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a signaling protein that is a RNA binding protein selected from Column A, Rows 307-364, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 307-364, of Table 1 (SEQ ID NOs: 308-365), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 307-364, of Table 1.
Among this subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following a RNA binding protein phosphorylation sites are: NCL (Y402), PABP 1(Y297), PSF (Y381) and SF2 (Y149) (see SEQ ID NOs: 310, 321, 334 and 351).
In yet another subset of embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a transcriptional regulator selected from Column A, Rows 367-400, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 367-400, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 367-400, of Table 1 (SEQ ID NOs: 368-401), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the transcriptional regulator when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a signaling protein that is a transcriptional regulator selected from Column A, Rows 367-400, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 367-400, of Table 1 (SEQ ID NOs: 368-401), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 367-400, of Table 1.
Among this subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following transcriptional regulator phosphorylation sites are: NFAT1 (Y860), NFkB-p105 (Y241), POLR2A (Y1916), POL2R1 (Y54) and REL (Y47) (see SEQ ID NOs: 371, 380, 393, 394 and 397).
In yet a further subset of embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a protein selected from the group consisting of N-cad (Y785), PARP1 (Y775), PLCL2 (Y896), MYH10 (Y1415), RPS3 (Y166) and Nice-4 (Y858) (Column A, Rows 29, 59, 196, 299, 405 and 422 of Table 1) only when phosphorylated at the tyrosine listed in corresponding Column D of Table 1), said tyrosine comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 28, 58, 196, 200, 406 and 423), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds a protein selected from the group consisting of N-cad (Y785), PARP1 (Y775), PLCL2 (Y896), MYH10 (Y1415), RPS3 (Y166) and Nice-4 (Y858) (Column A, Rows 29, 59, 195, 299, 405 and 422 of Table 1) when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a protein selected from the group consisting of selected from the group consisting of N-cad (Y785), PARP1 (Y775), PLCL2 (Y896), MYH10 (Y1415), RPS3 (Y166) and Nice-4 (Y858) (Column A, Rows 29, 59, 195, 299, 405 and 422 of Table 1), said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 28, 58, 196, 200, 406 and 423), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 29, 59, 195, 299, 405 and 422 of Table 1.
The invention also provides an immortalized cell line producing an antibody of the invention, for example, a cell line producing an antibody within any of the foregoing subsets of antibodies. In an embodiment, the immortalized cell line is a rabbit hybridoma or a mouse hybridoma.
In other embodiments, a heavy-isotope labeled peptide (AQUA peptide) of the invention (for example, an AQUA peptide within any of the foregoing subsets of AQUA peptides) comprises a disclosed site sequence wherein the phosphorylatable tyrosine is phosphorylated. In yet other embodiments, a heavy-isotope labeled peptide of the invention comprises a disclosed site sequence wherein the phosphorylatable tyrosine is not phosphorylated.
The foregoing subsets of reagents of the invention should not be construed as limiting the scope of the invention, which, as noted above, includes reagents for the detection and/or quantification of disclosed phosphorylation sites on any of the other protein type/group subsets (each a subset) listed in Column C of Table 1/
Also provided by the invention are methods for detecting or quantifying a Target signaling protein/polypeptide that is tyrosine phosphorylated, said method comprising the step of utilizing one or more of the above-described reagents of the invention to detect or quantify one or more Target Signaling Protein(s)/Polypeptide(s) selected from Column A of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D of Table 1. In certain embodiments of the methods of the invention, the reagents comprise a subset of reagents as described above. The antibodies according to the invention maybe used in standard (e.g., ELISA or conventional cytometric assays). The invention thus, provides compositions and methods for the detection and/or quantitation of a given target signaling protein or polypeptide in a sample, by contacting the sample and a control sample with one or more antibody of the invention under conditions favoring the binding and thus formation of the complex of the antibody with the protein or peptide. The formation of the complex is then detected according to methods well established and known in the art.
Also provided by the invention is a method for obtaining a phosphorylation profile of a certain protein type or group, for example adaptor/scaffold proteins or cell cycle regulation proteins (Rows 2-28 and Rows 41-54, respectively, of Table 1), that is phosphorylated in a disease signaling pathway, said method comprising the step of utilizing one or more isolated antibody that specifically binds the protein group selected from Column A of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, of Table 1, comprised within the phosphorylation site sequence listed in corresponding Column E, to detect the phosphorylation of one or more of said protein group, thereby obtaining a phosphorylation profile for said protein group.
The invention further contemplates compositions, foremost pharmaceutical compositions, containing one or a more antibody according to the invention formulated together with a pharmaceutically acceptable carrier. One of skill will appreciate that in certain instances the composition of the invention may further comprise other pharmaceutically active moieties. The compounds according to the invention are optionally formulated in a pharmaceutically acceptable vehicle with any of the well-known pharmaceutically acceptable carriers, including diluents and excipients (see Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, Mack Publishing Co., Easton, Pa. 1990 and Remington: The Science and Practice of Pharmacy, Lippincott, Williams & Wilkins, 1995). While the type of pharmaceutically acceptable carrier/vehicle employed in generating the compositions of the invention will vary depending upon the mode of administration of the composition to a mammal, generally pharmaceutically acceptable carriers are physiologically inert and non-toxic. Formulations of compositions according to the invention may contain more than one type of compound of the invention), as well any other pharmacologically active ingredient useful for the treatment of the symptom/condition being treated.
The invention also provides methods of treating a mammal comprising the step of administering such a mammal a therapeutically effective amount of a composition according to the invention.
As used herein, by “treating” is meant reducing, preventing, and/or reversing the symptoms in the individual to which a compound of the invention has been administered, as compared to the symptoms of an individual not being treated according to the invention. A practitioner will appreciate that the compounds, compositions, and methods described herein are to be used in concomitance with continuous clinical evaluations by a skilled practitioner (physician or veterinarian) to determine subsequent therapy. Hence, following treatment the practitioners will evaluate any improvement in the treatment of the pulmonary inflammation according to standard methodologies. Such evaluation will aid and inform in evaluating whether to increase, reduce or continue a particular treatment dose, mode of administration, etc. The term “therapeutic composition” refers to any compounds administered to treat or prevent a disease. It will be understood that the subject to which a compound (e.g., an antibody) of the invention is administered need not suffer from a specific traumatic state. Indeed, the compounds (e.g., antibodies) of the invention may be administered prophylactically, prior to any development of symptoms. The term “therapeutic,” “therapeutically,” and permutations of these terms are used to encompass therapeutic, palliative as well as prophylactic uses. Hence, as used herein, by “treating or alleviating the symptoms” is meant reducing, preventing, and/or reversing the symptoms of the individual to which a compound of the invention has been administered, as compared to the symptoms of an individual receiving no such administration.
The term “therapeutically effective amount” is used to denote treatments at dosages effective to achieve the therapeutic result sought. Furthermore, one of skill will appreciate that the therapeutically effective amount of the compound of the invention may be lowered or increased by fine tuning and/or by administering more than one compound of the invention, or by administering a compound of the invention with another compound. See, for example, Meiner, C. L., “Clinical Trials: Design. Conduct, and Analysis,” Monographs in Epidemiology and Biostatistics, Vol. 8 Oxford University Press, USA (1986). The invention therefore provides a method to tailor the administration/treatment to the particular exigencies specific to a given mammal. As illustrated in the following examples, therapeutically effective amounts may be easily determined for example empirically by starting at relatively low amounts and by step-wise increments with concurrent evaluation of beneficial effect.
The short name for each protein in which a phosphorylation site has presently been identified is provided in Column A, and its SwissProt accession number (human) is provided Column B. The protein type/group into which each protein falls is provided in Column C. The identified tyrosine residue at which phosphorylation occurs in a given protein is identified in Column D, and the amino acid sequence of the phosphorylation site encompassing the tyrosine residue is provided in Column E (lower case y=the tyrosine (identified in Column D)) at which phosphorylation occurs. Table 1 above is identical to
One of skill in the art will appreciate that, in many instances the utility of the instant invention is best understood in conjunction with an appreciation of the many biological roles and significance of the various target signaling proteins/polypeptides of the invention. The foregoing is illustrated in the following paragraphs summarizing the knowledge in the art relevant to a few non-limiting representative peptides containing selected phosphorylation sites according to the invention.
MYH10 (P35580), phosphorylated at Y13, Y194, Y1415, is among the proteins listed in this patent. MYH10, Myosin heavy chain 10 (non-muscle), a putative ATP- and actin-binding motor protein, expression of an alternative splice form is coincident with neuronal cell differentiation; mRNA is upregulated during coronary restenosis. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
NCK2 (O43639), phosphorylated at Y50, Y342, is among the proteins listed in this patent. NCK2, NCK adaptor protein 2, SH2/SH3 adaptor protein, binds PDGFR-beta (PDGFRB) and TrkB (NTRK2), inhibits EGF- and PDGF-stimulated DNA synthesis and PDGF-mediated actin polymerization, may modulate activity at promoters regulated by FOS and JUN. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
NFAT1 (Q13469), phosphorylated at Y346, Y860, is among the proteins listed in this patent. NFAT1, Nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 2, a calcineurin-dependent transcription factor that interacts with coactivators to regulate expression of cytokines and other genes, plays a role in T-cell activation. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
NFkB-p105 (P19838), phosphorylated at Y240, is among the proteins listed in this patent. NFkB-p105, Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, the p50 component of the NFkB transcription factor, involved in the immune response and inflammation; increased activation is linked to HIV infections and cancer. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Increased nucleus localization of NFKB1 may cause decreased induction of apoptosis by extracellular signals associated with prostatic neoplasms (Oncogene 21: 1759-67 (2002)). Increased transcription factor activity of NFKB1 may cause decreased cell death associated with squamous cell carcinoma (Cancer Res 59: 3468-74 (1999)). Increased transcription factor activity of NFKB1 correlates with arteriosclerosis (PNAS 101: 5634-9 (2004)). Increased nucleus localization of NFKB1 may cause abnormal smooth muscle cells function associated with arteriosclerosis (JBC 272: 15817-24 (1997)). Increased nucleus localization of NFKB1 correlates with advanced stage or high grade form of squamous cell carcinoma (Oncogene 22: 50-8 (2003)). Increased double-stranded DNA binding of NFKB1 may cause disease progression associated with prostatic neoplasms (MCB 22: 2862-70 (2002)). Increased transcription factor activity of NFKB1 correlates with increased cell proliferation associated with chronic B-cell leukemia (Leukemia 18: 1391-400 (2004)). Increased transcription factor activity of NFKB1 may cause inflammation associated with arteriosclerosis (Proc Natl Acad Sci USA 101: 5634-9 (2004)). Induced inhibition of the DNA binding of NFKB1 may prevent decreased response to drug associated with chronic B-cell leukemia (Blood 92: 990-5 (1998)). Induced inhibition of the DNA binding of NFKB1 may prevent decreased apoptosis associated with chronic B-cell leukemia (Blood 92: 990-5 (1998)). Increased DNA binding of NFKB1 correlates with decreased cell death associated with chronic B-cell leukemia (J Immunol 164: 2200-6 (2000)). Increased transcription factor activity of NFKB1 may cause increased interleukin-6 biosynthetic process associated with prostatic neoplasms (Cancer Res 63: 2206-15 (2003)). Increased nucleus localization of NFKB1 correlates with breast neoplasms (Oncogene 19: 1123-31 (2000)). Decreased transcription factor activity of NFKB1 may prevent decreased induction of apoptosis associated with pancreatic neoplasms (Oncogene 21: 6510-9 (2002)). Increased DNA binding of NFKB1 correlates with abnormal B-lymphocytes differentiation associated with chronic B-cell leukemia (J Immunol 164: 2200-6 (2000)). Increased DNA binding of NFKB1 correlates with increased severity of cervix neoplasms associated with squamous cell carcinoma (Oncogene 22: 50-8 (2003)). Decreased nucleus localization of NFKB1 may prevent decreased induction of apoptosis by extracellular signals associated with prostatic neoplasms (Anticancer Res 23: 3855-61 (2003)). Abnormal expression of NFKB1 in smooth muscle cells may cause increased 1-kappaB kinase/NF-kappaB cascade associated with arteriosclerosis (J Biol Chem 272: 15817-24 (1997)). Increased DNA binding of NFKB1 may correlate with head and neck neoplasms associated with squamous cell carcinoma (Cancer Res 59: 3468-74 (1999)). Increased nucleus localization of NFKB1 may cause increased 1-kappaB kinase/NF-kappaB cascade associated with melanoma (Cancer Res 59: 1372-7 (1999)). Increased transcription factor activity of NFKB1 may cause increased cytokine production associated with arteriosclerosis (Proc Natl Acad Sci USA 101: 5634-9 (2004)). Increased DNA binding of NFKB1 may cause increased cell proliferation associated with squamous cell carcinoma (Cancer Res 59: 3468-74 (1999)). Abnormal expression of NFKB1 in smooth muscle cells may cause increased inflammatory response associated with arteriosclerosis (J Biol Chem 272: 15817-24 (1997)). Increased nucleus localization of NFKB1 may cause increased positive regulation of transcription from RNA polymerase II promoter associated with melanoma (Cancer Res 59: 1372-7 (1999)). Abnormal expression of NFKB1 in smooth muscle cells may cause increased activation of NF-kappaB transcription factor associated with arteriosclerosis (J Biol Chem 272: 15817-24 (1997)). Decreased transcription factor activity of NFKB1 may prevent decreased response to drug associated with pancreatic neoplasms (Oncogene 21: 6510-9 (2002)). Abnormal expression of NFKB1 in smooth muscle cells may cause increased activation of NF-kappaB transcription factor associated with arteriosclerosis (JBC 272: 15817-24 (1997)). Increased transcription factor activity of NFKB1 may cause increased cytokine production associated with arteriosclerosis (PNAS 101: 5634-9 (2004)). Decreased nucleus localization of NFKB1 may correlate with increased response to drug associated with prostatic neoplasms (Oncogene 21: 1759-67 (2002)). Increased double-stranded DNA binding of NFKB1 may cause disease progression associated with prostatic neoplasms (Mol Cell Biol 22: 2862-70 (2002)). Increased transcription factor activity of NFKB1 may cause increased cell proliferation associated with squamous cell carcinoma (Cancer Res 59: 3468-74 (1999)). Increased DNA binding of NFKB1 may cause decreased cell death associated with squamous cell carcinoma (Cancer Res 59: 3468-74 (1999)). Increased DNA binding of NFKB1 correlates with increased cell-matrix adhesion associated with multiple myeloma (Oncogene 22: 2417-21 (2003)). Increased nucleus localization of NFKB1 may correlate with HIV infections (J Immunol 155: 4861-7 (1995)). Increased transcription factor activity of NFKB1 may cause inflammation associated with arteriosclerosis (PNAS 101: 5634-9 (2004)). Increased transcription factor activity of NFKB1 correlates with decreased apoptosis associated with chronic B-cell leukemia (Leukemia 18: 1391-400 (2004)). Abnormal expression of NFKB1 in smooth muscle cells may cause increased inflammatory response associated with arteriosclerosis (JBC 272: 15817-24 (1997)). Decreased nucleus localization of NFKB1 may prevent increased cell proliferation associated with prostatic neoplasms (Anticancer Res 23: 3855-61 (2003)). Increased transcription factor activity of NFKB1 correlates with arteriosclerosis (Proc Natl Acad Sci USA 101: 5634-9 (2004)). Increased double-stranded DNA binding of NFKB1 may cause disease progression associated with prostatic neoplasms (Mol. Cell. Biol 22: 2862-70 (2002)). Increased transcription factor activity of NFKB1 may cause thrombosis associated with arteriosclerosis (Proc Natl Acad Sci USA 101: 5634-9 (2004)). Increased nucleus localization of NFKB1 may cause increased I-kappaB kinase/NF-kappaB cascade associated with arteriosclerosis (JBC 272: 15817-24 (1997)). Increased nucleus localization of NFKB1 may cause increased inflammatory response associated with arteriosclerosis (J Biol Chem 272: 15817-24 (1997)). Increased transcription factor activity of NFKB1 may cause increased cytokine production associated with arteriosclerosis (Proc Natl Acad Sci USA 101: 5634-9 (2004)). Increased nucleus localization of NFKB1 may correlate with increased cytokine and chemokine mediated signaling pathway associated with arteriosclerosis (J Biol Chem 272: 15817-24 (1997)). Increased nitration of NFKB1 may prevent increased 1-kappaB kinase/NF-kappaB cascade associated with prostatic neoplasms (Oncogene 23: 4993-5003 (2004)). Increased transcription factor activity of NFKB1 may cause decreased occurrence of hormone-dependent neoplasms associated with prostatic neoplasms (J Cell Sci 115: 141-51 (2002)). Increased nucleus localization of NFKB1 may correlate with increased 1-kappaB kinase/NF-kappaB cascade associated with prostatic neoplasms (Mol Carcinog 39: 114-26 (2004)). Increased expression of NFKB1 in macrophages may correlate with HIV infections (J Virol 69: 1500-9 (1995)). Decreased expression of NFKB1 protein may prevent HIV infections (J Immunol 152: 4183-91 (1994)). Abnormal expression of NFKB1 in smooth muscle cells may correlate with increased cytokine and chemokine mediated signaling pathway associated with arteriosclerosis (J Biol Chem 272: 15817-24 (1997)). Increased DNA binding of NFKB1 correlates with advanced stage or high grade form of squamous cell carcinoma (Oncogene 22: 50-8 (2003)). Increased transcription factor activity of NFKB1 may correlate with head and neck neoplasms associated with squamous cell carcinoma (Cancer Res 59: 3468-74 (1999)). Increased transcription factor activity of NFKB1 may cause inflammation associated with arteriosclerosis (Proc Natl Acad Sci USA 101: 5634-9 (2004)). Increased nucleus localization of NFKB1 may cause abnormal smooth muscle cells function associated with arteriosclerosis (J Biol Chem 272: 15817-24 (1997)). Increased transcription factor activity of NFKB1 may cause increased interleukin-8 biosynthetic process associated with squamous cell carcinoma (Mol Carcinog 26: 119-29 (1999)). Increased transcription factor activity of NFKB1 may cause decreased occurrence of hormone-dependent neoplasms associated with prostatic neoplasms (J Cell Sci 115: 141-51 (2002)). Increased transcription factor activity of NFKB1 may cause thrombosis associated with arteriosclerosis (PNAS 101: 5634-9 (2004)). Increased DNA binding of NFKB1 correlates with increased release of cytoplasmic sequestered NF-kappaB associated with squamous cell carcinoma (Oncogene 22: 50-8 (2003)). Increased double-stranded DNA binding of NFKB1 may cause disease progression associated with prostatic neoplasms (Mol Cell Biol. 22: 2862-70 (2002)). Increased DNA binding of NFKB1 may cause increased cytokine production associated with squamous cell carcinoma (Cancer Res 59: 3468-74 (1999)). Increased DNA binding of NFKB1 may cause increased interleukin-8 biosynthetic process associated with squamous cell carcinoma (Mol Carcinog 26: 119-29 (1999)). Increased nucleus localization of NFKB1 may cause increased activation of NF-kappaB transcription factor associated with arteriosclerosis (J Biol Chem 272: 15817-24 (1997)). Increased nucleus localization of NFKB1 may cause increased inflammatory response associated with arteriosclerosis (JBC 272: 15817-24 (1997)). Increased transcription factor activity of NFKB1 may cause increased cytokine production associated with squamous cell carcinoma (Cancer Res 59: 3468-74 (1999)). Abnormal expression of NFKB1 in smooth muscle cells may correlate with increased cytokine and chemokine mediated signaling pathway associated with arteriosclerosis (JBC 272: 15817-24 (1997)). Increased double-stranded DNA binding of NFKB1 may cause disease progression associated with prostatic neoplasms (Mol. Cell. Biol. 22: 2862-70 (2002)). Increased expression of NFKB1 protein correlates with non-small-cell lung carcinoma (Oncogene 11: 999-1003 (1995)). Increased transcription factor activity of NFKB1 correlates with abnormal epidermal growth factor receptor signaling pathway associated with pancreatic neoplasms (Int J Cancer 105: 735-46 (2003)). Increased nucleus localization of NFKB1 may cause increased 1-kappaB kinase/NF-kappaB cascade associated with arteriosclerosis (J Biol Chem 272: 15817-24 (1997)). Increased nucleus localization of NFKB1 may cause increased activation of NF-kappaB transcription factor associated with arteriosclerosis (JBC 272: 15817-24 (1997)). Increased transcription factor activity of NFKB1 correlates with arteriosclerosis (Proc Natl Acad Sci USA 101: 5634-9 (2004)). Abnormal expression of NFKB1 in smooth muscle cells may cause increased 1-kappaB kinase/NF-kappaB cascade associated with arteriosclerosis (JBC 272: 15817-24 (1997)). Increased nucleus localization of NFKB1 correlates with increased severity of cervix neoplasms associated with squamous cell carcinoma (Oncogene 22: 50-8 (2003)). Increased expression of NFKB1 in monocytes may correlate with HIV infections (J Virol 69: 1500-9 (1995)). Increased nucleus localization of NFKB1 may correlate with increased cytokine and chemokine mediated signaling pathway associated with arteriosclerosis (JBC 272: 15817-24 (1997)). Increased nitration of NFKB1 may prevent decreased induction of apoptosis by extracellular signals associated with prostatic neoplasms (Oncogene 23: 4993-5003 (2004)). Increased DNA binding of NFKB1 may correlate with decreased induction of apoptosis associated with multiple myeloma (Blood 93: 3044-52 (1999)). Increased DNA binding of NFKB1 correlates with increased anti-apoptosis associated with multiple myeloma (Oncogene 22: 2417-21 (2003)). Increased transcription factor activity of NFKB1 may correlate with increased interleukin-1 alpha secretion associated with pancreatic neoplasms (J Biol Chem 279: 16452-62 (2004)). Increased transcription factor activity of NFKB1 may correlate with increased interleukin-1 alpha secretion associated with pancreatic neoplasms (JBC 279: 16452-62 (2004)). Increased nucleus localization of NFKB1 correlates with increased release of cytoplasmic sequestered NF-kappaB associated with squamous cell carcinoma (Oncogene 22: 50-8 (2003)). Increased DNA binding of NFKB1 may cause drug-resistant form of multiple myeloma (Blood 93: 3044-52 (1999)). Increased transcription factor activity of NFKB1 may cause thrombosis associated with arteriosclerosis (Proc Natl Acad Sci USA 101: 5634-9 (2004)). Increased nucleus localization of NFKB1 may correlate with increased 1-kappaB phosphorylation associated with melanoma (Cancer Res 61: 4901-9 (2001)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
NONO (Q15233), phosphorylated at Y265, is among the proteins listed in this patent. NONO, Non-POU-domain-containing octamer-binding, transcriptional co-activator with the androgen receptor, upregulated in prostate cancer, downregulated in EBV-infected nasopharyngeal carcinoma cells, fused with TFE3 in papillary renal cell carcinoma. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Translocation of the NONO gene may cause neoplastic cell transformation associated with renal cell carcinoma (Oncogene 15: 2233-9 (1997)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PABP 1 (P11940), phosphorylated at Y297, Y382, is among the proteins listed in this patent. PABP 1, Poly(A)-binding protein cytoplasmic 1, binds mRNA poly(A) tails, plays roles in regulating mRNA stability, translation, and perhaps transport from the nucleus to cytoplasm, degraded by specific viral proteases resulting in host protein synthesis shutoff. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Viral exploitation of the PABPC1 protein may cause increased suppression by virus of host termination of protein biosynthetic process associated with poliomyelitis (J Virol 73: 718-27 (1999)). Increased proteolysis of PABPC1 may cause increased suppression by virus of host termination of protein biosynthetic process associated with coxsackievirus infections (J Virol 73: 709-17 (1999)). Viral exploitation of the PABPC1 protein may cause increased suppression by virus of host termination of protein biosynthetic process associated with coxsackievirus infections (J Virol 73: 709-17 (1999)). Increased proteolysis of PABPC1 may cause increased suppression by virus of host termination of protein biosynthetic process associated with poliomyelitis (J Virol 73: 718-27 (1999)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PAK1 (Q13153), phosphorylated at Y142, Y153, is among the proteins listed in this patent. PAK1, p21 activated kinase 1, a serine-threonine kinase activated by GTPases CDC42 and RAC1, serves in MAP kinase cascade regulation, cytoskeletal organization, cell migration and apoptosis, increased activity may correlate with breast cancer invasiveness. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Decreased expression of PAK1 in frontal cortex correlates with encephalitis associated with HIV infections (J Neuroimmunol 157: 163-75 (2004)). Increased expression of PAK1 mRNA correlates with breast neoplasms (J Biol Chem 279: 1422-8 (2004)). Increased phosphorylation of PAK1 may cause increased severity of neoplastic cell transformation associated with breast neoplasms (J Biol Chem 276: 29403-9 (2001)). Increased expression of PAK1 mRNA correlates with breast neoplasms (JBC 279: 1422-8 (2004)). Loss of function mutation in the Protein kinase domain of PAK1 may prevent invasive form of breast neoplasms (JBC 275: 12041-50 (2000)). Loss of function mutation in the Protein kinase domain of PAK1 may prevent invasive form of breast neoplasms (J Biol Chem 275: 12041-50 (2000)). Increased protein kinase activity of PAK1 correlates with increased severity of invasive form of breast neoplasms (J Biol Chem 275: 36238-36244 (2000)). Absence of the protein kinase activity of PAK1 may cause increased actin filament polymerization associated with breast neoplasms (J Biol Chem 275: 12041-50 (2000)). Amplification of the PAK1 gene correlates with mycosis fungoides associated with skin neoplasms (Blood 101: 1513-9 (2003)). Amplification of the PAK1 gene correlates with Sezary syndrome associated with skin neoplasms (Blood 101: 1513-9 (2003)). Increased protein kinase activity of PAK1 may cause abnormal mitotic spindle organization and biogenesis associated with breast neoplasms (JBC 275: 36238-36244 (2000)). Absence of the protein kinase activity of PAK1 may prevent invasive form of breast neoplasms (JBC 275: 12041-50 (2000)). Increased phosphorylation of PAK1 may cause increased severity of neoplastic cell transformation associated with breast neoplasms (JBC 276: 29403-9 (2001)). Absence of the protein kinase activity of PAK1 may cause increased actin filament polymerization associated with breast neoplasms (JBC 275: 12041-50 (2000)). Absence of the protein kinase activity of PAK1 may prevent invasive form of breast neoplasms (J Biol Chem 275: 12041-50 (2000)). Increased protein kinase activity of PAK1 may cause abnormal mitotic spindle organization and biogenesis associated with breast neoplasms (J Biol Chem 275: 36238-36244 (2000)). Increased protein kinase activity of PAK1 may cause increased cell motility associated with breast neoplasms (J Biol Chem 275: 36238-36244 (2000)). Increased protein kinase activity of PAK1 may cause increased cell motility associated with breast neoplasms (JBC 275: 36238-36244 (2000)). Amplification of the PAK1 gene may correlate with breast neoplasms (Cytogenet Cell Genet. 79: 125-31 (1997)). Increased protein kinase activity of PAK1 correlates with increased severity of invasive form of breast neoplasms (JBC 275: 36238-36244 (2000)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PAK2 (Q13177), phosphorylated at Y252, is among the proteins listed in this patent. PAK2, p21-activated kinase 2, a protein serine-threonine kinase that autophosphorylates and autoactivates, acts as a modulator of Myc, RAC1 and myosin 2 activities, may be activated by HIV infection; gene is associated with 3q29 microdeletion syndrome. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Deletion mutation in the PAK2 gene correlates with chromosome deletion associated with mental retardation (Am J Hum Genet. 77: 154-60 (2005)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PARP1 (P09874), phosphorylated at Y774, is among the proteins listed in this patent. PARP1, Poly (ADP-ribose) polymerase family member 1, catalyzes formation of ADP ribose polymers in response to DNA damage, acts transcriptional regulation of nuclear-receptor dependent promoters, increased proteolysis may be therapeutic for colon cancer. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Increased cleavage of PARP1 may correlate with increased response to drug associated with prostatic neoplasms (Cancer Res 63: 4713-23 (2003)). Increased cleavage of PARP1 may correlate with increased response to drug associated with ovarian neoplasms (Oncogene 21: 4530-8 (2002)). Increased proteolysis of PARP1 may correlate with increased apoptosis associated with breast neoplasms (Exp Cell Res 255: 144-55 (2000)). Increased expression of PARP1 protein may correlate with leukemia (Cancer Lett 58: 131-5 (1991)). Increased cleavage of PARP1 may correlate with increased response to drug associated with ovarian neoplasms (Oncogene 21: 1-8 (2002)). Increased cleavage of PARP1 may correlate with increased response to drug associated with glioma (J Cell Physiol 201: 374-84 (2004)). Increased expression of PARP1 mutant protein may prevent prostatic neoplasms (Cancer Res 62: 6879-83 (2002)). Increased cleavage of PARP1 may correlate with increased apoptosis associated with prostatic neoplasms (Cancer Res 63: 4713-23 (2003)). Increased proteolysis of PARP1 may correlate with increased apoptosis associated with breast neoplasms (Oncogene 20: 8258-69 (2001)). Decreased expression of PARP1 mRNA correlates with disease progression associated with chronic lymphocytic leukemia (Leukemia 15: 1721-8 (2001)). Increased expression of PARP1 protein may correlate with ovarian neoplasms (Cancer Lett 58: 131-5 (1991)). Increased expression of PARP1 protein correlates with more severe form of B-cell lymphoma (Mol Carcinog 25: 256-61 (1999)). Increased cleavage of PARP1 may correlate with increased response to drug associated with prostatic neoplasms (Mol Carcinog 39: 114-26 (2004)). Increased proteolysis of PARP1 may correlate with increased apoptosis associated with lung neoplasms (Anticancer Res 21: 39-44 (2001)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PIK3C2A (O00443), phosphorylated at Y73, is among the proteins listed in this patent. PIK3C2A, Phosphoinositide-3-kinase class 2 alpha polypeptide, phosphorylates only PtdIns and PtdIns4P in the absence of phosphatidylserine but phosphorylates PtdIns(4,5)P2 in the presence of phosphatidylserine, exhibits insensitivity to wortmannin. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PIK3C2B (O00750), phosphorylated at Y1541, is among the proteins listed in this patent. PIK3C2B, Phosphoinositide-3-kinase class 2 beta polypeptide, a nuclear enzyme catalyzing phosphorylation of phosphatidylinositol and phosphatidylinositol 4 monophosphate, altered by nitrotyrosylation in platelets from patients with systemic sclerosis. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PIK3CA (P42336), phosphorylated at Y246, Y361, is among the proteins listed in this patent. PIK3CA, Phosphatidylinositol 3-kinase catalytic alpha polypeptide, heterodimerizes with an 85-kDa regulatory subunit that binds the kinase to receptors for signal transduction, altered expression and activity are involved in cancer progression. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Mutation in the PIK3CA gene correlates with medulloblastoma associated with brain neoplasms (Cancer Res 64: 5048-50 (2004)). Amplification of the PIK3CA gene may cause squamous cell carcinoma (Eur J Cancer 35: 641-6 (1999)). Amplification of the PIK3CA gene correlates with carcinoma tumors associated with cervix neoplasms (Int J Cancer 101: 427-33 (2002)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma associated with head and neck neoplasms (Gene Develop 16: 984-93 (2002)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma associated with lung neoplasms (Genes Dev 16: 984-93 (2002)). Increased phosphatidylinositol 3-kinase activity of PIK3CA may cause increased anti-apoptosis associated with head and neck neoplasms (Cancer Res 61: 4122-9 (2001)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma associated with head and neck neoplasms (Gene Develop 16: 984-93 (2002)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma associated with head and neck neoplasms (Genes Dev 16: 984-93 (2002)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma (Cancer Res 61: 4122-9 (2001)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma associated with esophageal neoplasms (Cancer Res 63: 5691-6 (2003)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma associated with head and neck neoplasms (Genes Dev 16: 984-93 (2002)). Increased expression of PIK3CA mRNA correlates with invasive form of ovarian neoplasms (Cancer Res 63: 4225-31 (2003)). Mutation in the PIK3CA gene correlates with carcinoma tumors associated with breast neoplasms (Cancer Res 65: 2554-9 (2005)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma associated with lung neoplasms (Gene Develop 16: 984-93 (2002)). Increased expression of PIK3CA mRNA correlates with carcinoma tumors associated with stomach neoplasms (Int J Cancer 104: 318-27 (2003)). Increased expression of PIK3CA mRNA correlates with carcinoma tumors associated with cervix neoplasms (Int J Cancer 101: 427-33 (2002)). Increased expression of PIK3CA mRNA correlates with increased severity of carcinoma associated with ovarian neoplasms (Cancer Res 63: 4225-31 (2003)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma associated with lung neoplasms (Genes Dev. 16: 984-93 (2002)). Increased expression of PIK3CA mRNA correlates with decreased apoptosis associated with ovarian neoplasms (Cancer Res 63: 4225-31 (2003)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma associated with lung neoplasms (Genes Dev 16: 984-93 (2002)). Amplification of the PIK3CA mRNA correlates with squamous cell carcinoma associated with head and neck neoplasms (Cancer Res 61: 4122-9 (2001)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma associated with lung neoplasms (Genes Dev. 16: 984-93 (2002)). Increased expression of PIK3CA protein may correlate with invasive form of colonic neoplasms (FASEB J 14: 2329-38 (2000)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma associated with lung neoplasms (Gene Develop 16: 984-93 (2002)). Amplification of the PIK3CA gene correlates with carcinoma tumors associated with stomach neoplasms (Int J Cancer 104: 318-27 (2003)). Mutation in the PIK3CA gene correlates with lymphatic metastasis associated with breast neoplasms (Cancer Res 65: 2554-9 (2005)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma associated with lung neoplasms (Genes Dev 16: 984-93 (2002)). Increased phosphatidylinositol 3-kinase activity of PIK3CA correlates with colonic neoplasms (Oncogene 19: 5083-90 (2000)). Increased expression of PIK3CA mRNA correlates with increased cell proliferation associated with ovarian neoplasms (Cancer Res 63: 4225-31 (2003)). Increased expression of PIK3CA mRNA correlates with esophageal neoplasms associated with squamous cell carcinoma (Cancer Res 63: 5691-6 (2003)). Increased expression of PIK3CA protein may cause increased cell proliferation associated with ovarian neoplasms (Nat Genet. 21: 99-102 (1999)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma associated with head and neck neoplasms (Genes Dev 16: 984-93 (2002)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma tumors associated with lung neoplasms (Eur J Cancer 35: 641-6 (1999)). Amplification of the PIK3CA gene correlates with papillomavirus infections associated with cervix neoplasms (Int J Cancer 101: 427-33 (2002)). Amplification of the PIK3CA gene correlates with ovarian neoplasms (Nat Genet. 21: 99-102 (1999)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma associated with non-small-cell lung carcinoma (Cancer Res 62: 3636-40 (2002)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma associated with head and neck neoplasms (Genes Dev. 16: 984-93 (2002)). Increased expression of PIK3CA mRNA may cause increased cell proliferation associated with cervix neoplasms (Oncogene 19: 2739-44 (2000)). Mutation in the PIK3CA gene correlates with glioblastoma associated with brain neoplasms (Cancer Res 64: 5048-50 (2004)). Increased phosphatidylinositol 3-kinase activity of PIK3CA may cause increased anti-apoptosis associated with squamous cell carcinoma (Cancer Res 61: 4122-9 (2001)). Increased expression of PIK3CA mRNA correlates with adenocarcinoma tumors associated with lung neoplasms (Cancer Res 61: 4122-9 (2001)). Increased expression of PIK3CA protein may correlate with invasive form of colonic neoplasms (FASEB 14: 2329-38 (2000)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma associated with head and neck neoplasms (Genes Dev. 16: 984-93 (2002)). Increased expression of PIK3CA mRNA may cause increased angiogenesis associated with ovarian neoplasms (Cancer Res 63: 4225-31 (2003)). Increased expression of PIK3CA mRNA correlates with papillomavirus infections associated with cervix neoplasms (Int J Cancer 101: 427-33 (2002)). Increased expression of PIK3CA protein may cause increased anti-apoptosis associated with ovarian neoplasms (Nat Genet. 21: 99-102 (1999)). Increased expression of PIK3CA protein correlates with colonic neoplasms (Oncogene 19: 5083-90 (2000)). Increased phosphatidylinositol 3-kinase activity of PIK3CA may cause increased anti-apoptosis associated with lung neoplasms (Cancer Res 61: 4122-9 (2001)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma associated with lung neoplasms (Genes Dev 16: 984-93 (2002)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma associated with lung neoplasms (Cancer Res 62: 3636-40 (2002)). Amplification of the PIK3CA gene correlates with squamous cell carcinoma associated with head and neck neoplasms (Genes Dev 16: 984-93 (2002)). Increased expression of PIK3CA mRNA correlates with squamous cell carcinoma associated with head and neck neoplasms (Cancer Res 61: 4122-9 (2001)). Mutation in the PIK3CA gene correlates with oligodendroglioma associated with brain neoplasms (Cancer Res 64: 5048-50 (2004)). Amplification of the PIK3CA gene correlates with cervix neoplasms (Oncogene 19: 2739-44 (2000)). Increased expression of PIK3CA mRNA may cause increased anti-apoptosis associated with cervix neoplasms (Oncogene 19: 2739-44 (2000)). Mutation in the PIK3CA gene correlates with astrocytoma associated with brain neoplasms (Cancer Res 64: 5048-50 (2004)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PIK3R1 (P27986), phosphorylated at Y426, Y504, Y657, is among the proteins listed in this patent. PIK3R1, Phosphoinositide-3-kinase regulatory subunit polypeptide 1 (p85 alpha), involved in insulin receptor signaling, inhibition may be therapeutic for invasive breast cancer; mutation of corresponding gene is associated with type II diabetes and some cancers. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Decreased insulin receptor signaling pathway associated with PIK3R1 correlates with type II diabetes mellitus (Diabetes 50: 1134-42 (2001)). Increased expression of PIK3R1 mutant protein may prevent increased cell motility associated with breast neoplasms (J Biol Chem 277: 3150-7 (2002)). Increased 1-kappaB kinase/NF-kappaB cascade associated with PIK3R1 may correlate with increased cell differentiation associated with colonic neoplasms (Biochem Biophys Res Commun 273: 853-8 (2000)). Deletion mutation in the PIK3R1 gene correlates with carcinoma tumors associated with colonic neoplasms (Cancer Res 61: 7426-9 (2001)). Decreased expression of PIK3R1 protein may prevent neoplasm metastasis associated with ovarian neoplasms (JBC 279: 6371-9 (2004)). Decreased expression of PIK3R1 protein may prevent neoplasm metastasis associated with ovarian neoplasms (J Biol Chem 279: 6371-9 (2004)). Decreased transmembrane receptor protein tyrosine kinase signaling pathway associated with PIK3R1 may correlate with decreased cell proliferation associated with breast neoplasms (Cancer Res 62: 4132-41 (2002)). Decreased expression of PIK3R1 protein may prevent increased cell migration associated with ovarian neoplasms (JBC 279: 6371-9 (2004)). Increased expression of PIK3R1 mutant protein may prevent increased cell motility associated with breast neoplasms (JBC 277: 3150-7 (2002)). Frameshift mutation in the PIK3R1 gene may correlate with Hodgkin's disease (Leukemia 16: 894-901 (2002)). Deletion mutation in the PIK3R1 gene correlates with carcinoma tumors associated with ovarian neoplasms (Cancer Res 61: 7426-9 (2001)). Decreased expression of PIK3R1 protein may prevent increased cell migration associated with ovarian neoplasms (J Biol Chem 279: 6371-9 (2004)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PIK3R2 (O00459), phosphorylated at Y423, Y577, is among the proteins listed in this patent. PIK3R2, Phosphoinositide-3-kinase regulatory polypeptide 2, a regulatory subunit of phosphatidylinositol 3-kinase that acts in signal transduction, cell motility and differentiation; tumorigenic fusion to USP8 gene may lead to chronic myeloproliferative disorder. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PIK3R3 (Q92569), phosphorylated at Y184, Y202, is among the proteins listed in this patent. PIK3R3, Phosphoinositide-3-kinase regulatory subunit 3, binds insulin receptor (INSR) and insulin-like growth factor receptor (IGF1R), acts in cell cycle regulation, expression is induced in highly tumorigenic breast cancer cells treated with doxorubicin. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PKCA (P17252), phosphorylated at Y194, is among the proteins listed in this patent. PKCA, Protein kinase C alpha isoform, important for cellular signaling, regulates cell proliferation and migration, and plays a role in RHO protein signal transduction; upregulated in liver of patients with non insulin dependent diabetes mellitus (NIDDM). This protein has potential diagnostic and/or therapeutic implications based on the following findings. Increased membrane localization of PRKCA may prevent increased cell migration associated with breast neoplasms (Biochem Biophys Res Commun 307: 839-46 (2003)). Decreased membrane localization of PRKCA may correlate with increased anti-apoptosis associated with prostatic neoplasms (Cell Growth Differ 7: 419-28 (1996)). Induced inhibition of the integrin binding of PRKCA may prevent increased cell migration associated with breast neoplasms (Mol Cell Biol. 22: 5897-911 (2002)). Decreased expression of PRKCA mRNA may cause decreased protein amino acid phosphorylation associated with breast neoplasms (J Clin Invest 95: 1906-15 (1995)). Increased expression of PRKCA protein may cause increased cell cycle arrest associated with pancreatic neoplasms (J Cell Sci 113: 3025-35 (2000)). Increased cytosol localization of PRKCA may correlate with adenoma associated with colonic neoplasms (Int J Cancer 80: 47-53 (1999)). Increased expression of PRKCA mRNA may cause increased cell proliferation associated with breast neoplasms (J Clin Invest 95: 1906-15 (1995)). Induced inhibition of the integrin binding of PRKCA may prevent increased cell migration associated with breast neoplasms (MCB 22: 5897-911 (2002)). Induced stimulation of the protein kinase C activity of PRKCA may cause increased apoptosis associated with prostatic neoplasms (J Biol Chem 278: 33753-62 (2003)). Increased expression of PRKCA protein may cause increased cell cycle arrest associated with pancreatic neoplasms (J Cell Sci 113: 3025-35 (2000)). Induced stimulation of the protein kinase C activity of PRKCA may cause drug-resistant form of colonic neoplasms (Biochem Pharmacol 48: 375-81 (1994)). Increased expression of PRKCA protein may correlate with increased Ras protein signal transduction associated with colonic neoplasms (Cancer Res 53: 2762-70 (1993)). Increased protein kinase C activity of PRKCA may correlate with increased cytokine and chemokine mediated signaling pathway associated with multiple myeloma (JBC 277: 7875-81 (2002)). Induced inhibition of the integrin binding of PRKCA may prevent increased cell migration associated with breast neoplasms (Mol Cell Biol 22: 5897-911 (2002)). Lack of expression of PRKCA protein correlates with basal cell carcinoma tumors associated with skin neoplasms (Cancer Res 63: 4692-7 (2003)). Increased expression of PRKCA mRNA correlates with drug-resistant form of breast neoplasms (Br J Cancer 88: 1400-2 (2003)). Increased proteolysis of PRKCA may correlate with Alzheimer disease (Proc Natl Acad Sci USA 95: 5562-7 (1998)). Decreased expression of PRKCA mRNA may cause drug-sensitive form of breast neoplasms (J Clin Invest 95: 1906-15 (1995)). Decreased expression of PRKCA protein may correlate with small cell carcinoma (Cancer Res 51: 5514-9 (1991)). Increased proteolysis of PRKCA may correlate with Alzheimer disease (PNAS 95: 5562-7 (1998)). Decreased expression of PRKCA protein may correlate with drug-resistant form of small cell carcinoma (Cell Growth Differ 7: 1507-12 (1996)). Decreased expression of PRKCA protein correlates with advanced stage or high grade form of colorectal neoplasms (Biochem Mol Biol Int 44: 523-8 (1998)). Induced inhibition of the integrin binding of PRKCA may prevent increased cell migration associated with breast neoplasms (Mol. Cell. Biol 22: 5897-911 (2002)). Increased membrane localization of PRKCA correlates with type II diabetes mellitus (J Clin Invest 95: 2938-44 (1995)). Decreased expression of PRKCA protein may correlate with drug-resistant form of ovarian neoplasms (Int J Cancer 62: 457-60 (1995)). Increased proteolysis of PRKCA may correlate with Alzheimer disease (Proc Natl Acad Sci USA 95: 5562-7 (1998)). Decreased expression of PRKCA protein may cause decreased cell proliferation associated with pancreatic neoplasms (Gut 39: 255-61 (1996)). Induced inhibition of the integrin binding of PRKCA may prevent increased cell migration associated with breast neoplasms (Mol. Cell. Biol. 22: 5897-911 (2002)). Induced stimulation of the protein kinase C activity of PRKCA may cause increased apoptosis associated with prostatic neoplasms (JBC 278: 33753-62 (2003)). Increased protein kinase C activity of PRKCA may correlate with increased cytokine and chemokine mediated signaling pathway associated with multiple myeloma (J Biol Chem 277: 7875-81 (2002)). Increased protein kinase C activity of PRKCA may correlate with increased cell migration associated with multiple myeloma (JBC 277: 7875-81 (2002)). Increased expression of PRKCA mRNA correlates with decreased cell proliferation associated with breast neoplasms (J Cell Physiol 172: 306-13 (1997)). Induced inhibition of the protein kinase C activity of PRKCA may correlate with drug-resistant form of colonic neoplasms (Br J Cancer 78: 1283-7 (1998)). Increased membrane localization of PRKCA may correlate with increased apoptosis associated with prostatic neoplasms (Cell Growth Differ 7: 419-28 (1996)). Increased protein kinase C activity of PRKCA may correlate with increased cell migration associated with multiple myeloma (J Biol Chem 277: 7875-81 (2002)). Increased membrane localization of PRKCA may correlate with small cell carcinoma (Cell Growth Differ 6: 1627-34 (1995)). Decreased expression of PRKCA mRNA may prevent increased cell proliferation associated with lung neoplasms (Exp Cell Res 250: 253-63 (1999)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PKCB (P05771), phosphorylated at Y194, is among the proteins listed in this patent. PKCB, Protein kinase C beta 1, serine/threonine kinase that acts in the glucose response and proliferation, expression is altered in ALS, colon adenoma, heart failure, Huntington's disease and diabetic nephropathy; rat Prkcb1 is involved in diabetic nephropathy. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Increased expression of PRKCB1 protein correlates with invasive form of stomach neoplasms (J Natl Cancer Inst 85: 402-7 (1993)). Increased expression of PRKCB1 protein may cause increased caspase activation associated with myeloid leukemia (Oncogene 19: 3941-7 (2000)). Decreased expression of PRKCB1 protein may prevent stomach neoplasms (Oncogene 21: 6113-22 (2002)). Increased expression of PRKCB1 in mast cells may correlate with increased phagocytosis, engulfment associated with Escherichia coli infections (J Leukoc Biol 66: 1031-8 (1999)). Decreased membrane fraction localization of PRKCB1 correlates with adenoma tumors associated with colonic neoplasms (Int J Cancer 80: 47-53 (1999)). Increased expression of PRKCB1 in nephron, glomerulus correlates with defective nephron, glomerulus development associated with diabetic nephropathies (Kidney Int 66: 1107-14 (2004)). Increased protein kinase C activity of PRKCB1 may cause increased hyaluronan biosynthetic process associated with Graves' disease (J Cell Biochem 82: 58-67 (2001)). Decreased expression of PRKCB1 mRNA correlates with colonic neoplasms (Mol Carcinog 11: 197-203 (1994)). Increased expression of PRKCB1 protein correlates with non-familial form of amyotrophic lateral sclerosis (J Neurochem 85: 432-42 (2003)). Increased protein kinase C activity of PRKCB1 causes increased entry of virus into host cell associated with influenza (J Virol 77: 460-9 (2003)). Increased expression of PRKCB1 in mast cells may correlate with increased detection of bacterium associated with Escherichia coli infections (J Leukoc Biol 66: 1031-8 (1999)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PKCD (Q05655), phosphorylated at Y374, is among the proteins listed in this patent. PKCD, Protein kinase C delta, calcium-independent serine-threonine kinase, promotes apoptosis, phospholipid scrambling, and lamin cleavage, inhibits histamine signaling in myeloid cells, may function as a tumor suppressor. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Induced inhibition of the protein kinase activity of PRKCD may prevent increased anti-apoptosis associated with non-small-cell lung carcinoma (Cancer Res 63: 780-6 (2003)). Decreased cytosol localization of PRKCD may correlate with increased cell proliferation associated with prostatic neoplasms (Biochem Biophys Res Commun 283: 806-12 (2001)). Decreased expression of PRKCD mRNA may correlate with malignant form of neuroblastoma (EMBO J. 10: 1119-25 (1991)). Increased expression of PRKCD mRNA may prevent increased cell proliferation associated with glioma (Biochem Biophys Res Commun 201: 363-72 (1994)). Increased protein binding of PRKCD may prevent increased insulin-like growth factor receptor signaling pathway associated with renal cell carcinoma (JBC 275: 20700-6 (2000)). Decreased endoproteolysis of PRKCD may prevent increased anti-apoptosis associated with prostatic neoplasms (J Clin Invest 109: 827-36 (2002)). Decreased endoproteolysis of PRKCD may prevent increased anti-apoptosis associated with prostatic neoplasms (Cancer Res 60: 6590-6 (2000)). Increased expression of PRKCD mRNA may prevent increased anti-apoptosis associated with prostatic neoplasms (J Biol Chem 275: 7574-82 (2000)). Increased protein binding of PRKCD may prevent increased insulin-like growth factor receptor signaling pathway associated with renal cell carcinoma (J Biol Chem 275: 20700-6 (2000)). Decreased membrane fraction localization of PRKCD may correlate with increased cell proliferation associated with prostatic neoplasms (Biochem Biophys Res Commun 283: 806-12 (2001)). Increased expression of PRKCD mRNA may prevent increased anti-apoptosis associated with prostatic neoplasms (JBC 275: 7574-82 (2000)). Increased membrane fraction localization of PRKCD correlates with increased response to hypoxia associated with anoxia (J Cell Physiol 188: 223-35 (2001)). Increased expression of PRKCD protein may cause decreased severity of neoplastic processes associated with colonic neoplasms (Int J Cancer 113: 42-53 (2005)). Decreased expression of PRKCD mRNA may correlate with malignant form of neuroblastoma (EMBO 10: 1119-25 (1991)). Increased proteolysis of PRKCD may cause increased cell differentiation associated with melanoma (Biochem Pharmacol 55: 1691-9 (1998)). Decreased expression of PRKCD mRNA may correlate with malignant form of neuroblastoma (EMBO J. 10: 1119-25 (1991)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PKCT (Q04759), phosphorylated at Y545, is among the proteins listed in this patent. PKCT, Protein kinase C theta, involved in T cell activation and protection from apoptosis, may play a role in insulin and multidrug resistance; rat Pkcq may play roles in hyperglycemia, hypertriglyceridemia and insulin resistance. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Increased expression of PRKCQ mRNA correlates with recurrence associated with acute myelocytic leukemia (Leukemia 10: 426-33 (1996)). Decreased expression of PRKCQ protein correlates with insulin resistance associated with type II diabetes mellitus (Endocrinology 141: 2773-8 (2000)). Increased expression of PRKCQ protein correlates with sarcoma associated with gastrointestinal neoplasms (Cancer Res 64: 5127-31 (2004)). Decreased expression of PRKCQ protein correlates with decreased glycogen biosynthetic process associated with type II diabetes mellitus (Endocrinology 141: 2773-8 (2000)). Decreased expression of PRKCQ in skeletal muscle correlates with insulin resistance associated with obesity (Diabetes 49: 1353-8 (2000)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PLCG1 (P19174), phosphorylated at Y186, Y210, Y217, Y379, Y428, Y496, Y506, Y509, Y833, is among the proteins listed in this patent. PLCG1, Phospholipase C gamma 1, catalyzes phosphatidylinositol 4,5-bisphosphate hydrolysis, involved in various growth factor and T-cell antigen receptor signaling pathways, upregulated in breast and colorectal carcinomas. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Increased expression of PLCG1 protein correlates with carcinoma tumors associated with colorectal neoplasms (Cancer 73: 36-41 (1994)). Increased phosphorylation of PLCG1 correlates with colorectal neoplasms associated with adenomatous polyposis coli (J Cell Biochem 55: 477-85 (1994)). Increased expression of PLCG1 protein correlates with carcinoma tumors associated with breast neoplasms (PNAS 88: 10435-9 (1991)). Increased expression of PLCG1 protein correlates with carcinoma tumors associated with breast neoplasms (Proc Natl Acad Sci USA 88: 10435-9 (1991)). Increased expression of PLCG1 protein correlates with carcinoma tumors associated with colonic neoplasms (Mol Carcinog 12: 146-52 (1995)). Increased expression of PLCG1 protein correlates with adenoma tumors associated with adenomatous polyposis coli (Cancer Res 54: 2240-4 (1994)). Increased expression of PLCG1 protein correlates with colorectal neoplasms associated with adenomatous polyposis coli (J Cell Biochem 55: 477-85 (1994)). Increased expression of PLCG1 protein correlates with carcinoma tumors associated with breast neoplasms (Proc Natl Acad Sci USA 88: 10435-9 (1991)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PLCG2 (P16885), phosphorylated at Y482, Y495, Y811, Y818, Y1137, is among the proteins listed in this patent. PLCG2, Phospholipase C gamma 2 (phosphatidylinositol-specific), hydrolyzes phosphatidyl inositol upon activation by tyrosine kinases, leading to Ca2+ release and PKC activation; plays a role in platelet activation, has a likely role in B cell receptor signaling. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PLK1 (P53350), phosphorylated at Y217, is among the proteins listed in this patent. PLK1, Polo-like kinase 1, a serine-threonine protein kinase that plays a role in mitotic cell cycle control, meiotic spindle assembly, maturation of mitotic centrosomes, and cell proliferation, upregulated in a wide variety of cancers. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with stomach neoplasms (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Increased expression of PLK1 mRNA correlates with leiomyosarcoma (PNAS 91: 1736-40 (1994)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with lung neoplasms (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with esophageal neoplasms (PNAS 91: 1736-40 (1994)). Decreased expression of PLK1 mRNA may prevent increased cell proliferation associated with breast neoplasms (Anticancer Res 24: 555-62 (2004)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with colonic neoplasms (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with lung neoplasms (PNAS 91: 1736-40 (1994)). Increased expression of PLK1 protein correlates with increased cell proliferation associated with thyroid neoplasms (Br J Cancer 90: 414-8 (2004)). Increased expression of PLK1 mRNA correlates with leiomyosarcoma (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Induced inhibition of PLK1 protein may prevent prostatic neoplasms (FASEB 18: 5-7 (2004)). Increased expression of PLK1 protein correlates with more severe form of ovarian neoplasms (Br J Cancer 90: 815-21 (2004)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with esophageal neoplasms (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with stomach neoplasms (PNAS 91: 1736-40 (1994)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with stomach neoplasms (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Decreased expression of PLK1 mRNA may prevent increased cell proliferation associated with non-small-cell lung carcinoma (Oncogene 21: 3162-71 (2002)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with colonic neoplasms (PNAS 91: 1736-40 (1994)). Increased expression of PLK1 mRNA correlates with increased occurrence of death associated with non-small-cell lung carcinoma (Oncogene 14: 543-9 (1997)). Decreased expression of PLK1 protein may prevent increased cell proliferation associated with neoplasms (J Natl Cancer Inst 94: 1863-77 (2002)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with esophageal neoplasms (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Increased expression of PLK1 mRNA correlates with non-Hodgkin's lymphoma (PNAS 91: 1736-40 (1994)). Decreased expression of PLK1 mRNA may prevent increased cell proliferation associated with bladder neoplasms (J Clin Invest 115: 978-85 (2005)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with lung neoplasms (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Increased expression of PLK1 mRNA correlates with non-Hodgkin's lymphoma (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Decreased expression of PLK1 protein may prevent increased cell proliferation associated with neoplasms (Oncogene 22: 69-80 (2003)). Increased expression of PLK1 protein correlates with carcinoma tumors associated with ovarian neoplasms (Br J Cancer 90: 815-21 (2004)). Increased expression of PLK1 protein correlates with invasive form of carcinoma (Cancer Lett 169: 41-9 (2001)). Induced inhibition of PLK1 protein may prevent prostatic neoplasms (FASEB J 18: 5-7 (2004)). Increased expression of PLK1 protein correlates with increased severity of lymphoma associated with thyroid neoplasms (Anticancer Res 24: 259-63 (2004)). Increased expression of PLK1 protein correlates with papillary carcinoma associated with thyroid neoplasms (Br J Cancer 90: 414-8 (2004)). Increased expression of PLK1 mRNA correlates with increased occurrence of death associated with hepatoblastoma (Oncogene 23: 5901-11 (2004)). Increased expression of PLK1 mRNA correlates with non-Hodgkin's lymphoma (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Increased expression of PLK1 protein correlates with disease progression associated with thyroid neoplasms (Br J Cancer 90: 414-8 (2004)). Decreased expression of PLK1 protein may prevent increased anti-apoptosis associated with neoplasms (J Natl Cancer Inst 94: 1863-77 (2002)). Increased expression of PLK1 mRNA correlates with leiomyosarcoma (Proc Natl Acad Sci USA 91: 1736-40 (1994)). Increased expression of PLK1 mRNA correlates with carcinoma tumors associated with colonic neoplasms (Proc Natl Acad Sci USA 91: 1736-40 (1994)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PRPF8 (Q6P2Q9), phosphorylated at Y394, Y1432, Y2062, Y2091, Y2102, is among the proteins listed in this patent. PRPF8, PRP8 pre-mRNA processing factor 8 homolog, a component of U5 snRNP complex, involved in spliceosome assembly and mRNA splice site selection; mutation of the gene is associated with autosomal dominant retinitis pigmentosa type 13. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Missense mutation in the PRPF8 gene correlates with autosomal dominant form of retinitis pigmentosa (Hum Mol Genet. 10: 1555-62 (2001)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
PTEN (P60484), phosphorylated at Y174, Y176, Y177, Y178, Y180, is among the proteins listed in this patent. PTEN, Phosphatase and tensin homolog, phosphatidylinositol phosphatase that acts as tumor suppressor and is involved in cell cycle control, development, and apoptosis; associated with Cowden disease, Bannayan-Zonana syndrome, diabetes II, and various cancers. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Mutation in the PTEN gene correlates with advanced stage or high grade form of renal cell carcinoma (Int J Cancer 91: 219-24 (2001)). Increased expression of PTEN mutant protein may cause decreased apoptosis associated with glioma (Br J Cancer 86: 1586-91 (2002)). Missense mutation in the PTEN gene may cause decreased apoptosis associated with glioma (Br J Cancer 86: 1586-91 (2002)). Lack of expression of PTEN protein may cause abnormal regulation of phosphoinositide 3-kinase activity associated with glioma (J Cell Physiol 201: 374-84 (2004)). Mutation in the PTEN gene correlates with breast neoplasms associated with multiple hamartoma syndrome (Am J Hum Genet. 61: 1254-60 (1997)). Increased expression of PTEN protein may cause increased cell cycle arrest associated with neuroblastoma (Proc Natl Acad Sci USA 95: 15406-11 (1998)). Mutation in the PTEN promoter correlates with multiple hamartoma syndrome (Am J Hum Genet. 73: 404-11 (2003)). Decreased expression of PTEN mRNA correlates with thyroid neoplasms (Oncogene 19: 3146-55 (2000)). Nonsense mutation in the PTEN gene correlates with chromosomal instability associated with colorectal neoplasms (Hum Mol Genet. 9: 283-7 (2000)). Decreased expression of PTEN mRNA correlates with advanced stage or high grade form of prostatic neoplasms (Proc Natl Acad Sci USA 95: 5246-50 (1998)). Mutation in the PTEN gene may cause glioblastoma (Proc Natl Acad Sci USA 95: 15587-91 (1998)). Decreased expression of PTEN mRNA correlates with advanced stage or high grade form of prostatic neoplasms (Proc Natl Acad Sci USA 95: 5246-50 (1998)). Increased expression of PTEN mutant protein may cause increased protein kinase B signaling cascade associated with glioma (Br J Cancer 86: 1586-91 (2002)). Single nucleotide polymorphism in the PTEN gene correlates with type II diabetes mellitus (FEBS Lett 554: 450-4 (2003)). Mutation in the PTEN gene may cause prostatic neoplasms (Proc Natl Acad Sci USA 95: 15587-91 (1998)). Haploinsufficiency of the PTEN gene correlates with carcinoma associated with stomach neoplasms (Int J Cancer 104: 318-27 (2003)). Loss of heterozygosity at the PTEN gene correlates with non-familial form of breast neoplasms (Br J Cancer 79: 718-23 (1999)). Decreased expression of PTEN protein may cause increased cell-cell adhesion associated with colonic neoplasms (Oncogene 21: 1450-60 (2002)). Decreased expression of PTEN protein causes increased protein kinase B signaling cascade associated with ganglioneuroma (Am J Hum Genet. 73: 1191-8 (2003)). Increased expression of PTEN protein may cause increased response to drug associated with glioma (Oncogene 18: 3936-43 (1999)). Decreased expression of PTEN protein correlates with increased occurrence of death associated with liver neoplasms (Int J Cancer 100: 152-7 (2002)). Increased expression of PTEN protein may prevent increased protein kinase B signaling cascade associated with neuroblastoma (PNAS 95: 15406-11 (1998)). Hypermethylation of the PTEN promoter correlates with increased incidence of malignant form of breast neoplasms (Int J Cancer 112: 407-10 (2004)). Splice site mutation in the PTEN gene correlates with hepatocellular carcinoma associated with liver neoplasms (Oncogene 18: 3181-5 (1999)). Increased expression of PTEN protein causes increased anoikis associated with glioma (Oncogene 20: 6669-78 (2001)). Decreased expression of PTEN protein may cause abnormal apoptosis associated with hematologic neoplasms (Hum Mol Genet. 8: 185-93 (1999)). Increased expression of PTEN protein may cause increased response to ionizing radiation associated with glioma (Oncogene 18: 3936-43 (1999)). Decreased expression of PTEN mRNA correlates with carcinoma associated with stomach neoplasms (Int J Cancer 104: 318-27 (2003)). Mutation in the PFEN promoter correlates with abnormal protein kinase B signaling cascade associated with multiple hamartoma syndrome (Am J Hum Genet. 73: 404-11 (2003)). Mutation in the PTEN gene correlates with adenocarcinoma associated with cervix neoplasms (Cancer Lett 210: 57-62 (2004)). Decreased expression of PTEN mRNA correlates with advanced stage or high grade form of prostatic neoplasms (PNAS 95: 5246-50 (1998)). Loss of function mutation in the PTEN gene correlates with chromosomal instability associated with colorectal neoplasms (Hum Mol Genet. 9: 283-7 (2000)). Decreased expression of PTEN protein correlates with hepatitis C associated with hepatocellular carcinoma (Int J Cancer 100: 152-7 (2002)). Increased expression of PTEN protein may cause increased apoptosis associated with breast neoplasms (Oncogene 18: 7034-45 (1999)). Increased expression of PTEN protein may cause increased cell death associated with breast neoplasms (Cancer Res 59: 5808-14 (1999)). Deletion mutation in the PFEN gene correlates with renal cell carcinoma (Int J Cancer 91: 219-24 (2001)). Decreased membrane localization of PTEN correlates with renal cell carcinoma (Int J Cancer 99: 53-7 (2002)). Deletion mutation in the PTEN gene correlates with non-familial form of breast neoplasms (Cancer Res 57: 3657-9 (1997)). Deletion mutation in the PFEN gene correlates with multiple hamartoma syndrome associated with breast neoplasms (Cancer Res 57: 3657-9 (1997)). Missense mutation in the PTEN gene may cause meningioma associated with glioma (Br J Cancer 86: 1586-91 (2002)). Increased expression of PTEN protein may prevent malignant form of melanoma (Mol Med 8: 451-61 (2002)). Mutation in the PTEN gene may cause glioblastoma (PNAS 95: 15587-91 (1998)). Increased expression of PTEN protein may prevent increased protein kinase B signaling cascade associated with neuroblastoma (Proc Natl Acad Sci USA 95: 15406-11 (1998)). Increased expression of PTEN protein may cause increased cell cycle arrest associated with bladder neoplasms (Oncogene 19: 5406-12 (2000)). Increased expression of PTEN protein correlates with papilloma associated with laryngeal neoplasms (Mol Med 9: 77-84 (2003)). Increased expression of PFEN protein may prevent invasive form of glioma (Cancer Lett 214: 205-13 (2004)). Mutation in the PTEN gene may cause prostatic neoplasms (Proc Natl Acad Sci USA 95: 15587-91 (1998)). Deletion mutation in the PFEN gene correlates with myelodysplastic syndromes (Leukemia 20: 230-8 (2006)). Frameshift mutation in the PTEN gene correlates with endometrial neoplasms associated with hereditary nonpolyposis colorectal neoplasms (Hum Mol Genet. 11: 445-50 (2002)). Missense mutation in the PTEN gene may cause increased protein kinase B signaling cascade associated with glioma (Br J Cancer 86: 1586-91 (2002)). Decreased expression of PTEN mRNA may correlate with small cell carcinoma associated with lung neoplasms (Oncogene 17: 1557-65 (1998)). Increased expression of PTEN protein may cause drug-resistant form of bladder neoplasms (Oncogene 19: 5406-12 (2000)). Increased expression of PTEN protein may prevent increased angiogenesis associated with melanoma (Mol Med 8: 451-61 (2002)). Decreased expression of PTEN protein correlates with non-small-cell lung carcinoma associated with lung neoplasms (Cancer 100: 1673-82 (2004)). Frameshift mutation in the PTEN gene correlates with colorectal neoplasms (Cancer Lett 174: 189-94 (2001)). Decreased expression of PTEN protein correlates with increased occurrence of death associated with hepatocellular carcinoma (Int J Cancer 100: 152-7 (2002)). Decreased expression of PTEN protein correlates with increased severity of hepatocellular carcinoma associated with liver neoplasms (Int J Cancer 100: 152-7 (2002)). Missense mutation in the PT EN gene correlates with hepatocellular carcinoma associated with liver neoplasms (Oncogene 18: 3181-5 (1999)). Decreased expression of PTEN protein correlates with liver cirrhosis associated with liver neoplasms (Int J Cancer 100: 152-7 (2002)). Increased expression of PTEN protein may prevent invasive form of bladder neoplasms (Oncogene 23: 6788-97 (2004)). Lack of expression of PTEN protein correlates with bone neoplasms associated with prostatic neoplasms (Cancer Res 62: 2942-50 (2002)). Mutation in the PTEN gene correlates with malignant form of renal cell carcinoma (Int J Cancer 91: 219-24 (2001)). Mutation in the PTEN gene may cause prostatic neoplasms (PNAS 95: 15587-91 (1998)). Missense mutation in the PTEN gene causes arteriovenous malformations associated with multiple abnormalities (Hum Mol Genet. 9: 765-8 (2000)). Decreased expression of PTEN protein correlates with non-familial form of colorectal neoplasms (Cancer Res 64: 3014-21 (2004)). Increased expression of PFEN protein may cause increased cell cycle arrest associated with endometrial neoplasms (Cancer Res 61: 4569-75 (2001)). Mutation in the PTEN gene may cause breast neoplasms (Proc Natl Acad Sci USA 95: 15587-91 (1998)). Lack of expression of PTEN protein correlates with increased protein kinase B signaling cascade associated with glioblastoma (Cancer Res 63: 2742-6 (2003)). Increased expression of PTEN protein may prevent increased protein kinase B signaling cascade associated with neuroblastoma (Proc Natl Acad Sci USA 95: 15406-11 (1998)). Decreased expression of PTEN protein causes late onset form of ganglioneuroma (Am J Hum Genet. 73: 1191-8 (2003)). Hypermethylation of the PTEN promoter correlates with non-familial form of colorectal neoplasms (Cancer Res 64: 3014-21 (2004)). Increased expression of PTEN protein may cause increased cell cycle arrest associated with neuroblastoma (Proc Natl Acad Sci USA 95: 15406-11 (1998)). Decreased expression of PTEN protein correlates with thyroid neoplasms (Oncogene 19: 3146-55 (2000)). Hypermethylation of the PTEN promoter correlates with chromosomal instability associated with colorectal neoplasms (Cancer Res 64: 3014-21 (2004)). Alternative form of PTEN mRNA correlates with non-familial form of breast neoplasms (Hum Mol Genet. 15: 777-87 (2006)). Decreased nucleus localization of PTEN may correlate with breast neoplasms (Cancer Res 63: 282-6 (2003)). Increased expression of PTEN protein may prevent decreased apoptosis associated with melanoma (Mol Med 8: 451-61 (2002)). Mutation in the PTEN gene causes late onset form of ganglioneuroma (Am J Hum Genet. 73: 1191-8 (2003)). Mutation in the PTEN gene correlates with multiple hamartoma syndrome associated with breast neoplasms (Am J Hum Genet. 61: 1254-60 (1997)). Mutation in the PTEN gene may cause breast neoplasms (Proc Natl Acad Sci USA 95: 15587-91 (1998)). Point mutation in the PTEN gene causes small cell carcinoma associated with lung neoplasms (Oncogene 17: 475-9 (1998)). Increased expression of PTEN protein may cause increased cell cycle arrest associated with neuroblastoma (PNAS 95: 15406-11 (1998)). Increased expression of PTEN protein may correlate with insulin resistance associated with type II diabetes mellitus (FEBS Lett 554: 450-4 (2003)). Loss of heterozygosity at the PTEN gene correlates with renal cell carcinoma (Int J Cancer 91: 219-24 (2001)). Mutation in the PTEN gene correlates with invasive form of renal cell carcinoma (Int J Cancer 91: 219-24 (2001)). Decreased expression of PTEN protein correlates with chromosomal instability associated with colorectal neoplasms (Cancer Res 64: 3014-21 (2004)). Nonsense mutation in the PFEN gene causes multiple hamartoma syndrome (Nat Genet. 16: 64-7 (1997)). Increased expression of PTEN protein may cause decreased cell proliferation associated with breast neoplasms (Hum Mol Genet. 10: 605-16 (2001)). Mutation in the PFEN gene may cause breast neoplasms (PNAS 95: 15587-91 (1998)). Missense mutation in the PTEN gene causes hypertrophy associated with multiple abnormalities (Hum Mol Genet. 9: 765-8 (2000)). Increased expression of PTEN protein may prevent increased protein kinase B signaling cascade associated with pancreatic neoplasms (Biochem Biophys Res Commun 301: 50-3 (2003)). Increased expression of PTEN mutant protein may cause increased cell proliferation associated with glioma (Br J Cancer 86: 1586-91 (2002)). Splice site mutation in the PTEN gene correlates with renal cell carcinoma (Int J Cancer 91: 219-24 (2001)). Alternative form of PTEN mRNA correlates with multiple hamartoma syndrome (Hum Mol Genet. 15: 777-87 (2006)). Decreased expression of PTEN protein correlates with renal cell carcinoma (Int J Cancer 99: 53-7 (2002)). Mutation in the PTEN gene may cause endometrial neoplasms (Cancer Res 57: 4736-8 (1997)). Splice site mutation in the PTEN gene correlates with hepatocellular carcinoma (Oncogene 18: 3181-5 (1999)). Deletion mutation in the PTEN gene correlates with increased incidence of advanced stage or high grade form of glioma (Oncogene 16: 3331-5 (1998)). Missense mutation in the PTEN gene causes multiple hamartoma syndrome (Nat Genet. 16: 64-7 (1997)). Decreased expression of PTEN protein correlates with liver cirrhosis associated with hepatocellular carcinoma (Int J Cancer 100: 152-7 (2002)). Increased expression of PTEN protein may cause decreased cell cycle associated with breast neoplasms (Hum Mol Genet. 10: 599-604 (2001)). Missense mutation in the PTEN gene correlates with renal cell carcinoma (Int J Cancer 91: 219-24 (2001)). Increased expression of PTEN protein may prevent increased cell proliferation associated with bladder neoplasms (Oncogene 19: 5406-12 (2000)). Missense mutation in the PTEN gene correlates with hepatocellular carcinoma (Oncogene 18: 3181-5 (1999)). Increased expression of PTEN protein may correlate with decreased insulin receptor signaling pathway associated with type II diabetes mellitus (FEBS Lett 554: 450-4 (2003)). Decreased expression of PTEN protein correlates with non-small-cell lung carcinoma (Cancer 100: 1673-82 (2004)). Decreased expression of PTEN mRNA may correlate with non-small-cell lung carcinoma associated with lung neoplasms (Oncogene 17: 1557-65 (1998)). Deletion mutation in the PTEN gene correlates with multiple hamartoma syndrome (Am J Hum Genet. 73: 404-11 (2003)). Mutation in the PTEN gene may cause glioblastoma (Proc Natl Acad Sci USA 95: 15587-91 (1998)). Decreased expression of PTEN protein correlates with multiple hamartoma syndrome (Am J Hum Genet. 73: 404-11 (2003)). Mutation in the PTEN gene correlates with non-familial form of colorectal neoplasms (Oncogene 23: 617-28 (2004)). Missense mutation in the PTEN gene causes lipomatosis associated with multiple abnormalities (Hum Mol Genet. 9: 765-8 (2000)). Increased expression of PTEN protein may prevent increased cell proliferation associated with prostatic neoplasms (Oncogene 23: 786-94 (2004)). Mutation in the PTEN gene correlates with chromosomal instability associated with colorectal neoplasms (Oncogene 23: 617-28 (2004)). Lack of expression of PTEN protein correlates with malignant form of prostatic neoplasms (Cancer Res 62: 2942-50 (2002)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
Rap1a (P62834), phosphorylated at Y159, is among the proteins listed in this patent. Rap1a, RAP1A member of RAS oncogene family, a monomeric GTPase that activates Rac and inhibits cell proliferation; corresponding gene acts as a tumor suppressor and is downregulated in fibrosarcomas and the adenocarcinoma of the salivary gland. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Decreased GTPase activity of RAP1A may cause tuberous sclerosis (J Biol Chem 270: 16409-14 (1995)). Decreased GTPase activity of RAP1A may cause tuberous sclerosis (JBC 270: 16409-14 (1995)). Increased expression of RAP1A mRNA may prevent drug-induced form of lung neoplasms (Mol Carcinog 17: 84-91 (1996)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
RAPGEF4 (Q8WZA2), phosphorylated at Y857, Y986, is among the proteins listed in this patent. RAPGEF4, Rap guanine nucleotide exchange factor 4, may play a role in the regulation of cell growth and differentiation. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
RPS3 (P23396), phosphorylated at Y166, Y167, is among the proteins listed in this patent. RPS3, Ribosomal protein S3, a putative small 40S ribosomal subunit component, has DNA endonuclease activity, binds DNA base excision repair proteins APEX1 and OGG1, endonuclease activity is absent in Xeroderma pigmentosum group D patients. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
RSK2 (P51812), phosphorylated at Y483, Y488, Y490, Y529, is among the proteins listed in this patent. RSK2, Ribosomal protein S6 kinase 90 kDa polypeptide 3, a histone H3-S10 specific kinase that plays a role in phosphorylation of multiple proteins in response to EGF or stress; mutation of the corresponding gene is associated with Coffin-Lowry syndrome. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Nonsense mutation in the RPS6KA3 gene causes multiple abnormalities associated with Coffin-Lowry syndrome (Am J Hum Genet. 63: 1631-40 (1998)). Missense mutation in the RPS6KA3 gene causes multiple abnormalities associated with Coffin-Lowry syndrome (Am J Hum Genet. 63: 1631-40 (1998)). Induced inhibition of the protein serine/threonine kinase activity of RPS6KA3 may prevent increased cell proliferation associated with prostatic neoplasms (Cancer Res 65: 3108-16 (2005)). Splice site mutation in the RPS6KA3 gene causes multiple abnormalities associated with Coffin-Lowry syndrome (Am J Hum Genet. 63: 1631-40 (1998)). Frameshift mutation in the RPS6KA3 gene causes multiple abnormalities associated with Coffin-Lowry syndrome (Am J Hum Genet. 63: 1631-40 (1998)). Increased expression of RPS6KA3 protein correlates with prostatic neoplasms (Cancer Res 65: 3108-16 (2005)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
Securin (O95997), phosphorylated at Y 111, is among the proteins listed in this patent. securin, Pituitary tumor-transforming 1 (securin), transcriptional activator, promotes cell proliferation and angiogenesis, involved in sister chromatin separation and euploidy maintenance, upregulated in pituitary, colorectal, thyroid and other cancers. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Increased expression of PTTG1 mRNA correlates with prolactinoma associated with pituitary neoplasms (J Clin Endocrinol Metab 84: 761-7 (1999)). Increased expression of PTTG1 mRNA correlates with adenoma (J Clin Endocrinol Metab 84: 761-7 (1999)). Increased expression of PTTG1 mRNA correlates with adenoma tumors associated with pituitary neoplasms (Oncogene 18: 5473-6 (1999)). Increased expression of PTTG1 protein correlates with invasive form of colorectal neoplasms (Lancet 355: 716-9 (2000)). Increased expression of PTTG1 mRNA correlates with adenoma tumors associated with pituitary neoplasms (J Clin Endocrinol Metab 84: 761-7 (1999)). Increased expression of PTTG1 protein correlates with pituitary neoplasms (J Clin Endocrinol Metab 85: 3409-16 (2000)). Increased expression of PTTG1 mRNA may cause abnormal fibroblast growth factor receptor signaling pathway associated with pituitary neoplasms (Mol Endocrinol 13: 156-66 (1999)). Decreased expression of PTTG1 protein may correlate with decreased adenoma tumors associated with pituitary neoplasms (J Clin Invest 109: 277-83 (2002)). Increased expression of PTTG1 mRNA correlates with adenocarcinoma tumors associated with breast neoplasms (Oncogene 18: 5473-6 (1999)). Increased expression of PTTG1 protein correlates with carcinoma tumors associated with colorectal neoplasms (Lancet 355: 716-9 (2000)). Increased expression of PTTG1 mRNA correlates with adenocarcinoma tumors associated with lung neoplasms (Oncogene 18: 5473-6 (1999)). Increased expression of PTTG1 mRNA correlates with invasive form of pituitary neoplasms (J Clin Endocrinol Metab 84: 761-7 (1999)). Increased expression of PTTG1 mRNA correlates with advanced stage or high grade form of pituitary neoplasms (J Clin Endocrinol Metab 84: 761-7 (1999)). Increased expression of PTTG1 mRNA correlates with prolactinoma (J Clin Endocrinol Metab 84: 761-7 (1999)). Increased expression of PTTG1 mRNA correlates with adenoma tumors associated with thyroid neoplasms (J Clin Endocrinol Metab 86: 5025-32 (2001)). Increased expression of PTTG1 mRNA may cause follicular papillary carcinoma associated with thyroid neoplasms (J Clin Endocrinol Metab 86: 5025-32 (2001)). Increased expression of PTTG1 mRNA correlates with carcinoma tumors associated with thyroid neoplasms (J Clin Endocrinol Metab 86: 5025-32 (2001)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
SFRS10 (P62995), phosphorylated at Y235, Y260, is among the proteins listed in this patent. SFRS10, Splicing factor arginine/serine rich 10, binds splicing enhancer elements and activates pre-mRNA splicing, involved in calcitonin (CALCA) splicing, may play roles in vascular disease and the pathogenesis of tauopathies. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Increased RNA splicing factor activity, transesterification mechanism of SFRS10 may cause abnormal RNA splicing associated with tauopathies (JBC 278: 18997-9007 (2003)). Increased RNA splicing factor activity, transesterification mechanism of SFRS10 may cause abnormal RNA splicing associated with tauopathies (J Biol Chem 278: 18997-9007 (2003)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
SHIP (Q92835), phosphorylated at Y555, Y643, Y795, Y943, is among the proteins listed in this patent. SHIP, Inositol polyphosphate-5-phosphatase D, hydrolyzes Ins-1,3,4,5-P4 and PtdIns-3,4,5-P3, interacts with SHC1 in signal transduction pathways, may play roles in erythrocyte differentiation and basophil secretion. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
SHP-1 (P29350), phosphorylated at Y301, is among the proteins listed in this patent. SHP-1, Protein tyrosine phosphatase non-receptor type 6, regulates signaling by many receptors, upregulated in breast cancer, downregulated in gallbladder cancer, lymphoma, multiple myeloma and chronic cholecystitis, localization is altered in prostate cancer. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Increased expression of PTPN6 protein correlates with prostatic neoplasms (J Clin Endocrinol Metab 87: 915-26 (2002)). Splice site mutation in the PTPN6 gene correlates with acute form of myeloid leukemia (Hum Mol Genet. 9: 2297-304 (2000)). Decreased expression of PTPN6 protein may correlate with Sezary syndrome associated with skin neoplasms (Leukemia 16: 1470-7 (2002)). Loss of heterozygosity at the PTPN6 gene may correlate with acute lymphocytic leukemia (Cancer Res 62: 6390-4 (2002)). Decreased expression of PTPN6 protein correlates with Burkitt Lymphoma (J Exp Med 186: 1575-83 (1997)). Increased expression of PTPN6 mRNA correlates with breast neoplasms (Int J Cancer 88: 363-8 (2000)). Increased membrane localization of PTPN6 may correlate with decreased cell proliferation associated with breast neoplasms (Endocrinology 137: 3461-8 (1996)). Increased protein tyrosine phosphatase activity of PTPN6 correlates with prostatic neoplasms (J Clin Endocrinol Metab 87: 915-26 (2002)). Increased protein tyrosine phosphatase activity of PTPN6 may cause increased response to drug associated with pancreatic neoplasms (Endocrinology 140: 765-77 (1999)). Decreased expression of PTPN6 protein may correlate with abnormal regulation of JAK-STAT cascade associated with Sezary syndrome (Leukemia 16: 1470-7 (2002)). Hypermethylation of the PTPN6 promoter correlates with multiple myeloma (Blood 103: 4630-5 (2004)). Decreased expression of PTPN6 mRNA may correlate with Sezary syndrome associated with skin neoplasms (Leukemia 16: 1470-7 (2002)). Lack of expression of PTPN6 protein correlates with invasive form of prostatic neoplasms (J Clin Endocrinol Metab 87: 915-26 (2002)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
SHP-2 (Q06124), phosphorylated at Y511, is among the proteins listed in this patent. SHP-2, Protein tyrosine phosphatase non-receptor type 11, acts in many receptor tyrosine kinase and PI3-kinase signaling pathways induced by growth factors, cytokines and immunoreceptors, exploited during Helicobacter infections; mutations cause Noonan syndrome. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Missense mutation in the PTPN11 gene causes defective several tissues development associated with Noonan syndrome (Am J Hum Genet. 70: 1555-63 (2002)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes lentigo associated with Noonan syndrome (Am J Hum Genet. 71: 389-94 (2002)). Bacterial exploitation of the protein tyrosine phosphatase activity of PTPN11 causes abnormal regulation of cell shape associated with Helicobacter infections (Science 295: 683-6 (2002)). Missense mutation in the PTPN11 gene causes deafness associated with Noonan syndrome (J Clin Endocrinol Metab 87: 3529-33 (2002)). Decreased protein binding of PTPN11 may cause abnormal NK cells function associated with lymphoproliferative disorders (J Immunol 165: 2932-6 (2000)). Increased protein tyrosine phosphatase activity of PTPN11 may cause neoplasm invasiveness associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Missense mutation in the PTPN11 gene causes familial form of Noonan syndrome (Am J Hum Genet. 70: 1555-63 (2002)). Increased protein binding of PTPN11 may cause increased chemotaxis associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Decreased protein binding of PTPN11 may cause lymphoproliferative disorders (Biochemistry 42: 14885-92 (2003)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes cafe-au-lait spots associated with Noonan syndrome (Am J Hum Genet. 71: 389-94 (2002)). Missense mutation in the PTPN11 gene causes abnormal in utero embryonic development associated with Noonan syndrome (Am J Hum Genet. 70: 1555-63 (2002)). Missense mutation in the PTPN11 gene causes pulmonary valve stenosis associated with Noonan syndrome (Am J Hum Genet. 70: 1555-63 (2002)). Increased phosphorylation of PTPN11 may cause increased chemotaxis associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Increased protein tyrosine phosphatase activity of PTPN11 may cause abnormal signal transduction associated with Noonan syndrome (Nat Genet. 29: 465-8 (2001)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes multiple abnormalities associated with Noonan syndrome (Nat Genet. 29: 465-8 (2001)). Increased phosphorylation of PTPN11 may cause neoplasm invasiveness associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Bacterial exploitation of the protein tyrosine phosphatase activity of PTPN11 causes abnormal signal transduction associated with Helicobacter infections (Science 295: 683-6 (2002)). Missense mutation in the PTPN11 gene correlates with early onset form of chronic myelomonocytic leukemia (Blood 103: 2325-31 (2004)). Increased protein binding of PTPN11 may cause neoplasm invasiveness associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes multiple abnormalities associated with lentigo (Am J Hum Genet. 71: 389-94 (2002)). Missense mutation in the PTPN11 gene causes hemorrhagic disorders associated with Noonan syndrome (J Clin Endocrinol Metab 87: 3529-33 (2002)). Decreased protein binding of PTPN11 may cause abnormal cell proliferation associated with lymphoproliferative disorders (Nature 395: 462-9 (1998)). Deletion mutation in the PTPN11 gene causes Noonan syndrome (J Clin Endocrinol Metab 89: 3359-64 (2004)). Mutation in the PTPN11 gene may cause myelodysplastic syndromes (Nat Genet. 34: 148-50 (2003)). Increased protein tyrosine phosphatase activity of PTPN11 may cause increased chemotaxis associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Decreased protein binding of PTPN11 may cause lymphoproliferative disorders (Biochemistry Usa 42: 14885-92 (2003)). Decreased protein binding of PTPN11 may cause abnormal natural killer cell activation associated with lymphoproliferative disorders (J Immunol 165: 2932-6 (2000)). Missense mutation in the PTPN11 gene causes Noonan syndrome (J Clin Endocrinol Metab 89: 3359-64 (2004)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes hemorrhagic disorders associated with Noonan syndrome (J Clin Endocrinol Metab 87: 3529-33 (2002)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes deafness associated with Noonan syndrome (J Clin Endocrinol Metab 87: 3529-33 (2002)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
SHP-2 (Q06124), phosphorylated at Y511, is among the proteins listed in this patent. SHP-2, Protein tyrosine phosphatase non-receptor type 11, acts in many receptor tyrosine kinase and PI3-kinase signaling pathways induced by growth factors, cytokines and immunoreceptors, exploited during Helicobacter infections; mutations cause Noonan syndrome. This protein has potential diagnostic and/or therapeutic implications based on the following findings. Missense mutation in the PTPN11 gene causes defective several tissues development associated with Noonan syndrome (Am J Hum Genet. 70: 1555-63 (2002)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes lentigo associated with Noonan syndrome (Am J Hum Genet. 71: 389-94 (2002)). Bacterial exploitation of the protein tyrosine phosphatase activity of PTPN11 causes abnormal regulation of cell shape associated with Helicobacter infections (Science 295: 683-6 (2002)). Missense mutation in the PTPN11 gene causes deafness associated with Noonan syndrome (J Clin Endocrinol Metab 87: 3529-33 (2002)). Decreased protein binding of PTPN11 may cause abnormal NK cells function associated with lymphoproliferative disorders (J Immunol 165: 2932-6 (2000)). Increased protein tyrosine phosphatase activity of PTPN11 may cause neoplasm invasiveness associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Missense mutation in the PTPN11 gene causes familial form of Noonan syndrome (Am J Hum Genet. 70: 1555-63 (2002)). Increased protein binding of PTPN11 may cause increased chemotaxis associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Decreased protein binding of PTPN11 may cause lymphoproliferative disorders (Biochemistry 42: 14885-92 (2003)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes cafe-au-lait spots associated with Noonan syndrome (Am J Hum Genet. 71: 389-94 (2002)). Missense mutation in the PTPN11 gene causes abnormal in utero embryonic development associated with Noonan syndrome (Am J Hum Genet. 70: 1555-63 (2002)). Missense mutation in the PTPN11 gene causes pulmonary valve stenosis associated with Noonan syndrome (Am J Hum Genet. 70: 1555-63 (2002)). Increased phosphorylation of PTPN11 may cause increased chemotaxis associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Increased protein tyrosine phosphatase activity of PTPN11 may cause abnormal signal transduction associated with Noonan syndrome (Nat Genet. 29: 465-8 (2001)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes multiple abnormalities associated with Noonan syndrome (Nat Genet. 29: 465-8 (2001)). Increased phosphorylation of PTPN11 may cause neoplasm invasiveness associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Bacterial exploitation of the protein tyrosine phosphatase activity of PTPN11 causes abnormal signal transduction associated with Helicobacter infections (Science 295: 683-6 (2002)). Missense mutation in the PTPN11 gene correlates with early onset form of chronic myelomonocytic leukemia (Blood 103: 2325-31 (2004)). Increased protein binding of PTPN11 may cause neoplasm invasiveness associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes multiple abnormalities associated with lentigo (Am J Hum Genet. 71: 389-94 (2002)). Missense mutation in the PTPN11 gene causes hemorrhagic disorders associated with Noonan syndrome (J Clin Endocrinol Metab 87: 3529-33 (2002)). Decreased protein binding of PTPN11 may cause abnormal cell proliferation associated with lymphoproliferative disorders (Nature 395: 462-9 (1998)). Deletion mutation in the PTPN11 gene causes Noonan syndrome (J Clin Endocrinol Metab 89: 3359-64 (2004)). Mutation in the PTPN11 gene may cause myelodysplastic syndromes (Nat Genet. 34: 148-50 (2003)). Increased protein tyrosine phosphatase activity of PTPN11 may cause increased chemotaxis associated with breast neoplasms (Oncogene 23: 157-67 (2004)). Decreased protein binding of PTPN11 may cause lymphoproliferative disorders (Biochemistry Usa 42: 14885-92 (2003)). Decreased protein binding of PTPN11 may cause abnormal natural killer cell activation associated with lymphoproliferative disorders (J Immunol 165: 2932-6 (2000)). Missense mutation in the PTPN11 gene causes Noonan syndrome (J Clin Endocrinol Metab 89: 3359-64 (2004)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes hemorrhagic disorders associated with Noonan syndrome (J Clin Endocrinol Metab 87: 3529-33 (2002)). Missense mutation in the Protein-tyrosine phosphatase domain of PTPN11 causes deafness associated with Noonan syndrome (J Clin Endocrinol Metab 87: 3529-33 (2002)). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)).
In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. As used in this specification, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. As used herein, unless specifically indicated otherwise, the word “or” is used in the “inclusive” sense of “and/or” and not the “exclusive” sense of “either/or.”
The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%.
As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable that is inherently discrete, the variable can be equal to any integer value of the numerical range, including the end-points of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any real value of the numerical range, including the end-points of the range. As an example, a variable that is described as having values between 0 and 2, can be 0, 1 or 2 for variables which are inherently discrete, and can be 0.0, 0.1, 0.01, 0.001, or any other real value for variables which are inherently continuous.
As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound or composition, the term “comprising” means that the compound or composition includes at least the recited features or components, but may also include additional features or components.
“Antibody” or “antibodies” refers to all classes of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including whole antibodies and any antigen biding fragment thereof (e.g., Fab) or single chains thereof, including chimeric, polyclonal, and monoclonal antibodies. Antibodies are antigen-specific protein molecules produced by lymphocytes of the B cell lineage. Following antigenic stimulation, B cells that have surface immunoglobulin receptors that bind the antigen clonally expand, and the binding affinity for the antigen increases through a process called affinity maturation. The B cells further differentiate into plasma cells, which secrete large quantities of antibodies in to the serum. While the physiological role of antibodies is to protect the host animal by specifically binding and eliminating microbes and microbial pathogens from the body, large amounts of antibodies are also induced by intentional immunization to produce specific antibodies that are used extensively in many biomedical and therapeutic applications.
Antibody molecules are shaped somewhat like the letter “Y”, and consist of 4 protein chains, two heavy (H) and two light (L) chains. Antibodies possess two distinct and spatially separate functional features. The ends of each of the two arms of the “Y” contain the variable regions (variable heavy (V(H)) and variable light (V(L)) regions), which form two identical antigen-binding sites. The variable regions undergo a process of “affinity maturation” during the immune response, leading to a rapid divergence of amino acids within these variable regions. The other end of the antibody molecule, the stem of the “Y”, contains only the two heavy constant (CH) regions, interacts with effector cells to determine the effector functions of the antibody. There are five different CH region genes that encode the five different classes of immunoglobulins: IgM, IgD, IgG, IgA and IgE. These constant regions, by interacting with different effector cells and molecules, determine the immunoglobulin molecule's biological function and biological response.
Each V(H) and V(L) region contains three subregions called complementarity determining regions. These include CDR1-3 of the V(H) domain and CDR1-3 of the V(L) domain. These six CDRs generally form the antigen binding surface, and include those residues that hypermutate during the affinity maturation phase of the immune response. The CDR3 of the V(H) domain seems to play a dominant role in generating diversity of both the B cell antigen receptor (BCR) and the T cell antigen receptor systems (Xu et al., Immunity 13:37-45 (2000)).
The term “antibody” or “antibodies” refers to all classes of polyclonal or monoclonal immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including whole antibodies and any antigen binding fragment thereof. This includes any combination of immunoglobulin domains or chains that contains a variable region (V(H) or V(L)) that retains the ability to bind the immunogen. Such fragments include F(ab)2 fragments (V(H)—C(H1), V(L)-C(L))2; monovalent Fab fragments (V(H)—C(H1), V(L)-C(L)); Fv fragment (V(H)—V(L); single-chain Fv fragments (Kobayashi et al., Steroids July; 67(8):733-42 (2002).
Monoclonal antibodies refer to clonal antibodies produced from fusions between immunized murine, rabbit, human, or other vertebrate species, and produced by classical fusion technology Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975 Aug. 7; 256(5517):495-7 or by alternative methods which may isolate clones of immunoglobulin secreting cells from transformed plasma cells.
When used with respect to an antibody's binding to one phospho-form of a sequence, the expression “does not bind” means that a phospho-specific antibody either does not apparently bind to the non-phospho form of the antigen as ascertained in commonly used experimental detection systems (Western blotting, IHC, Immunofluorescence, etc.). One of skill in the art will appreciate that the expression may be applicable in those instances when (1) a phospho-specific antibody either does not apparently bind to the non-phospho form of the antigen as ascertained in commonly used experimental detection systems (Western blotting, IHC, Immunofluorescence, etc.); (2) where there is some reactivity with the surrounding amino acid sequence, but that the phosphorylated residue is an immunodominant feature of the reaction. In cases such as these, there is an apparent difference in affinities for the two sequences. Dilutional analyses of such antibodies indicates that the antibodies apparent affinity for the phosphorylated form is at least 10-100 fold higher than for the non-phosphorylated form; or where (3) the phospho-specific antibody reacts no more than an appropriate control antibody would react under identical experimental conditions. A control antibody preparation might be, for instance, purified immunoglobulin from a pre-immune animal of the same species, an isotype- and species-matched monoclonal antibody. Tests using control antibodies to demonstrate specificity are recognized by one of skill in the art as appropriate and definitive.
“Target signaling protein/polypeptide” means any protein (or polypeptide derived therefrom) enumerated in Column A of Table 1/
“Heavy-isotope labeled peptide” (used interchangeably with AQUA peptide) means a peptide comprising at least one heavy-isotope label, which is suitable for absolute quantification or detection of a protein as described in WO/03016861, “Absolute Quantification of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry” (Gygi et al.), further discussed below.
“Protein” is used interchangeably with polypeptide, and includes protein fragments and domains as well as whole protein.
“Phosphorylatable amino acid” means any amino acid that is capable of being modified by addition of a phosphate group, and includes both forms of such amino acid.
“Phosphorylatable peptide sequence” means a peptide sequence comprising a phosphorylatable amino acid.
“Phosphorylation site-specific antibody” means an antibody that specifically binds a phosphorylatable peptide sequence/epitope only when phosphorylated, or only when not phosphorylated, respectively. The term is used interchangeably with “phospho-specific” antibody.
Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of recombinant DNA technology include Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989); Kaufman et al., Eds., Handbook of Molecular and Cellular Methods in Biology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991). Standard reference works setting forth the general principles of pharmacology include Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill Companies Inc., New York (2006).
A. Identification of Phosphorylation Sites. The Target signaling protein/polypeptide phosphorylation sites disclosed herein and listed in Table 1/
The IAP method employed generally comprises the following steps: (a) a proteinaceous preparation (e.g. a digested cell extract) comprising phosphopeptides from two or more different proteins is obtained from an organism; (b) the preparation is contacted with at least one immobilized general phosphotyrosine-specific antibody; (c) at least one phosphopeptide specifically bound by the immobilized antibody in step (b) is isolated; and (d) the modified peptide isolated in step (c) is characterized by mass spectrometry (MS) and/or tandem mass spectrometry (MS-MS). Subsequently, (e) a search program (e.g., Sequest) may be utilized to substantially match the spectra obtained for the isolated, modified peptide during the characterization of step (d) with the spectra for a known peptide sequence. A quantification step employing, e.g., SILAC or AQUA, may also be employed to quantify isolated peptides in order to compare peptide levels in a sample to a baseline.
In the IAP method as employed herein, a general phosphotyrosine-specific monoclonal antibody (commercially available from Cell Signaling Technology, Inc., Beverly, Mass., Cat. #9411 (p-Tyr-100)) was used in the immunoaffinity step to isolate the widest possible number of phospho-tyrosine containing peptides from the cell extracts.
Extracts from the following human cancer cell lines, tissues and patient samples were employed: 01364548-cll, 223-CLL, 293T, 3T3 TrkB, 3T3-Src, 3T3-TrkA, 3T3-wt, 577, A172, AML-4833, AML-6246, AML-6735, AML-7592, BaF3-10ZF, BaF3-4ZF, BaF3-APR, BaF3-FLT3(D842V), BaF3-FLT3(D842Y), BaF3-FLT3(K663Q), BaF3-FLT3(WT), BaF3-FLT3/ITD, BaF3-PRTK, BaF3-TDII, BaF3-Tel/FGFR3, Baf3, Baf3-V617F-jak2, Baf3/E255K, Baf3/H396P, Baf3/Jak2(IL-3 dep), Baf3/M351T, Baf3/T3151, Baf3/TpoR, Baf3/TpoR-Y98F, Baf3/Tyk2, Baf3/V617F-jak2 (IL-3), Baf3/Y253F, Baf3/cc-TpoR-IV, Baf3/p210wt, CHRF, CI-1, CMK, CTV-1, DMS 53, DND41, DU-528, DU145, ELF-153, EOL-1, GDM-1, H1703, H1734, H1793, H1869, H1944, H1993, H2023, H226, H3255, H358, H520, H82, H838, HCC1428, HCC1435, HCC1806, HCC1937, HCC366, HCC827, HCT116, HEL, HL107B, HL117B, HL131A, HL131B, HL133A, HL53B, HL59b, HL60, HL61a, HL61b, HL66B, HL68A, HL75A, HL84A, HL97B, HL98A, HT29, HU-3, HUVEC, Jurkat, K562, KG-1, KG1-A, KMS11, KMS18, KMS27, KOPT-K1, KY821, Karpas 299, Karpas-1106p, M-07e, M01043, M059K, MC-116, MCF-10A (Y561F), MCF-10A(Y969F), MDA-MB-453, MDA-MB-468, MEC-2, MKPL-1, ML-1, MO-91, MOLT15, MV4-11, Me-F2, Molm 14, Monomac 6, NCI-N87, Nomo-1, OCI-M1, OCI-ly4, OCI-ly8, OCI/AML2, OPM-1, PL21, Pfeiffer, RC-K8, RI-1, SCLC T1, SEM, SK-N-AS, SK-N-MC, SKBR3, SR-786, SU-DHL1, SUP-M2, SUPT-13, SuDHL5, T17, TRE-cll patient, TS, UT-7, VAL, Verona, Verona 1, Verona 4, WSU-NHL, XG2, Z-55, cs001, cs015, cs025, cs041, cs042, gz21, gz68, gz73, gz74, gzB1, hl1144b, hl1152b, lung tumor T26, lung tumor T57, normal human lung, pancreatic xenograft, patient 1, rat brain and sw480.
As described in more detail in the Examples, lysates were prepared from these cells and digested with trypsin after treatment with DTT and iodoacetamide to residue and alkylate cysteine residues. Before the immunoaffinity step, peptides were pre-fractionated by reversed-phase solid phase extraction using Sep-Pak C18 columns to separate peptides from other cellular components. The solid phase extraction cartridges were eluted with varying steps of acetonitrile. Each lyophilized peptide fraction was redissolved in MOPS IP buffer and treated with phosphotyrosine (P-Tyr-100, CST #9411) immobilized on protein G-Sepharose. Immunoaffinity-purified peptides were eluted with 0.1% TFA and a portion of this fraction was concentrated with Stage or Zip tips and analyzed by LC-MS/MS, using either a LCQ or ThermoFinnigan LTQ ion trap mass spectrometer. Peptides were eluted from a 10 cm×75 μm reversed-phase column with a 45-min linear gradient of acetonitrile. MS/MS spectra were evaluated using the program Sequest with the NCBI human protein database.
This revealed the tyrosine phosphorylation sites in signaling pathways affected by kinase activation or active in leukemia cells. The identified phosphorylation sites and their parent proteins are enumerated in Table 1/
As a result of the discovery of these phosphorylation sites, phospho-specific antibodies and AQUA peptides for the detection of and quantification of these sites and their parent proteins may now be produced by standard methods, as described below. These new reagents will prove highly useful in, e.g., studying the signaling pathways and events underlying the progression of leukemias and the identification of new biomarkers and targets for diagnosis and treatment of such diseases in a mammal.
The methods of the present invention are intended for use with any mammal that may experience the benefits of the methods of the invention. Foremost among such mammals are humans, although the invention is not intended to be so limited, and is applicable to veterinary uses. Thus, in accordance with the invention, “mammals” or “mammal in need” include humans as well as non-human mammals, particularly domesticated animals including, without limitation, cats, dogs, and horses.
B. Antibodies and Cell Lines. Isolated phosphorylation site-specific antibodies that specifically bind a Target signaling protein/polypeptide disclosed in Column A of Table 1 only when phosphorylated (or only when not phosphorylated) at the corresponding amino acid and phosphorylation site listed in Columns D and E of Table 1/
Polyclonal antibodies of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with a peptide antigen corresponding to the phosphorylation site of interest (i.e., a phosphorylation site enumerated in Column E of Table 1, which comprises the corresponding phosphorylatable amino acid listed in Column D of Table 1), collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, in accordance with known procedures. For example, a peptide antigen corresponding to all or part of the novel RACK1 adaptor/scaffold phosphorylation site disclosed herein (SEQ ID NO: 13=LTRDETNyGIPQR, encompassing phosphorylated tyrosine 52 (see Row 14 of Table 1)) may be employed to produce antibodies that only bind RACK1 when phosphorylated at Tyr 52. Similarly, a peptide comprising all or part of any one of the phosphorylation site sequences provided in Column E of Table 1 may employed as an antigen to produce an antibody that only binds the corresponding protein listed in Column A of Table 1 when phosphorylated (or when not phosphorylated) at the corresponding residue listed in Column D. If an antibody that only binds the protein when phosphorylated at the disclosed site is desired, the peptide antigen includes the phosphorylated form of the amino acid. Conversely, if an antibody that only binds the protein when not phosphorylated at the disclosed site is desired, the peptide antigen includes the non-phosphorylated form of the amino acid.
Peptide antigens suitable for producing antibodies of the invention may be designed, constructed and employed in accordance with well-known techniques. See, e.g., A
It will be appreciated by those of skill in the art that longer or shorter phosphopeptide antigens may be employed. See Id. For example, a peptide antigen may comprise the full sequence disclosed in Column E of Table 1/
Monoclonal antibodies of the invention may be produced in a hybridoma cell line according to the well-known technique of Kohler and Milstein. See Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976); see also, C
Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype are preferable for a particular application, particular isotypes can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Nat'l. Acad. Sci., 82: 8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)).
An epitope of a phosphorylation-site specific antibody of the invention is a peptide fragment consisting essentially of about 8 to 17 amino acids including the phosphorylatable tyrosine, wherein about 3 to 8 amino acids are positioned on each side of the phosphorylatable tyrosine (for example, the NARS tyrosine 539 phosphorylation site sequence disclosed in Row 84, Column E of Table 1), and antibodies of the invention thus specifically bind a Target Signal Protein/Polypeptide comprising such epitopic sequence. Epitopes bound by the antibodies of the invention comprise all or part of a phosphorylatable site sequence listed in Column E of Table 1, including the phosphorylatable amino acid.
Included in the scope of the invention are equivalent non-antibody molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a phospho-specific manner, to essentially the same phosphorylatable epitope to which the phospho-specific antibodies of the invention bind. See, e.g., Neuberger et al., Nature 312: 604 (1984). Such equivalent non-antibody reagents may be suitably employed in the methods of the invention further described below.
Antibodies provided by the invention may be any type of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including Fab or antigen-recognition fragments thereof. The antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrision et al., Proc. Nat'l. Acad. Sci. 81: 6851 (1984); Neuberger et al., Nature 312: 604 (1984)). The antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980.
The invention also provides immortalized cell lines that produce an antibody of the invention. For example, hybridoma clones, constructed as described above, that produce monoclonal antibodies to the protein phosphorylation sites disclosed herein are also provided. Similarly, the invention includes recombinant cells producing an antibody of the invention, which cells may be constructed by well known techniques; for example the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, e.g., A
Phosphorylation site-specific antibodies of the invention, whether polyclonal or monoclonal, may be screened for epitope and phospho-specificity according to standard techniques. See, e.g. Czernik et al., Methods in Enzymology, 201: 264-283 (1991). For example, the antibodies may be screened against the phospho and non-phospho peptide library by ELISA to ensure specificity for both the desired antigen (i.e. that epitope including a phosphorylation site sequence enumerated in Column E of Table 1) and for reactivity only with the phosphorylated (or non-phosphorylated) form of the antigen. Peptide competition assays may be carried out to confirm lack of reactivity with other phospho-epitopes on the given Target Signal Protein/Polypeptide. The antibodies may also be tested by Western blotting against cell preparations containing the signaling protein, e.g. cell lines over-expressing the target protein, to confirm reactivity with the desired phosphorylated epitope/target.
In an exemplary embodiment, phage display libraries containing more than 1010 phage clones are used for high-throughput production of monoclonal antibodies that target post-translational modification sites (e.g., phosphorylation sites) and, for validation and quality control, high-throughput immunohistochemistry is utilized to screen the efficacy of these antibodies. Western blots, protein microarrays and flow cytometry can also be used in high-throughput screening of phosphorylation site-specific polyclonal or monoclonal antibodies of the present invention. See, e.g., Blow N., Nature, 447: 741-743 (2007).
Specificity against the desired phosphorylated epitope may also be examined by constructing mutants lacking phosphorylatable residues at positions outside the desired epitope that are known to be phosphorylated, or by mutating the desired phospho-epitope and confirming lack of reactivity. Phosphorylation-site specific antibodies of the invention may exhibit some limited cross-reactivity to related epitopes in non-target proteins. This is not unexpected as most antibodies exhibit some degree of cross-reactivity, and anti-peptide antibodies will often cross-react with epitopes having high homology to the immunizing peptide. See, e.g., Czernik, supra. Cross-reactivity with non-target proteins is readily characterized by Western blotting alongside markers of known molecular weight. Amino acid sequences of cross-reacting proteins may be examined to identify sites highly homologous to the Target signaling protein/polypeptide epitope for which the antibody of the invention is specific.
In certain cases, polyclonal antisera may exhibit some undesirable general cross-reactivity to phosphotyrosine or phosphoserine itself, which may be removed by further purification of antisera, e.g., over a phosphotyramine column. Antibodies of the invention specifically bind their target protein (i.e., a protein listed in Column A of Table 1) only when phosphorylated (or only when not phosphorylated, as the case may be) at the site disclosed in corresponding Columns D/E, and do not (substantially) bind to the other form (as compared to the form for which the antibody is specific).
Antibodies may be further characterized via immunohistochemical (IHC) staining using normal and diseased tissues to evaluate phosphorylation and activation status in diseased tissue. IHC may be carried out according to well-known techniques. See, e.g., Antibodies: A Laboratory Manual, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g., tumor tissue) is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary antibody and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.
Antibodies may be further characterized by flow cytometry carried out according to standard methods. See Chow et al., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). Briefly and by way of example, the following protocol for cytometric analysis may be employed: samples may be centrifuged on Ficoll gradients to remove erythrocytes, and cells may then be fixed with 2% paraformaldehyde for 10 minutes at 37° C. followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with the primary phosphorylation-site specific antibody of the invention (which detects a Target Signal Protein/Polypeptide enumerated in Table 1), washed and labeled with a fluorescent-labeled secondary antibody. Additional fluorochrome-conjugated marker antibodies (e.g., CD45, CD34) may also be added at this time to aid in the subsequent identification of specific hematopoietic cell types. The cells would then be analyzed on a flow cytometer (e.g., a Beckman Coulter FC500) according to the specific protocols of the instrument used.
Antibodies of the invention may also be advantageously conjugated to fluorescent dyes (e.g., Alexa488, PE) for use in multi-parametric analyses along with other signal transduction (phospho-CrkL, phospho-Erk 1/2) and/or cell marker (CD34) antibodies.
Phosphorylation-site specific antibodies of the invention specifically bind to a target signaling protein/polypeptide only when phosphorylated at a disclosed site, but are not limited only to binding the human species, per se. The invention includes antibodies that also bind conserved and highly homologous or identical phosphorylation sites in respective target signaling protein/polypeptide from other species (e.g., mouse, rat, monkey, yeast), in addition to binding the human phosphorylation site. Highly homologous or identical sites conserved in other species can readily be identified by standard sequence comparisons, such as using BLAST, with the human target signaling protein/polypeptide phosphorylation sites disclosed herein.
C. Heavy-Isotope Labeled Peptides (AQUA Peptides). The phosphorylation sites disclosed herein now enable the production of corresponding heavy-isotope labeled peptides for the absolute quantification of such signaling proteins (both phosphorylated and not phosphorylated at a disclosed site) in biological samples. The production and use of AQUA peptides for the absolute quantification of proteins (AQUA) in complex mixtures has been described. See WO/03016861, Gerber et al., Proc. Natl. Acad. Sci. U.S.A. 100: 6940-5 (2003).
The AQUA methodology employs the introduction of a known quantity of at least one heavy-isotope labeled peptide standard (which has a unique signature detectable by LC-SRM chromatography) into a digested biological sample in order to determine, by comparison to the peptide standard, the absolute quantity of a peptide with the same sequence and protein modification in the biological sample. Briefly, the AQUA methodology has two stages: peptide internal standard selection and validation and method development; and implementation using validated peptide internal standards to detect and quantify a target protein in sample. The method is a powerful technique for detecting and quantifying a given peptide/protein within a complex biological mixture, such as a cell lysate, and may be employed, e.g., to quantify change in protein phosphorylation as a result of drug treatment, or to quantify differences in the level of a protein in different biological states.
Generally, to develop a suitable internal standard, a particular peptide (or modified peptide) within a target protein sequence is chosen based on its amino acid sequence and the particular protease to be used to digest. The peptide is then generated by solid-phase peptide synthesis such that one residue is replaced with that same residue containing stable isotopes (13C, 15N). The result is a peptide that is chemically identical to its native counterpart formed by proteolysis, but is easily distinguishable by MS via a 7-Da mass shift. A newly synthesized AQUA internal standard peptide is then evaluated by LC-MS/MS. This process provides qualitative information about peptide retention by reverse-phase chromatography, ionization efficiency, and fragmentation via collision-induced dissociation. Informative and abundant fragment ions for sets of native and internal standard peptides are chosen and then specifically monitored in rapid succession as a function of chromatographic retention to form a selected reaction monitoring (LC-SRM) method based on the unique profile of the peptide standard.
The second stage of the AQUA strategy is its implementation to measure the amount of a protein or modified protein from complex mixtures. Whole cell lysates are typically fractionated by SDS-PAGE gel electrophoresis, and regions of the gel consistent with protein migration are excised. This process is followed by in-gel proteolysis in the presence of the AQUA peptides and LC-SRM analysis. (See Gerber et al., supra.) AQUA peptides are spiked in to the complex peptide mixture obtained by digestion of the whole cell lysate with a proteolytic enzyme and subjected to immunoaffinity purification as described above. The retention time and fragmentation pattern of the native peptide formed by digestion (e.g., trypsinization) is identical to that of the AQUA internal standard peptide determined previously; thus, LC-MS/MS analysis using an SRM experiment results in the highly specific and sensitive measurement of both internal standard and analyte directly from extremely complex peptide mixtures. Because an absolute amount of the AQUA peptide is added (e.g., 250 fmol), the ratio of the areas under the curve can be used to determine the precise expression levels of a protein or phosphorylated form of a protein in the original cell lysate. In addition, the internal standard is present during in-gel digestion as native peptides are formed, such that peptide extraction efficiency from gel pieces, absolute losses during sample handling (including vacuum centrifugation), and variability during introduction into the LC-MS system do not affect the determined ratio of native and AQUA peptide abundances.
An AQUA peptide standard is developed for a known phosphorylation site sequence previously identified by the IAP-LC-MS/MS method within a target protein. One AQUA peptide incorporating the phosphorylated form of the particular residue within the site may be developed, and a second AQUA peptide incorporating the non-phosphorylated form of the residue developed. In this way, the two standards may be used to detect and quantify both the phosphorylated and non-phosphorylated forms of the site in a biological sample.
Peptide internal standards may also be generated by examining the primary amino acid sequence of a protein and determining the boundaries of peptides produced by protease cleavage. Alternatively, a protein may actually be digested with a protease and a particular peptide fragment produced can then sequenced. Suitable proteases include, but are not limited to, serine proteases (e.g., trypsin, hepsin), metallo proteases (e.g., PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin, carboxypeptidases, etc.
A peptide sequence within a target protein is selected according to one or more criteria to optimize the use of the peptide as an internal standard. Preferably, the size of the peptide is selected to minimize the chances that the peptide sequence will be repeated elsewhere in other non-target proteins. Thus, a peptide is preferably at least about 6 amino acids. The size of the peptide is also optimized to maximize ionization frequency. A workable range is about 7 to 15 amino acids. A peptide sequence is also selected that is not likely to be chemically reactive during mass spectrometry, thus sequences comprising cysteine, tryptophan, or methionine are avoided.
A peptide sequence that does not include a modified region of the target region may be selected so that the peptide internal standard can be used to determine the quantity of all forms of the protein. Alternatively, a peptide internal standard encompassing a modified amino acid may be desirable to detect and quantify only the modified form of the target protein. Peptide standards for both modified and unmodified regions can be used together, to determine the extent of a modification in a particular sample (i.e. to determine what fraction of the total amount of protein is represented by the modified form). For example, peptide standards for both the phosphorylated and unphosphorylated form of a protein known to be phosphorylated at a particular site can be used to quantify the amount of phosphorylated form in a sample.
The peptide is labeled using one or more labeled amino acids (i.e. the label is an actual part of the peptide) or less preferably, labels may be attached after synthesis according to standard methods. Preferably, the label is a mass-altering label selected based on the following considerations: the mass should be unique to shift fragment masses produced by MS analysis to regions of the spectrum with low background; the ion mass signature component is the portion of the labeling moiety that preferably exhibits a unique ion mass signature in MS analysis; the sum of the masses of the constituent atoms of the label is preferably uniquely different than the fragments of all the possible amino acids. As a result, the labeled amino acids and peptides are readily distinguished from unlabeled ones by the ion/mass pattern in the resulting mass spectrum. Preferably, the ion mass signature component imparts a mass to a protein fragment that does not match the residue mass for any of the 20 natural amino acids.
The label should be robust under the fragmentation conditions of MS and not undergo unfavorable fragmentation. Labeling chemistry should be efficient under a range of conditions, particularly denaturing conditions, and the labeled tag preferably remains soluble in the MS buffer system of choice. The label preferably does not suppress the ionization efficiency of the protein and is not chemically reactive. The label may contain a mixture of two or more isotopically distinct species to generate a unique mass spectrometric pattern at each labeled fragment position. Stable isotopes, such as 2H, 13C, 15N, 17O, 18O, or 34S, are suitable labels. Pairs of peptide internal standards that incorporate a different isotope label may also be prepared. Amino acid residues into which a heavy isotope label may be incorporated include leucine, proline, valine, and phenylalanine.
Peptide internal standards are characterized according to their mass-to-charge (m/z) ratio, and preferably, also according to their retention time on a chromatographic column (e.g. an HPLC column). Internal standards that co-elute with unlabeled peptides of identical sequence are selected as optimal internal standards. The internal standard is then analyzed by fragmenting the peptide by any suitable means, for example by collision-induced dissociation (CID) using, e.g., argon or helium as a collision gas. The fragments are then analyzed, for example by multi-stage mass spectrometry (MSn) to obtain a fragment ion spectrum, to obtain a peptide fragmentation signature. Preferably, peptide fragments have significant differences in m/z ratios to enable peaks corresponding to each fragment to be well separated, and a signature that is unique for the target peptide is obtained. If a suitable fragment signature is not obtained at the first stage, additional stages of MS are performed until a unique signature is obtained.
Fragment ions in the MS/MS and MS3 spectra are typically highly specific for the peptide of interest, and, in conjunction with LC methods, allow a highly selective means of detecting and quantifying a target peptide/protein in a complex protein mixture, such as a cell lysate, containing many thousands or tens of thousands of proteins. Any biological sample potentially containing a target protein/peptide of interest may be assayed. Crude or partially purified cell extracts may be employed. Generally, the sample has at least 0.01 mg of protein, typically a concentration of 0.1-10 mg/mL, and may be adjusted to a desired buffer concentration and pH.
A known amount of a labeled peptide internal standard, preferably about 10 femtomoles, corresponding to a target protein to be detected/quantified is then added to a biological sample, such as a cell lysate. The spiked sample is then digested with one or more protease(s) for a suitable time period to allow digestion. A separation is then performed (e.g., by HPLC, reverse-phase HPLC, capillary electrophoresis, ion exchange chromatography, etc.) to isolate the labeled internal standard and its corresponding target peptide from other peptides in the sample. Microcapillary LC is a method contemplated.
Each isolated peptide is then examined by monitoring of a selected reaction in the MS. This involves using the prior knowledge gained by the characterization of the peptide internal standard and then requiring the MS to continuously monitor a specific ion in the MS/MS or MSn spectrum for both the peptide of interest and the internal standard. After elution, the area under the curve (AUC) for both peptide standard and target peptide peaks are calculated. The ratio of the two areas provides the absolute quantification that can be normalized for the number of cells used in the analysis and the protein's molecular weight, to provide the precise number of copies of the protein per cell. Further details of the AQUA methodology are described in Gygi et al., and Gerber et al. supra.
In accordance with the present invention, AQUA internal peptide standards (heavy-isotope labeled peptides) may now be produced, as described above, for any of the phosphorylation sites disclosed herein. Peptide standards for a given phosphorylation site (e.g., the tyrosine 482 in PLCG2—see Row 116 of Table 1) may be produced for both the phosphorylated and non-phosphorylated forms of the site (e.g., see RENT1 site sequence in Column E, Row 128 of Table 1 (SEQ ID NO: 129) and such standards employed in the AQUA methodology to detect and quantify both forms of such phosphorylation site in a biological sample.
AQUA peptides of the invention may comprise all, or part of, a phosphorylation site peptide sequence disclosed herein (see Column E of Table 1/
The phosphorylation site peptide sequences disclosed herein (see Column E of Table 1/
Accordingly, the invention provides heavy-isotope labeled peptides (AQUA peptides) for the detection and/or quantification of any of the phosphorylation sites disclosed in Table 1/
Certain subsets of AQUA peptides provided by the invention are described above (corresponding to particular protein types/groups in Table 1, for example, tyrosine protein kinases or adaptor/scaffold proteins). Example 4 is provided to further illustrate the construction and use, by standard methods described above, of exemplary AQUA peptides provided by the invention. For example, the above-described AQUA peptides corresponding to both the phosphorylated and non-phosphorylated forms of the disclosed RICA G protein or regulator protein tyrosine 1188 phosphorylation site (see Row 162 of Table 1/
AQUA peptides of the invention may also be employed within a kit that comprises one or multiple AQUA peptide(s) provided herein (for the quantification of a target signaling protein/polypeptide disclosed in Table 1/
AQUA peptides provided by the invention will be useful in the further study of signal transduction anomalies associated with diseases such as for example cancer, including leukemias, and in identifying diagnostic/bio-markers of these diseases, new potential drug targets, and/or in monitoring the effects of test compounds on target signaling proteins/polypeptides and pathways.
D. Immunoassay Formats. Antibodies provided by the invention may be advantageously employed in a variety of standard immunological assays (the use of AQUA peptides provided by the invention is described separately above). Assays may be homogeneous assays or heterogeneous assays. In a homogeneous assay the immunological reaction usually involves a phosphorylation-site specific antibody of the invention), a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution. Immunochemical labels that may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
In a heterogeneous assay approach, the reagents are usually the specimen, a phosphorylation-site specific antibody of the invention, and suitable means for producing a detectable signal. Similar specimens as described above may be used. The antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the specimen. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, and so forth. For example, if the antigen to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.
Immunoassay formats and variations thereof that may be useful for carrying out the methods disclosed herein are well known in the art. See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see also, e.g., U.S. Pat. No. 4,727,022; U.S. Pat. No. 4,659,678; U.S. Pat. No. 4,376,110. Conditions suitable for the formation of reagent-antibody complexes are well described. See id. Monoclonal antibodies of the invention may be used in a “two-site” or “sandwich” assay, with a single cell line serving as a source for both the labeled monoclonal antibody and the bound monoclonal antibody. Such assays are described in U.S. Pat. No. 4,376,110. The concentration of detectable reagent should be sufficient such that the binding of a target signaling protein/polypeptide is detectable compared to background.
Phosphorylation site-specific antibodies disclosed herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Antibodies, or other target protein or target site-binding reagents, may likewise be conjugated to detectable groups such as radiolabels (e.g., 35S, 125I, 131I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
Antibodies of the invention may also be optimized for use in a flow cytometry (FC) assay to determine the activation/phosphorylation status of a target signaling protein/polypeptide in patients before, during, and after treatment with a drug targeted at inhibiting phosphorylation of such a protein at the phosphorylation site disclosed herein. For example, bone marrow cells or peripheral blood cells from patients may be analyzed by flow cytometry for target signaling protein/polypeptide phosphorylation, as well as for markers identifying various hematopoietic cell types. In this manner, activation status of the malignant cells may be specifically characterized. Flow cytometry may be carried out according to standard methods. See, e.g. Chow et al., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). Briefly and by way of example, the following protocol for cytometric analysis may be employed: fixation of the cells with 1% para-formaldehyde for 10 minutes at 37° C. followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with the primary antibody (a phospho-specific antibody of the invention), washed and labeled with a fluorescent-labeled secondary antibody. Alternatively, the cells may be stained with a fluorescent-labeled primary antibody. The cells would then be analyzed on a flow cytometer (e.g., a Beckman Coulter EPICS-XL) according to the specific protocols of the instrument used. Such an analysis would identify the presence of activated Target Signaling Protein(s)/Polypeptide(s) in the malignant cells and reveal the drug response on the targeted protein.
Alternatively, antibodies of the invention may be employed in immunohistochemical (IHC) staining to detect differences in signal transduction or protein activity using normal and diseased tissues. IHC may be carried out according to well-known techniques. See, e.g., A
Antibodies of the invention may be also be optimized for use in other clinically-suitable applications, for example bead-based multiplex-type assays, such as IGEN, Luminex™ and/or Bioplex™ assay formats, or otherwise optimized for antibody array formats, such as reversed-phase array applications (see, e.g., Paweletz et al., Oncogene 20(16): 1981-89 (2001)). Accordingly, in another embodiment, the invention provides a method for the multiplex detection of phosphorylation in a biological sample, the method comprising utilizing two or more antibodies or AQUA peptides of the invention to detect the presence of two or more phosphorylated proteins enumerated in Column A of Table 1/
Antibodies and/or AQUA peptides of the invention may also be employed within a kit that comprises at least one phosphorylation site-specific antibody or AQUA peptide of the invention (which binds to or detects a target signaling protein/polypeptide disclosed in Table 1/
Reference is made hereinafter in detail to specific embodiments of the invention. While the invention will be described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the invention to such specific embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. In the description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail, in order not to unnecessarily obscure the present invention.
The following examples are intended to further illustrate certain embodiments of the invention and are not limiting in nature. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein.
Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention. However, materials and methods are described. Materials, reagents and the like to which reference is made in the following description and examples are obtainable from commercial sources, unless otherwise noted.
IAP isolation techniques were employed to identify phosphotyrosine containing peptides in cell extracts from the following human cancer cell lines, tissues and patient cell lines: 01364548-cll, 223-CLL, 293T, 3T3 TrkB, 3T3-Src, 3T3-TrkA, 3T3-wt, 577, A172, AML-4833, AML-6246, AML-6735, AML-7592, BaF3-10ZF, BaF3-4ZF, BaF3-APR, BaF3-FLT3(D842V), BaF3-FLT3(D842Y), BaF3-FLT3(K663Q), BaF3-FLT3(WT), BaF3-FLT3/ITD, BaF3-PRTK, BaF3-TDII, BaF3-Tel/FGFR3, Baf3, Baf3-V617F-jak2, Baf3/E255K, Baf3/H396P, Baf3/Jak2(IL-3 dep), Baf3/M35 IT, Baf3/T3151, Baf3/TpoR, Baf3/TpoR-Y98F, Baf3/Tyk2, Baf3/V617F-jak2 (IL-3), Baf3/Y253F, Baf3/cc-TpoR-IV, Baf3/p210wt, CHRF, CI-1, CMK, CTV-1, DMS 53, DND41, DU-528, DU145, ELF-153, EOL-1, GDM-1, H1703, H1734, H1793, H1869, H1944, H1993, H2023, H226, H3255, H358, H520, H82, H838, HCC1428, HCC1435, HCC1806, HCC1937, HCC366, HCC827, HCT116, HEL, HL107B, HL117B, HL131A, HL131B, HL133A, HL53B, HL59b, HL60, HL61a, HL61b, HL66B, HL68A, HL75A, HL84A, HL97B, HL98A, HT29, HU-3, HUVEC, Jurkat, K562, KG-1, KG1-A, KMS11, KMS18, KMS27, KOPT-K1, KY821, Karpas 299, Karpas-1106p, M-07e, M01043, M059K, MC-116, MCF-10A (Y561F), MCF-10A(Y969F), MDA-MB-453, MDA-MB-468, MEC-2, MKPL-1, ML-1, MO-91, MOLT15, MV4-11, Me-F2, Molm 14, Monomac 6, NCI-N87, Nomo-1, OCI-M1, OCI-ly4, OCI-ly8, OCI/AML2, OPM-1, PL21, Pfeiffer, RC-K8, RI-1, SCLC T1, SEM, SK-N-AS, SK-N-MC, SKBR3, SR-786, SU-DHL1, SUP-M2, SUPT-13, SuDHL5, T17, TRE-cll patient, TS, UT-7, VAL, Verona, Verona 1, Verona 4, WSU-NHL, XG2, Z-55, cs001, cs015, cs025, cs041, cs042, gz21, gz68, gz73, gz74, gzB1, hl144b, hl152b, lung tumor T26, lung tumor T57, normal human lung, pancreatic xenograft, patient 1, rat brain and sw480.
Tryptic phosphotyrosine containing peptides were purified and analyzed from extracts of each of the cell lines mentioned above, as follows. Cells were cultured in DMEM medium or RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin.
Suspension cells were harvested by low speed centrifugation. After complete aspiration of medium, cells were resuspended in 1 mL lysis buffer per 1.25×108 cells (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplemented or not with 2.5 mM sodium pyro-phosphate, 1 mM β-glycerol-phosphate) and sonicated.
Sonicated cell lysates were cleared by centrifugation at 20,000×g, and proteins were reduced with DTT at a final concentration of 4.1 mM and alkylated with iodoacetamide at 8.3 mM. For digestion with trypsin, protein extracts were diluted in 20 mM HEPES pH 8.0 to a final concentration of 2 M urea and soluble TLCK®-trypsin (Worthington® Biochemcial Corporation, Lakewood, N.J.) was added at 10-20 μg/mL. Digestion was performed for 1-2 days at room temperature.
Trifluoroacetic acid (TFA) was added to protein digests to a final concentration of 1%, precipitate was removed by centrifugation, and digests were loaded onto Sep-Pak® C18 columns (provided by Waters Corporation, Milford, Mass.) equilibrated with 0.1% TFA. A column volume of 0.7-1.0 ml was used per 2×108 cells. Columns were washed with 15 volumes of 0.1% TFA, followed by 4 volumes of 5% acetonitrile (MeCN) in 0.1% TFA. Peptide fraction I was obtained by eluting columns with 2 volumes each of 8, 12, and 15% MeCN in 0.1% TFA and combining the eluates. Fractions II and III were a combination of eluates after eluting columns with 18, 22, 25% MeCN in 0.1% TFA and with 30, 35, 40% MeCN in 0.1% TFA, respectively. All peptide fractions were lyophilized.
Peptides from each fraction corresponding to 2×108 cells were dissolved in 1 ml of IAP buffer (20 mM Tris/HCl or 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl) and insoluble material was removed by centrifugation. IAP was performed on each peptide fraction separately. The phosphotyrosine monoclonal antibody P-Tyr-100 (Cell Signaling Technology®, Inc., Danvers, Mass. catalog number 9411) was coupled at 4 mg/ml beads to protein G or protein A agarose (Roche®, Basel, Switzerland), respectively. Immobilized antibody (15 μl, 60 μg) was added as 1:1 slurry in IAP buffer to 1.4 ml of each peptide fraction, and the mixture was incubated overnight at 40° C. with gentle rotation. The immobilized antibody beads were washed three times with 1 ml IAP buffer and twice with 1 ml water, all at 4° C. Peptides were eluted from beads by incubation with 75 μl of 0.1% TFA at room temperature for 10 minutes.
Alternatively, one single peptide fraction was obtained from Sep-Pak C18 columns by elution with 2 volumes each of 10%, 15%, 20%, 25%, 30%, 35% and 40% acetonitirile in 0.1% TFA and combination of all eluates. IAP on this peptide fraction was performed as follows: After lyophilization, peptide was dissolved in 1.4 ml IAP buffer (MOPS pH 7.2, mM sodium phosphate, 50 mM NaCl) and insoluble material was removed by centrifugation. Immobilized antibody (40 μl, 160 μg) was added as 1:1 slurry in IAP buffer, and the mixture was incubated overnight at 4° C. with gentle shaking. The immobilized antibody beads were washed three times with 1 ml IAP buffer and twice with 1 ml water, all at 4° C. Peptides were eluted from beads by incubation with 40 μl of 0.15% TFA at room temperature for 10 min (eluate 1), followed by a wash of the beads (eluate 2) with 40 μl of 0.15% TFA. Both eluates were combined.
Analysis by LC-MS/MS Mass Spectrometry.
40 μl or more of IAP eluate were purified by 0.2 μl StageTips (Proxeon, Staermosegaardsvej 6,DK-5230 Odense M, Denmark) or ZipTips® (produced by Millipore®, Billerica Mass.). Peptides were eluted from the microcolumns with 1 μl of 40% MeCN, 0.1% TFA (fractions I and II) or 1 μl of 60% MeCN, 0.1% TFA (fraction III) into 7.6 μl of 0.4% acetic acid/0.005% heptafluorobutyric acid. This sample was loaded onto a 10 cm×75 μm PicoFrit® capillary column (produced by New Objective, Woburn, Mass.) packed with Michrom Magic Bullets® C18 AQ reversed-phase resin (Michrom Bioresources, Auburn Calif.) using a Famos™ autosampler with an inert sample injection valve (Dionex®, Sunnyvale, Calif.). The column was then developed with a 45-min linear gradient of acetonitrile delivered at 200 nl/min (using an Ultimate® pump, Dionex®, Sunnyvale, Calif.), and tandem mass spectra were collected in a data-dependent manner with an LTQ® (produced by Thermo® Finnigan® San, Jose, Calif.), ion trap mass spectrometer essentially as described by Gygi et al., supra.
Database Analysis & Assignments.
MS/MS spectra were evaluated using TurboSequest™ in the Sequest® (owned by Thermo® Finnigan® San Jose, Calif.) Browser package (v. 27, rev. 12) supplied as part of BioWorkS™ 3.0 (Thermo® Finnigan®, San Jose, Calif.). Individual MS/MS spectra were extracted from the raw data file using the Sequest® Browser program CreateDta™ (owned by Thermo® Finnigan® San Jose, Calif.), with the following settings: bottom MW, 700; top MW, 4,500; minimum number of ions, 20; minimum TIC, 4×105; and precursor charge state, unspecified. Spectra were extracted from the beginning of the raw data file before sample injection to the end of the eluting gradient. The IonQuest™ and VuDta™ (owned by Thermo® Finnigan® San Jose, Calif.) programs were not used to further select MS/MS spectra for Sequest® analysis. MS/MS spectra were evaluated with the following TurboSequest™ parameters: peptide mass tolerance, 2.5; fragment ion tolerance, 0.0; maximum number of differential amino acids per modification, 4; mass type parent, average; mass type fragment, average; maximum number of internal cleavage sites, 10; neutral losses of water and ammonia from b and y ions were considered in the correlation analysis. Proteolytic enzyme was specified except for spectra collected from elastase digests.
Searches were performed against the NCBI human protein database (as released on Aug. 24, 2004 and containing 27, 960 protein sequences). Cysteine carboxamidomethylation was specified as a static modification, and phosphorylation was allowed as a variable modification on serine, threonine, and tyrosine residues or on tyrosine residues alone. It was determined that restricting phosphorylation to tyrosine residues had little effect on the number of phosphorylation sites assigned. Furthermore, it should be noted that certain peptides were originally isolated in mouse and later normalized to human sequences as shown by Table 1/
In proteomics research, it is desirable to validate protein identifications based solely on the observation of a single peptide in one experimental result, in order to indicate that the protein is, in fact, present in a sample. This has led to the development of statistical methods for validating peptide assignments, which are not yet universally accepted, and guidelines for the publication of protein and peptide identification results (see Carr et al., Mol. Cell. Proteomics 3: 531-533 (2004)), which were followed in this Example. However, because the immunoaffinity strategy separates phosphorylated peptides from unphosphorylated peptides, observing just one phosphopeptide from a protein is a common result, since many phosphorylated proteins have only one tyrosine-phosphorylated site. For this reason, it is appropriate to use additional criteria to validate phosphopeptide assignments. Assignments are likely to be correct if any of these additional criteria are met: (i) the same sequence is assigned to co-eluting ions with different charge states, since the MS/MS spectrum changes markedly with charge state; (ii) the site is found in more than one peptide sequence context due to sequence overlaps from incomplete proteolysis or use of proteases other than trypsin; (iii) the site is found in more than one peptide sequence context due to homologous but not identical protein isoforms; (iv) the site is found in more than one peptide sequence context due to homologous but not identical proteins among species; and (v) sites validated by MS/MS analysis of synthetic phosphopeptides corresponding to assigned sequences, since the ion trap mass spectrometer produces highly reproducible MS/MS spectra. The last criterion is routinely employed to confirm novel site assignments of particular interest.
All spectra and all sequence assignments made by Sequest were imported into a relational database. The following Sequest scoring thresholds were used to select phosphopeptide assignments that are likely to be correct: RSp<6, XCorr≧2.2, and DeltaCN>0.099. Further, the assigned sequences could be accepted or rejected with respect to accuracy by using the following conservative, two-step process.
In the first step, a subset of high-scoring sequence assignments should be selected by filtering for XCorr values of at least 1.5 for a charge state of +1, 2.2 for +2, and 3.3 for +3, allowing a maximum RSp value of 10. Assignments in this subset should be rejected if any of the following criteria were satisfied: (i) the spectrum contains at least one major peak (at least 10% as intense as the most intense ion in the spectrum) that can not be mapped to the assigned sequence as an a, b, or y ion, as an ion arising from neutral-loss of water or ammonia from a b or y ion, or as a multiply protonated ion; (ii) the spectrum does not contain a series of b or y ions equivalent to at least six uninterrupted residues; or (iii) the sequence is not observed at least five times in all the studies conducted (except for overlapping sequences due to incomplete proteolysis or use of proteases other than trypsin).
In the second step, assignments with below-threshold scores should be accepted if the low-scoring spectrum shows a high degree of similarity to a high-scoring spectrum collected in another study, which simulates a true reference library-searching strategy.
Polyclonal antibodies that specifically bind a Target Signal Protein/Polypeptide only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 1/
A. NCK2 (Tyrosine 50).
An 11 amino acid phospho-peptide antigen, TGy*VPSNYVER (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 50 phosphorylation site in human NCK2 adaptor/scaffold protein (see Row 5 of Table 1; SEQ ID NO: 4), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See A
B. Securin (Tyrosine 111)
A 15 amino acid phospho-peptide antigen, SSVPASDDAy*PEIEK (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 261 phosphorylation site in human securin cell cycle regulation protein (see Row 52 of Table 1 (SEQ ID NO: 51)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See A
C. p47phox (Tyrosine 48)
A 9 amino acid phospho-peptide antigen, FTEIy*EFHK (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 48 phosphorylation site in human p47phox enzyme protein (see Row 91 of Table 1 (SEQ ID NO: 92), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See A
Immunization/Screening.
A synthetic phospho-peptide antigen as described in A-C above is coupled to KLH, and rabbits are injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (500 μg antigen per rabbit). The rabbits are boosted with same antigen in incomplete Freund adjuvant (250 μg antigen per rabbit) every three weeks. After the fifth boost, bleeds are collected. The sera are purified by Protein A-affinity chromatography by standard methods (see A
The isolated antibody is then tested for phospho-specificity using Western blot assay using an appropriate cell line that expresses (or overexpresses) target phospho-protein (i.e. phosphorylated NCK2, securin or p47phox), for example, MO-91, Jurkat and Nomo-1 cells, respectively. Cells are cultured in DMEM or RPMI supplemented with 10% FCS. Cell are collected, washed with PBS and directly lysed in cell lysis buffer. The protein concentration of cell lysates is then measured. The loading buffer is added into cell lysate and the mixture is boiled at 100° C. for 5 minutes. 20 μl (10 μg protein) of sample is then added onto 7.5% SDS-PAGE gel.
A standard Western blot may be performed according to the Immunoblotting Protocol set out in the C
In order to confirm the specificity of the isolated antibody, different cell lysates containing various phosphorylated signal transduction proteins other than the target protein are prepared. The Western blot assay is performed again using these cell lysates. The phospho-specific polyclonal antibody isolated as described above is used (1:1000 dilution) to test reactivity with the different phosphorylated non-target proteins on Western blot membrane. The phospho-specific antibody does not significantly cross-react with other phosphorylated signal transduction proteins, although occasionally slight binding with a highly homologous phosphorylation-site on another protein may be observed. In such case the antibody may be further purified using affinity chromatography, or the specific immunoreactivity cloned by rabbit hybridoma technology.
Monoclonal antibodies that specifically bind a Target Signal Protein/Polypeptide only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 1/
A. Rap1a (Tyrosine 159)
A 12 amino acid phospho-peptide antigen, IMVNEIFy*DLVR (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 159 phosphorylation site in human Rap1a G protein or regulator protein (see Row 151 of Table 1 (SEQ ID NO: 152)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See A
B. PIK3R3 (Tyrosine 184)
An 11 amino acid phospho-peptide antigen, LQEy*HSQYQEK (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 184 phosphorylation site in human PIK3R3 kinase (non-protein) (see Row 185 of Table 1 (SEQ ID NO: 186)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See A
C. PIK4CA (Tyrosine 973)
A 13 amino acid phospho-peptide antigen, DQPy*YDIPDAPYR (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 973 phosphorylation site in human PIK4CA kinase (non-protein) (see Row 188 of Table 1 (SEQ ID NO: 189), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See A
Immunization/Fusion/Screening.
A synthetic phospho-peptide antigen as described in A-C above is coupled to KLH, and BALB/C mice are injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (e.g. 50 μg antigen per mouse). The mice are boosted with same antigen in incomplete Freund adjuvant (e.g. 25 μg antigen per mouse) every three weeks. After the fifth boost, the animals are sacrificed and spleens are harvested.
Harvested spleen cells are fused to SP2/0 mouse myeloma fusion partner cells according to the standard protocol of Kohler and Milstein (1975). Colonies originating from the fusion are screened by ELISA for reactivity to the phospho-peptide and non-phospho-peptide forms of the antigen and by Western blot analysis (as described in Example 1 above). Colonies found to be positive by ELISA to the phospho-peptide while negative to the non-phospho-peptide are further characterized by Western blot analysis. Colonies found to be positive by Western blot analysis are subcloned by limited dilution. Mouse ascites are produced from a single clone obtained from subcloning, and tested for phospho-specificity (against the Rap1a, PIK3R3 or PIK4CA phospho-peptide antigen, as the case may be) on ELISA. Clones identified as positive on Western blot analysis using cell culture supernatant as having phospho-specificity, as indicated by a strong band in the induced lane and a weak band in the uninduced lane of the blot, are isolated and subcloned as clones producing monoclonal antibodies with the desired specificity.
Ascites fluid from isolated clones may be further tested by Western blot analysis. The ascites fluid should produce similar results on Western blot analysis as observed previously with the cell culture supernatant, indicating phospho-specificity against the phosphorylated target (e.g. PIK4CA phosphorylated at tyrosine 973).
Heavy-isotope labeled peptides (AQUA peptides (internal standards)) for the detection and quantification of a Target Signal Protein/Polypeptide only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 1/
A. MYH10 (Tyrosine 1415).
An AQUA peptide comprising the sequence, ALAy*DKLEK (y*=phosphotyrosine; sequence incorporating 14C/15N-labeled leucine (indicated by bold L), which corresponds to the tyrosine 1415 phosphorylation site in human MYH10 motor or contractile protein (see Row 199 in Table 1 (SEQ ID NO: 200)), is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.) as further described below in Synthesis & MS/MS Signature. The MYH10 (tyr 1415) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated MYH10 (tyr 1415) in the sample, as further described below in Analysis & Quantification.
B. PPP6C (Tyrosine 261)
An AQUA peptide comprising the sequence LVTVWSAPNy*CYR (y*=phosphotyrosine; sequence incorporating 14C/15N-labeled leucine (indicated by bold L), which corresponds to the tyrosine 261 phosphorylation site in human PPP6C phosphatase (see Row 222 in Table 1 (SEQ ID NO: 223)), is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.) as further described below in Synthesis & MS/MS Signature. The PPP6C (tyr261) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated PPP6C (tyr261) in the sample, as further described below in Analysis & Quantification.
C. PKCT (Tyrosine 545)
An AQUA peptide comprising the sequence TNTFCGTPDy*IAPEILLGQK (y*=phosphotyrosine; sequence incorporating 14C/15N-labeled phenylalanine (indicated by bold F), which corresponds to the tyrosine 545 phosphorylation site in human G-alpha-s protein kinase (Ser/Thr) (see Row 271 in Table 1 (SEQ ID NO: 272)), is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.) as further described below in Synthesis & MS/MS Signature. The PKCT (tyr545) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated PKCT (tyr545) in the sample, as further described below in Analysis & Quantification.
D. PLK1 (Tyrosine 217)
An AQUA peptide comprising the sequence, TLCGTPNy*IAPEVLSK (y*=phosphotyrosine; sequence incorporating 14C/15N-labeled proline (indicated by bold P), which corresponds to the tyrosine 217 phosphorylation site in human PLK1 receptor/channel/transporter/cell surface protein (see Row 274 in Table 1 (SEQ ID NO: 175)), is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.) as further described below in Synthesis & MS/MS Signature. The PLK1 (tyr217) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated PLK1 (tyr217) in the sample, as further described below in Analysis & Quantification.
Synthesis & MS/MS Spectra.
Fluorenylmethoxycarbonyl (Fmoc)-derivatized amino acid monomers may be obtained from AnaSpec (San Jose, Calif.). Fmoc-derivatized stable-isotope monomers containing one 15N and five to nine 13C atoms may be obtained from Cambridge Isotope Laboratories (Andover, Mass.). Preloaded Wang resins may be obtained from Applied Biosystems. Synthesis scales may vary from 5 to 25 μmol. Amino acids are activated in situ with 1-H-benzotriazolium, 1-bis(dimethylamino) methylene]-hexafluorophosphate (1-),3-oxide: 1-hydroxybenzotriazole hydrate and coupled at a 5-fold molar excess over peptide. Each coupling cycle is followed by capping with acetic anhydride to avoid accumulation of one-residue deletion peptide by-products. After synthesis peptide-resins are treated with a standard scavenger-containing trifluoroacetic acid (TFA)-water cleavage solution, and the peptides are precipitated by addition to cold ether. Peptides (i.e. a desired AQUA peptide described in A-D above) are purified by reversed-phase C18 HPLC using standard TFA/acetonitrile gradients and characterized by matrix-assisted laser desorption ionization-time of flight (Biflex III, Bruker Daltonics, Billerica, Mass.) and ion-trap (ThermoFinnigan, LCQ DecaXP) MS.
MS/MS spectra for each AQUA peptide should exhibit a strong y-type ion peak as the most intense fragment ion that is suitable for use in an SRM monitoring/analysis. Reverse-phase microcapillary columns (0.1 Ř150-220 mm) are prepared according to standard methods. An Agilent 1100 liquid chromatograph may be used to develop and deliver a solvent gradient [0.4% acetic acid/0.005% heptafluorobutyric acid (HFBA)/7% methanol and 0.4% acetic acid/0.005% HFBA/65% methanol/35% acetonitrile] to the microcapillary column by means of a flow splitter. Samples are then directly loaded onto the microcapillary column by using a FAMOS inert capillary autosampler (LC Packings, San Francisco) after the flow split. Peptides are reconstituted in 6% acetic acid/0.01% TFA before injection.
Analysis & Quantification.
Target protein (e.g., a phosphorylated protein of A-D above) in a biological sample is quantified using a validated AQUA peptide (as described above). The IAP method is then applied to the complex mixture of peptides derived from proteolytic cleavage of crude cell extracts to which the AQUA peptides have been spiked in.
LC-SRM of the entire sample is then carried out. MS/MS may be performed by using a ThermoFinnigan (San Jose, Calif.) mass spectrometer (LTQ ion trap or TSQ Quantum triple quadrupole). On the LTQ, parent ions are isolated at 1.6 m/z width, the ion injection time being limited to 100 ms per microscan, with one microscans per peptide, and with an AGC setting of 1×105; on the Quantum, Q1 is kept at 0.4 and Q3 at 0.8 m/z with a scan time of 200 ms per peptide. On both instruments, analyte and internal standard are analyzed in alternation within a previously known reverse-phase retention window; well-resolved pairs of internal standard and analyte are analyzed in separate retention segments to improve duty cycle. Data are processed by integrating the appropriate peaks in an extracted ion chromatogram (60.15 m/z from the fragment monitored) for the native and internal standard, followed by calculation of the ratio of peak areas multiplied by the absolute amount of internal standard (e.g., 500 fmol).
This is a National Stage Application of International Application No. PCT/US07/073,540 filed Jul. 13, 2007, which itself claims priority to U.S. Ser. No. 60/830,550 filed Jul. 13, 2006 now abandoned, both disclosures of which are incorporated herein, in their entirety, by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2007/073540 | 7/13/2007 | WO | 00 | 1/13/2009 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/009002 | 1/17/2008 | WO | A |
| Number | Date | Country | |
|---|---|---|---|
| 20090258436 A1 | Oct 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60830550 | Jul 2006 | US |
