Identification and functional characterization of a novel ribosomal S6 protein kinase

Abstract

A novel S6 kinase, p70βS6k, is described, along with methods of making and using p70βS6k and related nucleic acids. The invention also discloses methods of identifying agents which modulate the activity of p70βS6k and/or its ligands.

Description

FIELD OF THE INVENTION

The present invention relates to a novel S6 kinase (p70β Sk6 ), mutant variants thereof methods of making and using this S6 kinase, and related nucleic acids and Ski antibodies. The invention also relates to binding partners of the S6 kinase, methods of identifying the binding partners and antibodies thereto.

BACKGROUND OF THE INVENTION

The 40S ribosomal protein S6 is a component of the 40S subunit of eukaryotic ribosomes. The ribosomes are part of the cellular machinery responsible for translation of mRNA and protein synthesis. The S6 protein is phosphorylated in response to certain cellular signaling events such as hormone or growth factor induced cellular proliferation. p70 S6 kinase (p70 S6k ) is responsible for S6 phosphorylation and is believed to be the major physiological S6 kinase in mammalian cells (Proud, 1996 Trends Biochem. Sci. 21:181-185).

1. p70α S6 Kinase

A. Structure and Function

The first p70 S6 kinase identified was the alpha (α) form. The gene encoding the human p70α S6 kinase (70 S6k )was isolated in 1991 (Grove et al., 1991 Mol. Cell. Biol. 11:5541-5550). Other p70α S6 kinase sequences have been described in Mus musculus (GenBank Accession No. SEG_AB015196S, AB015197, and AB015196), Xenopus laevis (GenBank Accession No. X66179), and rat (GenBank Accession No. M57428).

Two p70α S6 kinase isoforms were identified: p70α-I GenBank Accession No. M60724) and p70α-II (GenBank Accession No. M60725). The two p70α S6 kinase isoforms differ only in their amino tenmini by 23 amino acid residues resulting in a 70 kD protein and a 85 kD protein. The isoforms are referred to in the literature as p70 S6k /p85 S6k or p70α S6 kinase. Both isoforms share similar activity towards ribosomal protein S6 in vitro but are expressed in different cells and tissues. The two isoforms are produced by two mRNA products and are not a result of post-translational modifications. They are serine/threonine kinases and are known to act on the substrate KKRNRTLSVA (SEQ ID No. 7) (Pai et al., 1994 Eur. J. Immunol. 24:2364-8; and Leighton et al., 1995 FEBS Letters 375:289-93).

The p70α S6 kinase plays an important role in the progression of cells from G1 to S phase of the cell cycle and in the initiation of protein synthesis. Recently, p70α S6 kinase has been demonstrated to regulate the translation of a class of mRNAs containing an oligopyrimidine tract in their 5′ untranslated region. This class of mRNAs, termed 5′TOP mRNAs, represent up to 20% of the a cell's total mRNA. Many of the proteins encoded by 5′TOP mRNAs are translational apparatus proteins and cell-cycle progression proteins.

The p70α S6 kinase has four identified interdependent domains: (1) a catalytic domain, (2) a kinase extension domain, (3) a pseudosubstrate autoinhibitory domain, and (4) the N-terminal domain. The catalytic domain is located in the middle of the protein and is followed by the kinase extension domain, which is a unique feature for the PKA family. The pseudosubstrate autoinhibitory domain is also unique for the p70α S6 kinase, not having been observed in any other known kinases. It possesses 5 phosphorylation sites which are responsible for the p70α S6 kinase regulation. The N-terminal domain mediates the sensitivity for rapamycin, which strongly inhibits serum-induced phosphorylation and activation of the p70α S6 kinase. This domain may also mediate the interaction with a yet unknown phosphatase.

B. Regulators and Cascades

Growth factors, such as insulin, and mitogens are known to activate in vivo p70α S6 kinase (Alessi et al., 1998 Curr. Biol. 8:69-81). Heat shock also activates p70α S6 kinase (Lin et al., 1997 J. Biol. Chem. 272:31196-31202). Certain drugs have been identified that regulate p70α S6 kinase activity including: rapamycin, wortmannin, Ro31-8220, GF109203X, LY294002, phenylephrine (PE), PD098059, SQ20006, polymerized collagen, forskolin, interleukin-10 (IL-10), demethoxyviridin, phorbol 12-myristate 13-acetate (PMA), A23187, bombesin and antibodies which recognize the p70α S6 kinase (Proud, 1996; Morreale et al., 1997 FEBS Letters 417:38-42; Kanda et al., 1997 J. Biol. Chem, 272:23347-23353; Boluyt et al., 1997 Circ Res. 81:176-186; Coolican et al, 1997 J. Biol. Chem. 272:6653-6662; Koyama et al., 1996 Cell 87: 1069-1078; Busca et al., 1996 J. Biol. Chem. 271:31824-31830; Crawley et al., 1996 J. Biol. Chem. 271:16357-16362; and Petritsch et al., 1995 Eur, J. Biochem. 230:431-8). The immunosuppressant rapamycin (Rap) is the most potent inhibitor of p70α S6 kinase described (Pullen et al., 1997 FEBS Letters 410:78-82).

p70α S6 kinase is an enzyme which lies downstream of phosphoinositide 3-kinases (P13-kinase). The mechanisms regulating the p70α S6 kinase have not been fully elucidated. P13-kinase has recently been shown to activate another phosphoinositide-dependent protein kinase, termed PDK-1. So far, only PDK-1 has been shown to phosphorylate p70α S6 kinase in vivo, and this phosphorylation is essential for p70α S6k activity towards ribosomal S6 protein. Wortmannin, a fungal inhibitor which down-regulates the p70α S6 kinase, is believed to act by inhibiting PI-3 kinase. In contrast, another fungal inhibitor, rapamycin, inhibits the p70α S6 kinase by another cascade pathway involving the mammalian target of rapamycin (mTOR; also known as RAFT or FRAP) (Proud, 1996; Stewart et al., 1994 BioEssays 16:809-815). mTOR is a member of the PIK-related family of protein kinases (Pullen et al., 1997). Additional regulators of the p70α S6 kinase include, but are not limited to protein kinase B (PKB), Cdc42, and Rac. The role of most of these proteins as p70α S6 kinase regulators has yet to be fully elucidated.

SUMMARY OF THE INVENTION

The present invention is based on our discovery of a new gene which encodes a novel S6 kinase (p70β S6k ). The invention includes isolated nucleic acid molecules selected from the group consisting of an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID No.2, (e.g., SEQ ID No.1) an isolated nucleic acid molecule that encodes a fragment of SEQ ID No.2, an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule comprising SEQ ID No.1 under conditions of sufficient stringency to produce a clear signal and an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule that encodes the amino acid sequence of SEQ ID No.2 under conditions of sufficient stringency to produce a clear signal.

The present invention further includes the nucleic acid molecules operably linked to one or more expression control elements, including vectors comprising the isolated nucleic acid molecules. The invention further includes host cells transformed to contain the nucleic acid molecules of the invention and methods for producing a protein comprising the step of culturing a host cell transformed with the nucleic acid molecule of the invention under conditions in which the protein is expressed.

The invention further provides an isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID No.2, an isolated polypeptide comprising a fragment of SEQ ID No.2, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID No.2 and naturally occurring amino acid sequence variants of SEQ ID No.2.

The invention further provides an isolated antibody that binds to a polypeptide of the invention, including monoclonal and polyclonal antibodies and fragments thereof.

The invention further provides methods of identifying an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2 comprising the steps of: exposing cells which express the nucleic acid to the agent; and determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2.

The invention further provides methods of identifying an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2 comprising the steps of: exposing cells which express the protein to the agent; and determining whether the agent modulates at least on activity of said protein, thereby identifying an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2.

The invention further provides methods of identifying binding partners for a protein comprising the sequence of SEQ ID No.2 or activated variants thereof, comprising for example, the steps of: exposing said protein to a potential binding partner, and determining if the potential binding partner binds to said protein, thereby identifying binding partners for a protein comprising the sequence of SEQ ID No.2. Exposing may be accomplished by expressing the protein in a cell.

The present invention further provides methods of modulating the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2 comprising the step of: administering an effective amount of an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2.

The invention also provides methods of modulating at least one activity of a protein comprising the sequence of SEQ ID No.2 comprising the step of: administering an effective amount of an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Nucleic acid sequence of p70β S6k (SEQ ID No.1) and comparison with p70α S6k (SEQ ID No. 3). Sequence analysis of cDNA encoding the p70β S6 kinase. p70β cDNA may encode two isoforms with the use of alternative start codon. The longer form may encode a protein of 495 amino acid residues and the shorter form, 482 amino acids (aa). Alternative start codons and a stop codon are highlighted

FIGS. 2A-2B . Amino Acid Sequence of p70β S6k (SEQ ID No.2) and comparison with p70α S6K (SEQ ID No. 4).

FIG. 2 A. Alignment of predicted protein sequences corresponding to the p70β S6K and p70β S6 kinases. Identical amino acids are boxed.

FIG. 2 B. Comparative analysis of regulatory domains and phosphorylation sites between p70α and β S6 kinases.

FIGS. 3A-3B . Tissue Specific Expression of p70β S6k . Northern blot analysis of poly(A)+ RNA from human tissues ( FIG. 3A ) and tumor cell lines (FIG. 3 B).

FIG. 3 A. Nylon membrane containing 2 μg of gel-separated, pre-bound poly(A)+ RNA samples from various human tissues was hybridized with cDNA fragments of p70α S6k , p70β S6k or β-actin labeled by random priming. The upper, middle and lower panels are autoradiographs probed with p70β S6k , p70α S6k and β-actin, respectively. Each lane contains mRNA prepared from: heart (lane 1), brain (lane 2), placenta (lane 3). lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7), pancreas (lane 8), spleen (lane 9), thymus (lane 10), prostate (lane 11), testis (lane 12), ovary (lane 13), small intestine (lane 14), mucosal lining of the colon (lane 15), and peripheral blood leukocytes (lane 16).

FIG. 3 B. Nylon membrane containing 2 μg of mRNA isolated from tumor cell lines was probed with the 3′ cDNA fragment from p70β S6k , which was labeled by random-prime labeling. Specific binding was determined by autoradiography. Promyelocytic leukemia HL-60 (lane 1), HeLa cell S3 (lane 2, chronic myelogenous leukemia K562 (lane 3), lymphoblastic leukemia MOLT-4 (lane 4), Burkitt's lymphoma Raji (lane 5), colorectal adenocarcinoma SW480 (lane 6), lung carcinoma (lane 7), and melanoma G361 (lane 8).

FIGS. 4A-4B . Phosphorylation of the ribosomal protein S6 ( FIG. 4A ) and its C-terminal synthetic peptide ( FIG. 4B ) by p70α S6k and p70β S6k . Ribosomal S6 protein (purified ribosomal 40S subunits from liver) and synthetic peptides corresponding to the S6 protein C-terminus (e.g., KEAKEKRQEQIARRRLSSLRASTSKSESSQK-long form (SEQ ID No. 5) and RRRLSSLRASTSKSESSQK-(SEQ ID No. 6) short form) were used to measure the activity of the p70α S6k and p70β S6k . HEK293 cells were transfected with plasmids containing Flag-tag or EE-tag versions of p70α S6k or p70β S6k . Recombinant proteins were immunoprecipitated with anti-EE or anti-Flag antibodies and an in vitro kinase reaction performed in the presence of the ribosomal 40S subunits or synthetic peptides. After SDS-PAGE analysis, phosphorylation of the S6 protein and synthetic peptides was measured by PhosphoImager and expressed in arbitrary units (PI units).

FIGS. 5A-5B . Stimulation of p70β S6k Activity by insulin, serum and TPA.

FIG. 5 A. CHO—IR cells were transfected with mock (lane1) or plasmids containing cDNAs of FLAG-tagged p70α-I (lanes 2 and 3) or FLAG-tagged p70β-II (lanes 4 to 7). After serum starvation for 16 hrs, cells were treated with the vehicle (lanes 2 and 4), 10 −7 M insulin for 10 min (lanes 3 and 5), 15% serum for 10 min (lane 6) or 500 nM TPA for 30 min (lane7). After cell lysis and subsequent immunoprecipitation with anti-FLAG antibodies, immunoprecipitates were subjected to a p70 S6 kinase assay using 40S subunits as substrates. The reaction mixture was separated by SDS-PAGE, transferred onto PVDF membrane. The membrane was analyzed by autoradiography (upper panel) and then immunoblotted with anti-FLAG antibodies (lower panel). A representative of three experiments is shown. 32 P incorporation into S6 was quantified by Molecular Dynamics PhosphorImager™ and is expressed in arbitrary units (PI units).

FIG. 5 B. Stimulation of the p70α S6k and p70β S6k activity towards ribosomal S6 protein by PDGF in PAE-PDGF-R cells. PAE-PDGF-R cells were transfected with EE-tagged p70α S6k and p70β S6k plasmids using lipofectAMINE. After 24 hr, transfected cells were serum-starved for 16 hr and stimulated with 20 ng/ml PDGF BB (Calbiochem) for 20 min. Control cells were treated with the vehicle under the same conditions. After immunoprecipitation with anti-EE antibodies, an in vitro kinase reaction was carried out in the presence of 40S subunits, containing the S6 protein. Reaction mixtures were separated by SDS-PAGE and incorporation of 32 P into S6 protein was measured by PhosphoImager.

FIGS. 6A-6B . Effects of rapamycin and wortmannin on p70β S6k and p70α S6k . Effects of rapamycin and wortmannin on S6 phosphorylation activity of p70α-I or p70β-II. HEK293 cells were transfected with mock cDNA or plasmids containing FLAG-tagged p70α1 or p70β2 isoforms. After 48 h of transfection, cells were treated with vehicle or indicated concentrations of rapamycin or wortmannin for 30 min. After immunoprecipitation with anti-FLAG antibodies, the kinase activity was determined by a p70 S6 kinase assay using 40S subunits as substrates. The proteins of the reaction mixture were separated by SDS-PAGE, transferred onto PVDF membrane and analyzed by autoradiography ( FIGS. 6A and 6B , upper panels). Subsequent immunoblotting with anti-FLAG antibody confirmed the expression of p70α-I ( FIG. 6A , lower panel) and p70β-II ( FIG. 6B , lower panel). A representative of three experiments is shown. 32 P incorporation into S6 was quantified by PhosphorImager and is expressed in arbitrary units (PI units).

FIG. 7 . Interaction of the p70β S6k with different GST/SH3 fusion proteins. HEK293 cells were transiently transfected with EE-tag/p70β S6k . Two days later, cells were lysed and the lysates were immunoprecipitated with anti-EE antibodies. GST/SH3 fusion proteins (1.5 μg each) were incubated with anti-EE tag immunoprecipitates. Specific interaction with p70β S6k was measured by anti-GST immunoblotting, SH3 domains from different signaling and cytoskeletal proteins were expressed in bacteria as GST fusion proteins and purified nearly to homogeneity using glutathione-Sepharose beads. The GST/SH3 fusion proteins used are: GST (lane 1), p80α subunit of the PI3-kinase (lane 2), GAP (lane 3), PLCγ (lane 4), spectrin (lane 5), crk (lane 6), n-grb2 (lane 7), c-grb2 (lane 8), grb2 full (lane 9), csk (lane 10), fgr (lane 11), fyn (lane 12), src (lane 13), ruk a (lane 14), ruk b (lane 15), ruk c (lane 16), p15 (lane 17), profilin (lane 18) and GST/GAP control (lane 19). “I” indicates instances of binding between p70β S6k and a SH3 containing fusion protein.

FIG. 8 Immunoprecipitation and Western blot analysis of the p70β-I and p70β-II isoforms transiently over expressed in HEK293 cells with anti-p70β S6k antibodies. The lanes are the same for each panel: mock transfected (lane 1), Flag-p70α-I transfected (lane 2), Flag-p70β-I transfected (lane 3), and Flag-p70β-II transfected (lane 4).

FIG. 9. A model for the activation of p70 S6 Kinase. Schematic presentation of the p70α S6k structure, protein—protein interactions, activation levels and phosphorylation state.

FIG. 10. p 70β S6k mutations. Schematic presentation of substitution mutations engineered into p70β S6k , including a change of Threonine at amino acid 401 to Aspartic acid (T401D).

FIG. 11. p 70β S6k (T401D) activity. Activity of p70β S6k (T401D) variant as compared to wt p70β S6k under S6 Kinase and autophosphorylation assays.

FIG. 12 : p70α S6k (T412D) activity. Activity of p70α S6k (T412D) variant as compared to wt p70α S6k under S6 Kinase and autophosphorylation assays.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The terms “p70α”, “p70α S6k ” and “p70α S6 kinase” are meant to include the two isoforms, p70 and p85, both of which phosphorylate the ribosomal protein S6. By “p70α-1” and “p70α-I” are meant the p85 isoform of the p70α S6 kinase. By “p70α-2” and “p70α-II” are meant the p70 isoform of the p70α S6 kinase.

The terms “p70β”, “p70β S6k ” and “p70β S6 kinase” include the newly identified S6 kinase and all its isoforms. I. General Description

The present invention is based in part on identifying a new gene that encodes a novel S6 kinase (p70β S6k ). This new gene and the protein that it encodes are members of the family of S6 kinases, of which the p70α-I and -II (also referred to as p70α-1 and p70α-2) isoforms have already been reported.

The protein can serve as a target for agents that can be used to modulate the expression or activity of the protein. For example, agents may be identified which modulate biological processes associated with ribosomal activity.

The present invention is further based on the development of methods for isolating binding partners that bind to the protein or its activated variants. Probes based on the protein are used as capture probes to isolate potential binding partners, such as other proteins. Dominant negative proteins, DNAs encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function. Additionally, these proteins provide a novel target for screening of synthetic small molecules and combinatorial or naturally occurring compound libraries to discover novel therapeutics to regulate ribosomal function.

II. Specific Embodiments

A. The Ribosome Associated Protein

The present invention provides isolated protein, allelic variants of the protein, and conservative amino acid substitutions of the protein, including substitutions that activate the protein. As used herein, the protein or polypeptide refers to a protein that has the human amino acid sequence of depicted in SEQ ID No.2. The invention includes naturally occurring allelic variants and proteins that have a slightly different amino acid sequence than that specifically recited above. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the same or similar biological functions associated with the disclosed protein.

As used herein, the family of proteins related to the disclosed protein refer to proteins that have been isolated from organisms in addition to humans. The methods used to identify and isolate other members of the family of proteins related to the disclosed protein are described below.

The proteins of the present invention are preferably in isolated form. As used herein, a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated protein.

The proteins of the present invention further include conservative variants of the proteins herein described. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the protein. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the protein. For example, the overall charge, structure or hydrophobic/hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein. Conservative substitutions may irreversibly activate the protein.

Ordinarily, the allelic variants, the conservative substitution variants, the members of the protein family, will have an amino acid sequence having at least 71% about 75% amino acid sequence identity with the human sequence set forth in SEQ ID No.2, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95%. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.

Homology or identity is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblasto and tblastx (Karlin, et al. Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and Altschul S. F. J. Mol. Evol. 36:290-300(1993), fully incorporated by reference) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al. (Nature Genetics 6:119-129 (1994)) which is fully incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff, et al. Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992), fully incorporated by reference). For blastn, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N USA 87:2264-2268 (1990) and (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and 4, respectively.

Thus, the proteins of the present invention include molecules having the amino acid sequence disclosed in SEQ ID No.2; fragments thereof having a consecutive sequence of at least about 3, 5, 10 or 15 amino acid residues of the disclosed protein; amino acid sequence variants of such sequence wherein an amino acid residue has been inserted N- or C-terminal to, or within, the disclosed sequence; amino acid sequence variants of the disclosed sequence, or their fragments as defined above, that have been substituted by another residue. Contemplated variants further include those containing predetermined mutations by, e.g. homologous recombination, site-directed or PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to rabbit, rat, murine, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope). Proteins of the invention may include fusion proteins comprising any of the foregoing.

As described below, members of the family of proteins can be used: 1) to identify agents which modulate at least one activity of the protein, including agents which may modulate phosphorylation mediated by the protein; 2) in methods of identifying binding partners for the protein, 3) as an antigen to raise polyclonal or monoclonal antibodies, and 4) as a therapeutic agent.

B. Nucleic Acid Molecules

The present invention further provides nucleic acid molecules that encode the protein having SEQ ID No.2 and the related proteins herein described, preferably in isolated form. As used herein, “nucleic acid” is defined as RNA or DNA that encodes a peptide as defined above, or is complementary to nucleic acid sequence encoding such peptides, or hybridizes to such nucleic acid and remains stably bound to it under appropriate stringency conditions, or encodes a polypeptide sharing at least 75% sequence identity, preferably at least 80%, and more preferably at least 85%, with the peptide sequences. Specifically contemplated are genomic DNA, cDNA, mRNA and antisense molecules, as well as nucleic acids based on alternative backbone or including alternative bases whether derived from natural sources or synthesized. Such hybridizing or complementary nucleic acids, however, are defined further as being novel and nonobvious over any prior art nucleic acid including that which encodes hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a protein according to the present invention.

“Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl, 0.0015 M sodium titrate, 0.1% SDS at 50° C.; or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C. Another example is use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS or at 68° C. in 0.1×SSC and 0.5% SDS. A skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal.

As used herein, a nucleic acid molecule is said to be “isolated” when the nucleic acid molecule is substantially separated from contaminant nucleic acid encoding other polypeptides from the source of nucleic acid.

The present invention further the provides fragments of the encoding nucleic acid molecule. As used herein, a fragment of an encoding nucleic acid molecule refers to a small portion of the entire protein encoding sequence. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the protein, the fragment will need to be large enough to encode the functional region(s) of the protein. If the fragment is to be used as a nucleic acid probe or PCR primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming.

Fragments of the encoding nucleic acid molecules of the present invention (i.e., synthetic oligonucleotides) that are used as probes or specific primers for the polymerase chain reaction (PCR), or to synthesize gene sequences encoding proteins of the invention can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., ( J. Am. Chem. Soc 103:3185-3191, 1981) or using automated synthesis methods. In addition, larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build the complete modified gene.

The encoding nucleic acid molecules of the present invention may further be modified so as to contain a detectable label for diagnostic and probe purposes. A variety of such labels are known in the art and can readily be employed with the encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides and the like. A skilled artisan can employ any of the art known labels to obtain a labeled encoding nucleic acid molecule.

Modifications to the primary structure itself by deletion, addition, or alteration of the amino acids incorporated into the protein sequence during translation can be made without destroying the activity of the protein. Such substitutions or other alterations result in proteins having an amino acid sequence encoded by a nucleic acid failing within the contemplated scope of the present invention.

C. Isolation of Other Related Nucleic Acid Molecules

As described above, the identification of the human nucleic acid molecule having SEQ ID No.1 allows a skilled artisan to isolate nucleic acid molecules that encode other members of the p70β S6k family in addition to the human sequence herein described. Further, the presently disclosed nucleic acid molecules allow a skilled artisan to isolate nucleic acid molecules that encode other members of the p70β S6k family of proteins in addition to the disclosed protein having SEQ ID No.2.

Essentially, a skilled artisan can readily use the amino acid sequence of SEQ ID No.2 to generate antibody probes to screen expression libraries prepared from appropriate cells. Typically, polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a mammalian cDNA or genomic expression library, such as λgtll library, to obtain the appropriate coding sequence for other members of the protein family. The cloned cDNA sequence can be expressed as a fusion protein expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the enzyme.

Alternatively, a portion of the coding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the protein family from any mammalian organism. Oligomers containing approximately 18-20 or 21 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.

Additionally, pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively clone an encoding nucleic acid molecule. A PCR denature/anneal/extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other encoding nucleic acid molecules.

D. rDNA molecules Containing a Nucleic Acid Molecule

The present invention further provides recombinant DNA molecules (rDNAs) that contain a coding sequence. As used herein, a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al. (1989). In the preferred rDNA molecules, a coding DNA sequence is operably linked to expression control sequences and/or vector sequences.

The choice of vector and/or expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is well known in the at on the functional properties desired, e.g., protein expression, and the host cell to be transformed. A vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the struck gene included in the rDNA molecule.

Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promotes, constitutive promoters, secretion signals and other regulatory elements. Preferably, the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.

In one embodiment, the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.

Vectors that include a prokarotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli . A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, Calif.), pPL and pKK223 available from Pharmacia, Piscataway. N.J.

Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can also be used to form a rDNA molecules the contains a coding sequence. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors.

Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene (Southern et al., J. Mol. Anal. Genet. 1:327-341, 1982). Alternatively, the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co-transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.

E. Host Cells Containing an Exogenously Supplied Coding Nucleic Acid Molecule

The present invention further provides host cells transformed with a nucleic acid molecule that encodes a protein of the present invention. The host cell can be either prokaryotic or eukaryotic. Eukaryotic cells useful for expression of a protein of the invention are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product. Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line. Preferred eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines.

Any prokaryotic host can be used to express a rDNA molecule encoding a protein of the invention. The preferred prokaryotic host is E. coli.

Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al., Proc. Natl. Acad, Sci. USA 69:2110, 1972; and Maniatis et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). With regard to transformation of vertebrate cells with vectors containing rDNAs, electroporation, cationic lipid or salt treatment methods are typically employed, see, for example, Graham et al., Virol. 52:456, 1973; Wigler et al., Proc. Natl. Acad. Sci. USA 76:1373-76, 1979.

Successfully transformed cells, i.e., cells that contain a rDNA molecule of the present invention, can be identified by well known techniques including the selection for a selectable marker. For example, cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J. Mol. Biol. 98:503, 1975, or Berent et al., Biotech 3:208, 1985 or the proteins produced from the cell assayed via an immunological method.

Recombinant p70β S6k DNA can also be utilized to analyze the function of coding and non-coding sequences. For example, the 5′ untranslated region of the p70β S6k clone contains a GA repeat (nucleotides 1-66 of P70β S6k ), that may modulate the initiation of translation of its mRNA. This sequence can be utilized in an affinity matrix system to purify proteins obtained from cell lysates that associate with the p70β S6k GA sequence. Synthetic oligonucleotides would be coupled to the beads and probed with the lysates, as is commonly known in the art. Associated proteins could then be separated using, for example, a two dimensional SDS-PAGE system. Proteins thus isolated could be further identified using mass spectroscopy or protein sequencing.

F. Production of Recombinant Proteins using a rDNA Molecule

The present invention further provides methods for producing a protein of the invention using nucleic acid molecules herein described. In general tams, the production of a recombinant form of a protein typically involves the following steps:

First, a nucleic acid molecule is obtained that encodes a protein of the invention, such as the nucleic acid molecule depicted in SEQ ID No. 1, or particularly for the p70β S6k nucleotides encoding the proline rich domain or the amino terminus of p70β S6k . The coding sequence is directly suitable for expression in any host, as it is not interrupted by introns. The sequence can be transfected into host cells such as eukaryotic cells or prokaryotic cells. Eukaryotic hosts include mammalian cells (e.g., HEK293 cells, CHO cells and PAE-PDGF-R cells) as well as insect cells such as Sf9 cells using recombinant baculovirus. Alternatively, fragments encoding only portion of p70β S6k can be expressed alone or in the form of a fusion protein For example, the C-terminal fragment of p70β S6k containing the proline-rich domain, was expressed in bacteria as a GST- or His-tag fusion protein. These fusion proteins were then purified and used to generate polyclonal antibodies.

The nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame. The expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein. Optionally the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.

Each of the foregoing steps can be done in a variety of ways. For example, the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts. The construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce recombinant protein.

G. In Vitro Methods to Identify Binding Partners

Another embodiment of the present invention provides methods for use in isolating and identifying binding partners of proteins of the invention In detail, a protein of the invention is nixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein of the invention. After mixing, peptides, polypeptides, proteins or other molecules that have become associated with a protein of the invention are separated from the mixture. The binding partner that bound to the protein of the invention can then be removed and further analyzed. To identify and isolate a binding partner, the entire protein, for instance the entire disclosed protein of SEQ ID No.2 can be used. Alternatively, a fragment of the protein can be used.

As used herein, a cellular extract refers to a preparation or fraction which is made from a lysed or disrupted cell.

A variety of methods can be used to obtain cell extracts. Cells can be disrupted using either physical or chemical disruption methods. Examples of physical disruption methods include, but are not limited to, sonication and mechanical shearing. Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis. A skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.

Once an extract of a cell is prepared, the extract is mixed with the protein of the invention under conditions in which association of the protein with the binding partner can occur. A variety of conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell. Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner.

After mixing under appropriate conditions, the bound complex is separated from the mixture. A variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used.

After removal of non-associated cellular constituents found in the extract, the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.

To aid in separating associated binding partner pairs from the mixed extract, the protein of the invention can be immobilized on a solid support For example, the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide/binding partner pairs from other constituents found in the extract. The identified binding partners can be either a single protein or a complex made up of two or more proteins.

Alternatively, the nucleic acid molecules of the invention can be used in a yeast two-hybrid system. The yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described.

One preferred in vitro binding assay for p70β S6k would comprise a mixture of a polypeptide comprising at least the kinase domain of p70β S6k and one or more candidate binding targets or substrates. After incubating the mixture under appropriate conditions, one would determine whether p70β S6k or a polypeptide fragment thereof containing the kinase region either bound with the candidate substrate or phosphorylated the candidate substrate. For cell-free binding assays, one of the components usually comprises or is coupled to a label. The label may provide for direct detection, such as radioactivity, luminescence, optical or electron density, etc., or indirect detection such as an epitope tag, an enzyme, etc. A variety of methods may be employed to detect the label depending on the nature of the label and other assay components. For example, the label may be detected bound to the solid substrate or a portion of the bound complex containing the label may be separated from the solid substrate, and the label thereafter detected.

H. Methods to Identify Agents that Modulate the Expression a Nucleic Acid Encoding the S6 Kinase Protein.

Another embodiment of the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding a protein of the invention such as a protein having the amino acid sequence of SEQ ID No.2. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention. As used herein, an agent is said to modulate the expression of a nucleic acid of the invention, for instance a nucleic acid encoding the protein having the sequence of SEQ ID No.2, if it is capable of up- or down-regulating expression of the nucleic acid in a cell.

In one assay format, cell lines that contain reporter gene fusions between the open reading frame defined by p70β S6k nucleotides 77-1,564 or 116-1,564 of SEQ ID No.1 and any assayable fusion partner may be prepared. Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al., 1990 Anal. Biochem 188:245-254). Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No2.

Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a nucleic acid encoding a protein of the invention such as the protein having SEQ ID No.2. For instance, mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention. Cell lines are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. Cold Spring Harbor Laboratory Press, 1989).

Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids of the invention. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid Stringency should be chosen to maximize the difference in stability between the probe:target hybrid and potential probe:non-target hybrids.

Probes may be designed from the nucleic acids of the invention through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available in Sambrook et al. (1989) or Ausubel el al. (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Co., NY, 1995).

Hybridization conditions are modified using known methods, such as those described by Sambrook et al. (1989) and Ausubel et al. (1995) as required for each probe. Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize. Alternatively, nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a porous glass wafer. The glass wafer can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize. Such glass wafers and hybridization methods are widely available, for example, those disclosed by Beattie (WO 95/11755). By examining for the ability of a given probe to specifically hybridize to an RNA sample from an untreated cell population and from a cell population exposed to the agent, agents which up or down regulate the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2 are identified.

I. Cell-Based Methods to Identify Binding Partners and Agents that Modulate at Least One Activity of the S6 Kinase Protein and Related Antibodies.

Another embodiment of the present invention provides methods for identifying agents that modulate at least one activity of a protein of the invention such as the protein having the amino acid sequence of SEQ ID No.2. Such methods or assays may utilize any means of monitoring or detecting the desired activity.

In one format, the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an unexposed control cell population may be assayed. In this format, probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time. Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.

For example, N- and C-terminal fragments of p70β S6k can be expressed in bacteria and used to search for proteins which bind to these fragments. Fusion proteins, such as His-tag or GST fusion to the N- or C-terminal regions of p70β S6k can be prepared for use as a p70β S6k fragment substrate. These fusion proteins can be coupled to Talon or Glutathione-Sepharose beads and then probed with cell lysates. Prior to lysis, the cells may be treated with rapamycin or other drugs which may modulate p70β S6k or proteins that interact with p70β S6k . Lysate proteins binding to the fusion proteins can be resolved by SDS-PAGE, isolated and identified by protein sequencing or mass spectroscopy, as is known in the art. It is likely that signaling molecules containing one or more SH3 domains may bind directly to the C-terminal region of p70β S6k . The N-terminal domain may have a p70β S6k -specific phosphatase as a binding partner.

Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins of the invention, such as p70β S6k , variants and isolated binding partners, if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., may be desirable to provide accessibility to the hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a Cys residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.

Anti-peptide antibodies can be generated using synthetic peptides corresponding to, for example, the carboxy terminal 15 amino acids p70β S6k . Synthetic peptides can be as small as 1-3 amino acids in length, but are preferably at least 4 or more amino acid residues long. The peptides are coupled to KLH using standard methods and can be immunized into animals such as rabbits. Polyclonal anti-p70β S6k peptide antibodies can then be purified, for example using Actigel beads containing the covalently bound peptide.

While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred. Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid. Of particular interest, are monoclonal antibodies which recognize the proline-rich domain of p70β S6k .

The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as Fav, sc FV, Fab, Fab′, or F(ab′) 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin. Antibodies may preferably also be human, humanized or chimeric variants of the foregoing. Such antibodies can be less immunogenic when administered to a subject. Methods of producing humanized or chimeric antibodies are well known in the art. The antibodies contemplated also include different isotypes and isotype subclasses (e.g. IgG 1 , IgG 2 , IgM, to name a few). These antibodies can be prepared by raising them in vertebrates, in hybridoma cell lines or other cell lines, or by recombinant means. For references on how to prepare these antibodies, see E. Harlow and D. Lane, ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988); Kohler and Mistein, (1976) E. J. Immunol. 6:511; Queen et al. U.S. Pat. No. 5,585,089; and Riechmann et al., Nature 332:323 (1988).

The antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of receptor can also be produced in the context of chimeras with multiple species origin.

In an alternative format, a specific activity of a protein of the invention may be assayed, such as the ability of the protein to phosphorylate a substrate such as polypeptides of the S6 protein. For example, p70β S6k has been demonstrated to phosphorylate the S6 protein and a synthetic peptide, RRLSSLRASTSKSESSQK (SEQ ID No.8). The sequence comprising the synthetic peptide is located in the C-terminus of the S6 protein and is known to contain the five phosphorylation sites targeted by p70β S6k . Cell lines or populations are exposed under appropriate conditions to the agent to be tested. Agents which modulate the kinase activity of the protein of the invention are identified by assaying the kinase activity of the protein from the exposed cell line or population and a control unexposed cell line or population, thereby identifying agents which modulate the kinase activity of the protein. Polypeptides of the S6 protein, such as the above examples, are useful positive controls in identifying additional p70β S6k substrates.

Kinase assays to measure the ability of the agent to modulate the kinase activity of a protein of the invention are widely available such as the assays disclosed by Mishima et al. (1996) J. Biochem 119:906-913) and Michnoff et al. (1986) J. Biol. Chem. 261:8320-8326. Alternative assay formats include actin-myosin motility assays such as those disclosed by Kohama et al. (1996)TIPS 17:284-287 or Warrick et al. (1987) Ann Rev. Cell. Biol. 3:379-421.

Agents that arm assayed in the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the a protein of the invention alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.

As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a non-random basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action. As described in the Examples, there are proposed binding sites for ATP/GTP and calmodulin as well as cAMP/cGMP kinase sites, TyrP sites and Ser/Thr kinase (catalytic) sites in the protein having SEQ ID No.2. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to the ATP or calmodulin binding sites or domains.

The agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.

The peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene coded amino acids are to be included.

Another class of agents of the present invention are antibodies immunoreactive with critical positions of proteins of the invention. Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies.

J. Uses for Agents that Modulate at Least One Activity of the S6 Kinase Protean.

As provided in the Examples, the proteins and nucleic acids of the invention, such as the protein having the amino acid sequence of SEQ ID No.2, are involved in ribosomal function. Agents that modulate or down-regulate the expression of the protein or agents such as agonists or antagonists of at least one activity of the protein may be used to modulate biological and pathologic processes associated with the protein's function and activity.

As used herein, a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein of the invention. The term “mammal” is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects.

As used herein, a biological or pathological process mediated by a protein of the invention may include binding of substrates such as ATP, GTP or calmodulin or phosphorylation of a substrate, such as the S6 protein.

Pathological processes refer to a category of biological processes which produce a deleterious effect For example, expression or up-regulation of expression of a protein of the invention may be associated with certain diseases. As used herein, an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process. For instance, a disease may be prevented or disease progression modulated by the administration of agents which reduce or modulate in some way the expression or at least one activity of a protein of the invention.

The agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process. As used herein, two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.

The agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered ill be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

The present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise about 0.1 to 100 μg/kg body weight. The preferred dosages comprise about 0.1 to 10 μg/kg body weight. The most preferred dosages comprise about 0.1 to 1 μg/kg body weight. In tissue culture, optimal dosage ranges for drugs such as wortmannin and rapamycin range from about 500 pM to about 1000 nM. Less optimum ranges include about 10 pM to about 10 mg.

In addition to the pharmacologically active agent, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.

Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

In practicing the methods of this invention, the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice. The compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES

Example 1

Amino Acid Sequence of p70β S6k and comparison with p70α S6k

Materials and Methods Restriction enzymes and DNA modification enzymes were obtained from standard commercial sources and used according to manufacturer's recommendations. Oligonucleotides which were used for sequencing of p70β S6k and various PCR fragments were synthesis by Genosys or Japan Bioservice, Inc. The pcDNA1 and pcDNA3 mammalian expression vectors were from Invitrogen. The pGEX4T vector, glutathione-Sepharose4B and HiTrapQ columns were purchased from Pharmacia. cDNA of rat p70α-I was a gift from Dr. Joseph Avruch (Diabetes Unit, Massachusetts General Hospital). Rapamycin and PDGF BB were purchased form Calbiochem. Wortmannin was purchased from Sigma.

Cell cultures and Antibodies. A porcine aortic endothelial cell line (PAE-PDGF-R), stably expressing the human PDGF-β receptor, was maintained in HAM's F12 medium containing 10% fetal calf serum (FCS). CHO cells stably overexpressing human insulin receptors (CHO-IR cells) and HEK293 cells were maintained and cultured as described earlier (Hara et al., 1998 J. Biol. Chem. 273:14484-14494) in HAM's F12 medium or Dulbecco's modified Eagles minimal essential medium (DMEM) supplemented with 10% FCS, respectively. Anti-FLAG monoclonal M2 antibody was purchased from Eastman Kodak Corp. Anti-phosphopeptide antibodies against proline-directed site Ser434 of p70α-I were purchased from New England Biolabs. Polyclonal antibodies raised against the C-terminal 104 amino acids fragment of p70α S6k were from Dr. Joseph Avruch. A GST fusion protein containing amino acids 443-495 of p70β S6k (p70βC Ab) was used to raise polyclonal antibodies specific for p70β S6k . Immunoreactive sera were affinity-purified on an Affigel matrix containing the GST/p70β S6k -terminal fusion protein.

Fractionation of cell extracts. HEK293 cells were starved in DMEM medium for 16 h and then treated with 15% FCS for 10 min or 200 nM rapamycin for 30 min. After treatment, cells were lysed in ice-cold buffer A (20 mM Tris/HCl pH 7.5, 20 mM NaCl, 1 mM EDTA, 5 mM EGTA, 20 mM β-glycerophosphate, 1 mM-DTT, 1 mM PMSF, 2 mg/ml aprotinin, 10 mg/ml leupeptin) and the lysates centrifuged at 4° C. for min at 10,000×g. Supernatants were filtered through a 0.45 μM filter and then loaded onto a HighTrapQ Sepharose column (1.0 ml column volume) equilibrated in Buffer A. The column was washed extensively in buffer A and bound proteins eluted with a linear gradient of NaCl (20-500 mM). Aliquots of eluted proteins were subjected to SDS—PAGE and immunoblotted with anti-phosphopeptide antibodies directed against proline-directed site Ser434 of p70α S6k or anti-peptide antibodies against the carboxyl terminal end of p70α S6k .

Construction and screening of a HEK293 Uni-ZAP library and DNA sequencing analysis. Total RNA was isolated from HEK293 cells as described (Hara et al., 1998; Chomczynski et at., 1987 Analytical Biochem. 162:156-159), and poly(A)+mRNA was selected by using Dynabeads mRNA purification kit (Dynal). An oligo(dT)-primed library was constructed in UNI-ZAP XR vector from 5 mg of HEK293 mRNA, using the Uni-ZAP cDNA synthesis kit (Stratagene). Packaging into phages was carried out by using Gigapack III Gold Packaging extracts (Stratagene). The cDNA encoding full length p70β S6k kinase was isolated by screening 1×10 6 primary phages from HEK293 Uni-ZAP library with a 32 P-labeled 0.65 Kb EcoRI/NotI fragment derived from the EST clone GenBank Accession No. AA410355 (Hillier et al., published on GenBank, 1997). Positive clones were confirmed, isolated in second-round screening and rescued as Bluescript plasmids by in vivo excision (Stratagene). PCR amplification and restriction mapping were used for primary characterization of isolated clones. Sequencing analysis of selected clones was performed on an Applied Biosystem 373A DNA automatic sequencer (PE Applied Biosystems).

Results. The p70β S6k is activated by multiple phosphorylation within the pseudosubstrate and catalytic domains in response to extracellular stimuli, including serum, growth factors and hormones. Phosphospecific antibodies directed against phosphorylated sites of p70α S6k have been recently developed: Phospho-p70α S6k (Ser434) and Phospho-p70α S6k (Thr444/Ser447). Both antibodies were shown to recognize specifically phosphorylated forms of p70α S6k , and this recognition was found to be sensitive to rapamycin. To compare the phosphorylation state and chromatographic behavior of p70α S6k from cells stimulated with serum and treated with or without rapamycin, HEK293 cells were starved in DMEM medium for 16 h and then stimulated with 15% serum for 10 min prior treatment with or without 200 nM of rapamycin for 30 min. Cell extracts were fractionated using HighTrapQ Sepharose columns. Proteins were resolved on SDS-PAGE and immunoblotted with anti-phosphopeptide antibodies Ser434 and S444/T447 or antibodies specific for p70α S6k . In total lysates of serum stimulated HEK293 cells, this antibody recognizes specifically phosphorylated versions of p70α S6k isoforms (p70 and p85). However, when cell lysates were fractionated and separated on SDS-PAGE, several additional bands appeared on the immunoblot together with the p70 and p85 isoforms of p70α S6k , including p190, p110, p90 and p60 (not shown). It is important to note that the recognition of these proteins by phosphospecific S434 antibodies was sensitive to rapamycin, indicating the specificity for the phosphorylated epitope. As expected, the anti-peptide antibodies specific for the p70α S6k at recognized 85-kDa and 70-kDa bands, which correspond to p70α-I and p70α-II, respectively. S434 is located in the autoinhibitory region of the p70α S6k , which is highly conserved among different species of p70α S6k , including Drosophila (Stewart et al., 1996 Proc. Natl. Acad. Sci, USA 93:10791-10796; Watson et al., 1996 Proc. Natl Acad. Sci. USA 93:13694-13698). As phosphorylation of the S434 site is sensitive to rapamycin, it is possible to speculate that p190, p110, p90 and/or p60 may represent novel rapamycin-sensitive p70β S6k -related kinases.

Molecular cloning of novel p70α S6k . Peptide sequences which were used for raising anti-phosphopeptide antibodies S434 of p70α-I were taken to search the expressed sequence tag (EST) databases. This search generated hundreds of EST clones that showed high degree of homology to the query sequence. Extensive analysis of these clones allowed us to isolate several clones which were highly homologous to the 434 peptide, but did not match to cDNA clones from EMBL or Swissprot databases. Further characterization of these sequences indicated that two nearly identical clones (GenBak Accession Nos. AA284234 and AA410355) exhibited strong homology to the kinase extension domain of the protein kinase A (PKA) family of serine/threonine (S/T) kinases. Additionally, the homology in the kinase extension domain extended into the putative autoinhibitory domain, which is unique for the p70α S6k (less than 75% identity between p70α S6k and the unknown potential S6 kinase). However, the homology with p70α S6k dropped significantly downstream of the kinase extension and autoinhibitory domains suggesting that these cones encoded a novel kinase of this family. Based an these data, we decided to further characterize these clones. Both EST clones were obtained from the UK HGMP Resource Center. Restriction mapping indicated that the GenBank Accession Nos. AA284234 and AA410355 clones contain short inserts of 0.6 kB and 0.65 kB respectively. Sequence analysis showed that these clones are identical to each other in an overlapping region and may encode a partial open reading frame (ORF), which shows very strong homology to the kinase extension and autoinhibitory domains of the p70α S6k . These ESTs did not contain a full gene nor was there a pin coding sequence previously identified in these ESTs. Furthermore, four of the five proline directed Ser/Thr phosphorylation sites located in an autoinhibitory pseudo-substrate domain of p70α S6k were conserved in the clones of p70β S6k . Immediately after the autoinhibitory pseudo-substrate domain, the homology between p70β S6k and p7β S6k was very low (12% identity). Taking in account these findings, we proceeded to clone the full length cDNA clone encoding this potentially novel kinase.

Since several rapamycin-sensitive bands were found in the HEK293 cells with the use of anti-Ser434 phosphospecific antibodies, a library from this cell line was created. We screened 10 4 primary clones from the Uni-Zap/HEK293 library with a full length insert from EST clone AA410355 and isolated 12 positive clones. Sequence analysis of rescued plasmids allowed us to identify one clone, which contained an open reading frame of 495 amino acids (FIG. 2 A). The C-terminus of this clone was found to be identical to the sequence of the EST clone AA410355, which was used for screening.

By analogy to p70α S6k , the novel cDNA, encoding p70β S6k , could potentially encode two isoforms as a result of alternative start codons. If this is the case, the shorter isoform may utilize an ATG codon which is 13 amino acids (aa) downstream of the first methionine and may encode a protein of 482 amino acids. Two potential isoforms were termed p70β-I (495 aa long) and p70β-II (482 aa long). The presence of additional 13 aa at the N-terminus of p70β-I isoform may determine its subcellular localization in the nucleus due to the presence of a putative nuclear localization sequence (RGRRARG, amino acid numbers 3-9 of SEQ ID No. 2). The overall structure of p70β S6k is similar to that of p70α S6k . p70α S6k and p70β S6k share 70% identity and 85% similarity on protein level. The p70β S6k kinase consists of the amino-terminal non-catalytic region, a catalytic domain, a kinase extension and a carboxyl-terminal non-catalytic tail, whose amino acid identity corresponds to domains of p70α S6k is 40%, 83%, 80%, and 47%, respectively (FIG. 2 B). The strong argument that this clone encodes a novel p70 S6 kinase is the presence of the autoinhibitory pseudosubstrate domain, which is not present in any other known kinases.

p70α S6k undergoes a multi-site phosphorylation in response to stimulation by insulin or mitogens. Such multiple phosphorylation sites are also well conserved in p70β S6k (FIG. 2 B). It also contains 3 sets of phosphorylation sites, similar to p70α S6k : (i) a set of Ser/Thr-Pro motifs clustered in an autoinhibitory pseudosubstrate domain (Ser423, Ser430, Ser436, Ser441 in p70β S6k correspond to Ser 434, 441, and 447, Ser 452 in p70α S6k ; (ii) a second set includes Ser383 and Thr401 which is located in the kinase extension domain and corresponds to Ser394 and Thr412 in p70α S6k ; (iii) a third set consists of Thr251 which resides in the activation T-loop of kinase domain and corresponds to Thr252 in p70α S6k . The greatest difference between the p70α S6k and the p70β S6k sequences arm in the amino-terminal non-catalytic region (40% identity and 60% similarity) and the carboxyl-terminal non-catalytic tail (47% identity and 66% similarity). p70β S6k also contains proline-rich sequences at the C-terminus, that may mediate the interaction with SH3domain-containing molecules.

Example 2

Tissue Specific Expression of p70β S6k

Materials and Methods. Northern blot analysis was performed using commercial nylon membranes pre-bound with 2 μg of gel-separated poly(A)+ RNA samples obtained from various human tissues or tumor cell lines from Clontech. The following probes were used for the detection of p70α S6k and p70β S6k messages: (i) a 476-base pair (bp) HindIII fragment spanning 56 bp of 3′ end coding region and 420 bp of 3′non-coding region of the human p70α S6k (EST clone, AA425599), (ii) a 650-bp fragment spanning 518 bp upstream of the stop codon and about 130 bp of non-coding region of the human p70β S6k (EST clone, AA410355). Human β-actin cDNA probe was used as a negative control (Clontech). These probes were labeled by Multiprime DNA labeling system (Amersham) and separated from unincorporated [γ 32 P]dCTP by Nuctrap push columns (Stratagene). Northern blots were pre-hybridized with ExpressHyb solution and hybridized with labeled probes according to manufacturer's recommendations. After extensive washing with 2×SSC, 0.05% sodium dodecyl sulfate (SDS) at room temperature, and twice with 0.1×SSC, 0.1% SDS at 50° C., the localization of bound probes on membranes was identified by autoradiography or with the use of the PhosphoImager.

Result. In order to compare expression patterns of p70α S6k and p70β S6k in human tissues and cell lines, 3′ prime coding and non-coding regions, which exhibit low level of homology between both the α and β S6 kinases, were used as probes. Northern blot analysis using poly(A)+ RNA isolated from human tissues revealed a single 2.2 kb transcript for p70β S6k , while p70α S6k probe specifically hybridized to 3.4 kb and 7.4 kb transcripts (FIG. 3 A). The expression pattern of p70α S6k and p70β S6k transcripts is remarkably similar, showing ubiquitous expression in all tissues. Highest expression levels were found in spleen, skeletal muscle and peripheral blood leukocytes, whereas brain, lung and kidney showed the lowest expression of transcripts for both S6 kinases. The only significant difference on the level of mRNA expression between p70α S6k and p70β S6k was found in liver. The expression of p70β S6k mRNA in liver is 2-3 times higher than that of p70α S6k . Liver possesses a high concentration of p70α S6k and was originally used by several groups to purify p70α S6k for biochemical studies and protein sequencing analysis (Banerjee et al., 1990 Proc. Natl Acad. Sci. USA 87:8550-8554; Koza et al., 1990 Proc. Natl. Acad. Sci. USA 87:7365-7369).

We also analyzed the expression of p70β S6k mRNA in tumor cell lines using the same probe as for the analysis of tissue distribution. A single transcript of the same size as in human tissues, 2.2 kb, was found to be highly expressed in HeLa and K562 cells, but was barely detectable in HL-60, MOLT-4 and melanoma G361 cell lines (FIG. 3 B).

Example 3

Phosphorylation of the ribosomal protein S6 and its C-terminal synthetic peptide by p70α S6k and p70β S6k

Materials and Methods. Expression of GST/p70β S6k fusion protein in bacteria. A PCR-based strategy was used to make a bacterial expression plasmid for GST/p70β S6k fusion protein. A cDNA fragment encoding 443-495 amino acids of p70β S6k was amplified by PCR and cloned into the pGEX-4T expression vector (Pharmacia). This construct was transformed into E. coli XL1-Blue competent cells (Stratagene) and the expression of the GST/p70β S6k fusion protein was induced by isopropyl β-D-thiogalactopyranoside (IPTG). The GST/p70β S6k fusion protein was purified by using glutathione-Sepharose4B beads according to manufacturer's recommendation (Pharmacia). After SDS-PAGE analysis, affinity purified fusion proteins were dialyzed against 20 mM (Tris pH 7.4), 150 mM NaCl, 50% Glycerol and stored at −20° C. This preparation of the GST/p70β S6k C-terminal fragment was used for the production of polygonal antibodies specific for p70β S6k .

Construction of mammalian expression plasmids. The full length coding sequence, corresponding to the p70β-I (I-495 amino acids) was amplified by PCR using human cDNA clone N53 isolated from HEK293 library as a template and a panel of specific oligonucleotides. Amplified constructs were digested with appropriate enzymes, gel purified and cloned into the pcDNA1 vector in-frame with N-terminal FLAG epitope.

Amino-terminal EE-tagged p70α-II and p70β-II constructs were created by a PCR-based cloning strategy. This was achieved by using specific oligonucleotides containing EE-tag sequence and appropriate restriction sites. The cDNA encoding full length human p70β S6k (clone 53) and rat p70α S6k were used as templates. The resulting PCR fragments were digested with restriction enzymes, gel purified and cloned into the pcDNA3 expression vector. The construction of the pMT2 FLAG p70α-I was described previously (Hara et al., 1998). All construct generated by a PCR-based approach were verified by sequencing. A Qiagen plasmid Midi kit was used to purify plasmid DNAs for transient transfections. The introduction of the FLAG-tag and the EE-tag sequel at the N-terminus of p70α S6k and p70β S6k allows the study of recombinant proteins via the use of specific antibodies.

Results. To test if the isolated p70β S6k cDNA would encode a functional kinase capable of phosphorylating ribosomal protein S6, a cDNA fragment encoding a short version of p70β S6k (p70β-II) was subcloned into mammalian expression vector in frame with EE-tag or Flag-tag epitopes. These constructs were transfected into HEK293 cells using lipofectAMINE under conditions recommended by the manufacturer. The expression of recombinant p70β S6k was analyzed by immunoprecipatation or western blotting with EE-tag or Flag-tag antibodies. Both constructs express the protein of approximately 60 kDa. The expression level of p70β S6k was comparable to that of p70α-I and p70α-II isoforms, when expressed in HEK293 cells.

Anti-p70β S6k polyclonal antibodies were generated using synthetic peptides corresponding to the carboxy terminal 15 amino acids of p70β S6k . These peptides were coupled to KLH and then injected into rabbits using standard procedures. Immune sera was purified using Affigel beads containing covalently cross-linked carboxy terminal peptide.

To determine whether the putative p70β S6k was indeed a novel ribosomal protein S6 kinase, the recombinant p70β S6k was expressed in HEK293 cells, immunoprecipitated with anti-EE-tag antibodies and an in vitro kinase reaction performed in the presence of purified 40S ribosomal subunit. As &own in FIG. 4A , p70β S6k (p70β-II isoform) phosporylates the S6 protein in vitro nearly as efficiently as p70α S6k . We also tested the ability of p70β S6k (p70β-II isoform) to phosphorylate a synthetic peptide representing C-terminus of S6 protein, which contains all sites known to be phosphorylated by p70α S6k . FIG. 4B demonstrates that p70β S6k (p70β-II isoform) also phosphorylates this peptide but with slightly lower efficiency than observed with p70α S6k . Therefore, p70β S6k (p70β-II isoform) may not phosphorylate the same sites of the ribosomal protein S6 as does p70α S6k .

The intrinsic activity of the p70β S6k is significantly lower than p70α S6k . The absence of one phosphorylation site in the autoinhibitory domain of p70β S6k may be responsible for this decrease in intrinsic activity.

Example 4

Stimulation of p70β S6k Activity by insulin, serum and TPA

Materials and Methods. HMK293 cells or CHO-IR cell were transfected with plasmids containing p70α S6k and p70β S6k sequences using lipofectAMINE under conditions recommended by the manufacturer (Gibco-BRL). Two days later, transfected cells were frozen in liquid nitrogen and stored until lysis. After cell extraction, the lysates were subjected to immunoprecipitation and/or immunoblot analysis. If cells were to be stimulated, they were starved in medium without FCS for 16 h and then stimulated with 10 −7 M insulin for 10 min, 15% FCS for 10 min, 500 nM TPA for 30 min or vehicle alone. PAE-PDGF-R cells were transfected with appropriate plasmids using lipofectAMINE under conditions recommended by the manufacturer. After 24 h transfected cells were serum starved for 16 h and stimulated with 20 ng/ml PDGF BB (Calbiochem) for 20 min. Control cells were treated with the vehicle under the same conditions.

Results. The effect of various extracellular stimuli on the p70β S6k activity was studied in different cell lines, transiently transfected with Flag-tagged or EE-tagged versions of p70β S6k . The activation of p70β S6k (p70β-II isoform) by insulin was N analyzed in CHO-IR cells, which stably over expresses the insulin receptor. As shown mill in FIG. 5A , treatment of CHO-IR cells with insulin induces p70β S6k (p70β-II isoform) activity towards ribosomal S6 protein by 2.8 fold. In the same cell line and under the same conditions, the activity of p70α S6k was activated 3.5 fold with insulin treatment (FIG. 5 A). Almost equal amounts of p70α S6k and p70β S6k (p70β-II isoform) were expressed in cells and used in an in vitro kinase assay after immunoprecipitation. In addition, both serum and TPA also stimulated p70β S6k (p70β-II isoform) phosphorylation of the ribosomal protein S6 (FIG. 5 A).

Using PAE-PDGF-R cells, the activation of the p70α S6k and p70β S6k (p70β-II isoform) by PDGF was examined. This cell line stably over expresses the PDGF receptor, and the activation of p70α S6k in response to PDGF was shown to be very efficient (FIG. 5 B). We found that PDGF stimulation of these cells leads to a rapid activation of recombinant p70β S6k (p70β-II isoform).

These data indicate that p70 β S6k is activated by a number of extracellular stimuli in a very similar way as p70α S6k . However, activation in the PAE-PDGF-R cells was 30 fold for p70β S6k and only 3.4 fold for p70α S6k .

Example 5

Effects of rapamycin and wortmannin on p70β S6k and p70α S6k

Materials and Methods. Treatment of transfected cells with rapamycin or wortmannin was performed as follows: 48 h after transfection, the cells were treated with various concentrations of rapamycin or wortmannin for 30 min.

Results. These data in Examples 4 and 5 indicate that p70β S6k is activated by a number of extracellular stimuli in a similar fashion to p70α S6k . The two fungal inhibitors, wortmannin and rapamycin, specifically inhibit activation of p70α S6k via PI3-kinase- and mTOR-dependent pathways respectively. Thus, the effects of both inhibitors on p70β S6k activity were examined. After p70α-I and p70β-II were transiently expressed in HBK293 cells, cells were maintained in DMEM containing 10% FCS and then treated with various concentrations of rapamycin or wortmannin. We found that the activity of p70α S6k and p70β S6k were inhibited by rapamycin and wortmannin in a dose dependent manner ( FIGS. 6A and B , upper panel). However, it appears that p70β S6k is less sensitive to rapamycin and wortmannin, when compared with p70α S6k . This difference is more obvious at lower concentration of inhibitors. In the presence of 20 nM rapamycin the inhibition of the p70α S6k is 92%, while only 46% for p70β S6k . Addition of 100 nM of wortmannin inhibits 86% of p70α S6k activity and 62% of p70β S6k activity. The inhibition of p70β S6k by rapamycin and wortmannin is lower than that observed for p70α S6k indicating different mechanisms of regulation exist for p70β S6k .

Example 6

Interaction of the p70β S6k with different GST/SH3 fusion proteins

Materials and Methods. The EEtag/p70β S6k was transiently over expressed in HEK293 cells as described above. Transfected cells were lysed in buffer A (50 mM-Tris/HCl pH=8.0, 1% NP-40, 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 20 mM sodium β-glycerophosphate, 1 mM DTT, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin and 2 μg/ml aprotinin) two days after transfection. After centrifugation at 12,000 rpm for 20 min, the supernatants were incubated for 2 hrs with Protein A beads pre-coupled with anti-EE antibodies. Beads were washed with lysis buffer before different GST/SH3 fusion proteins (1.5 mg each) were added to separate immunoprecipitation reactions. Two hours later, beads were washed extensively in lysis buffer, bound proteins separated on the SDS-PAGE and transferred to the PVDF membrane. Specific interaction of the GST/SH3 domains with pre-bound EE-tag/p70β S6k was assessed by immunoblotting with the anti-GST antibodies.

Results. The C-terminus of the p70β S6k contains proline-rich sequences which are not present in p70α S6k . Src-homology region 3 domains (SH3 domain) are present in many signaling and cytoskeletal molecules and interact specifically with proline-rich sequences which form left-handed helixes. Sequence analysis of the proline-rich region in p70β S6k indicates the presence of several putative SH3 domain binding motifs. Therefore, the ability of p70β S6k (p70β-II isoform) to interact with a panel of SH3 domains was examined. In this experiment, the EE-tag/p70β S6k was transiently over expressed in HEK293 cells and immunoprecipitated with anti-EE antibodies coupled to Protein G Sepharose. The resulting immunoprecipitates were incubated with different GST/SH3 domain fission proteins. After extensive washing, specific interaction between p70β S6k and SH3 domains was analyzed by SDS-PAGE and immunoblotting with anti-GST antibodies. As shown in FIG. 7 , several SH3 domains, including those of GAP, Src, Fgr exhibited specific interaction towards p70β S6k .

Example 7

Immunoprecipitation and Western blot analysis of transiently expressed p70β-I and p70β-II

Materials and Methods. Anti-p70β S6k polyclonal antibodies were generated using a synthetic peptide corresponding to the C-terminal tail of p70β S6k . The peptide was coupled to KLH, and rabbits were immunized using standard procedures Immune sera harvested obtained from the immunized rabbits was purified using affinity chromatography on Affigel beads containing covalently cross-linked C-terminal peptides of p70β S6k .

HEK293 cells were transfected with pcDNA1 along, pcDNA1/Flag-p70α-I, pcDNA1/Flag-p70β-I, or pcDNA1/Flag-p70β-II. Two days after transfection with one of these plasmids, cell lysates were prepared. Proteins were immunoprecipitated using the p70β S6k C-terminal affinity purified polyclonal antibodies or anti-Flag monoclonal antibodies. Immunoprecipitates were resolved on SDS PAGE and proteins transferred to PVDF membranes. The PVDF membranes were immunoblotted using anti-Flag monoclonal antibodies or p70β S6k C-terminal antibodies as indicated in FIG. 8 .

Results. The expression of both p70β S6k isoforms was analyzed in HEK293 cells using anti-p70β S6k C-terminus specific polyclonal antibodies. Both p70β S6k isoforms were found to be specifically immunoprecipitated with anti-p70β S6k antibodies, but not p70α-I, as confirmed by anti-Flag and anti-p70β S6k immunoblotting. It was found that the p70β-I isoform which encodes a 495 amino acid protein, is translated into a protein which mires in a SDS-PAGE gel at approximately 70 kD. The p70β-II isoform, which is a truncated form of p70β-I lacking 13 amino acids at the amino terminus of p70β-II, migrates in a SDS-PAGE gel at approximately 60 kD.

Example 8

Generation of activated variants of p70β S6k (T401D) and p70α S6k (T412D)

Materials and Method. Activated variants of p70β S6k and p70α S6k were made by site-directed mutagenesis. Oligonucleotide primers, specific to the site to be mutated and complimentary to opposite strands of p70β S6k and p70α S6k sequences were generated as recommended by manufacturer (Stratagene). Site-directed mutagenesis was carried out using pcDNA3/Glu-tag-p70β S6k and pcDNA3/Glu-tag-p70α S6k expression vector/plasmids as templates, according to the recommended protocol (Stratagene). All mutations were verified by DNA sequencing. Expression of mutated forms of both kinases was analyzed by immunoblotting with anti-Glu-tag antibodies. The activity of normal and mutated forms of both kinases was measured by in vitro S6 kinase assay. 40S ribosomal subunit was used as a substrate in this reaction.

Results. Studies on p70α S6k demonstrate that this kinase is activated by multiple phosphorylations in response to growth factors or hormones (FIG. 9 ). A number of kinases that can phosphorylate p70α s6k in vitro and in vivo have been identified, including cdc2, MAPK, SAPK, p38, TOR and PDK1. However, very little is known about the process of dephosphorylation, which is essential for the inactivation of the kinase.

We have generated an activated form of p70β S6k by substituting putative phosphorylation site Thr 401 with Aspartic acid (Asp) (“p70β S6k (T401D)”) as shown in FIG. 10 . Transfection studies in H293 cells and S6 kinase assays indicated that p70β S6k (T401D) mutant is in an activated state in unstimulated cells, when compared with wild type kinase (3 times higher activity, as shown in FIG. 11 ). We have also created an activated version of p70α S6k (T412D), which showed a greater state of activation (18 fold activation, FIG. 12 ).

It will be apparent to the skilled artisan that activated variants of both kinases such as disclosed above can be used in the search for binding partners. Binding partners or molecules, such as phosphatases, are considered to form tighter and more stable complexes with such irreversibly activated kinases.

Example 9

Identification of p70β S6k -binding partners

Materials and Methods. HEK293 cells are transfected using lipofectAMINE (as recommended by manufacturer, Gibco-BRL) with pcDNA3 expression vectors encoding activated variants of p70β S6k (T401D) and p70α S6k (T412D). Binding partners that preferentially associate with p70β S6k may be identified by comparing the profile of proteins precipitated from activated p70β S6k expressing cells to the profile of activated p70α S6k as expressing cells and/or negative control cells. Cells transfected with the pcDNA3 plasmid alone, may be used as a negative control in this experiment. Two days after transfection, cells are lysed in extraction buffer. 50 mM Tris/HCl (pH8.0); 120 mM NaCl; 20 mM NaF; 20 mM b-glyerophosphate; 1 mM EDTA, (pH 8.0); 6 mM EGTA; 1% NP-40; 1 mM DTT. The following protease and phosphatase inhibitors are added to the extraction buffer just before cell lysis: 5 mM Benzamidine; 1 mM PMSF; 1 mg/ml of aprotinin; 0.125 mM NaVO4; Pepstatin; and Leupeptin.

The resulting cell lysate is centrifuged at 14,000 rpm for 20 min at 40° C. to remove the insoluble fraction. If the lysate is not used immediately it is stored at −80° C. until needed. The protein concentration of the samples is measured using a Coomassie Protein Assay reagent (Pierce) at 595 nm. An equal amount of supernatant from each sample is added to fresh 1.5 ml tubes and the volumes is equalized using lysis buffer. Affinity purified anti-Glu antibody is added to the supernatant and incubated on the wheel for 1 hr at 4° C. Protein-G sepharose beads, pre-washed in lysis buffer, are used to bring down immune complexes.

After extensive washing in lysis buffer (4×), 2× sample buffer is added to the beads. Bound proteins are eluted from the beads by boiling and separated by SDS-PAGE electrophoresis. Separated proteins are silver stained and the pattern of associated proteins is analyzed.

Results. The pattern of associated proteins are compared between activated variants of p70β S6k and p70α S6k kinases. Mutated variants of both kinases are transiently expressed in HEK293 cells as Glu-tag fusion proteins. The presence of a Glu-tag epitope at the N-terminus of p70β S6k and p70α S6k allows specific immunoprecipitation of activated kinases from transfected cells. The skilled practioner will recognize that the Glu-tag fusion is not necessary to the invention and that similar results could be obtained with antibodies specific to each or both of the activated variants in the absence of a fusion epitope.

The skilled practioner will recognize that binding partners or polypeptides that preferentially bind to activated P70β S6k can be isolated by one or more standard techniques such as immunoprecipitation, hplc, fplc, column chromatography or preparative electrophoresis.

Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety.

8 1 1816 DNA Homo sapiens CDS (77)..(1561) p70(beta) S6 Kinase gene 1gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga 60gagagactcg tgccga atg gca cga ggc cga cgg gcc cgc ggg gcc ggc gcc 112 Met Ala Arg Gly Arg Arg Ala Arg Gly Ala Gly Ala 1 5 10gcc atg gcg gcc gtg ttt gat ttg gat ttg gag acg gag gaa ggc agc 160Ala Met Ala Ala Val Phe Asp Leu Asp Leu Glu Thr Glu Glu Gly Ser 15 20 25gag ggc gag ggc gag cca gag ctc agc ccc gcg gac gca tgt ccc ctt 208Glu Gly Glu Gly Glu Pro Glu Leu Ser Pro Ala Asp Ala Cys Pro Leu 30 35 40gcc gag ttg agg gca gct ggc cta gag cct gtg gga cac tat gaa gag 256Ala Glu Leu Arg Ala Ala Gly Leu Glu Pro Val Gly His Tyr Glu Glu 45 50 55 60gtg gag ctg act gag acc agc gtg aac gtt ggc cca gag cgc atc ggg 304Val Glu Leu Thr Glu Thr Ser Val Asn Val Gly Pro Glu Arg Ile Gly 65 70 75ccc cac tgc ttt gag ctg ctg cgt gtg ctg ggc aag ggg ggc tat ggc 352Pro His Cys Phe Glu Leu Leu Arg Val Leu Gly Lys Gly Gly Tyr Gly 80 85 90aag gtg ttc cag gtg cga aag gtg caa ggc acc aac ttg ggc aaa ata 400Lys Val Phe Gln Val Arg Lys Val Gln Gly Thr Asn Leu Gly Lys Ile 95 100 105tat gcc atg aaa gtc cta agg aag gcc aaa att gtg cgc aat gcc aag 448Tyr Ala Met Lys Val Leu Arg Lys Ala Lys Ile Val Arg Asn Ala Lys 110 115 120gac aca gca cac aca cgg gct gag cgg aac att cta gag tca gtg aag 496Asp Thr Ala His Thr Arg Ala Glu Arg Asn Ile Leu Glu Ser Val Lys125 130 135 140cac ccc ttt att gtg gaa ctg gcc tat gcc ttc cag act ggt ggc aaa 544His Pro Phe Ile Val Glu Leu Ala Tyr Ala Phe Gln Thr Gly Gly Lys 145 150 155ctc tac ctc atc ctt gag tgc ctc agt ggt ggc gag ctc ttc acg cat 592Leu Tyr Leu Ile Leu Glu Cys Leu Ser Gly Gly Glu Leu Phe Thr His 160 165 170ctg gag cga gag ggc atc ttc ctg gaa gat acg gcc tgc ttc tac ctg 640Leu Glu Arg Glu Gly Ile Phe Leu Glu Asp Thr Ala Cys Phe Tyr Leu 175 180 185gct gag atc acg ctg gcc ctg ggc cat ctc cac tcc cag ggc atc atc 688Ala Glu Ile Thr Leu Ala Leu Gly His Leu His Ser Gln Gly Ile Ile 190 195 200tac cgg gac ctc aag ccc gag aac atc atg ctc agc agc cag ggc cac 736Tyr Arg Asp Leu Lys Pro Glu Asn Ile Met Leu Ser Ser Gln Gly His205 210 215 220atc aaa ctg acc gac ttt gga ctc tgc aag gag tct atc cat gag ggc 784Ile Lys Leu Thr Asp Phe Gly Leu Cys Lys Glu Ser Ile His Glu Gly 225 230 235gcc gtc act cac acc ttc tgc ggc acc att gag tac atg gcc cct gag 832Ala Val Thr His Thr Phe Cys Gly Thr Ile Glu Tyr Met Ala Pro Glu 240 245 250att ctg gtg cgc agt ggc cac aac cgg gct gtg gac tgg tgg agc ctg 880Ile Leu Val Arg Ser Gly His Asn Arg Ala Val Asp Trp Trp Ser Leu 255 260 265ggg gcc ctg atg tac gac atg ctc act gga tcg ccg ccc ttt acc gca 928Gly Ala Leu Met Tyr Asp Met Leu Thr Gly Ser Pro Pro Phe Thr Ala 270 275 280gag aac cgg aag aaa acc atg gat aag atc atc agg ggc aag ctg gca 976Glu Asn Arg Lys Lys Thr Met Asp Lys Ile Ile Arg Gly Lys Leu Ala285 290 295 300ctg ccc ccc tac ctc acc cca gat gcc cgg gac ctt gtc aaa aag ttt 1024Leu Pro Pro Tyr Leu Thr Pro Asp Ala Arg Asp Leu Val Lys Lys Phe 305 310 315ctg aaa cgg aat ccc agc cag cgg att ggg ggt ggc cca ggg gat gct 1072Leu Lys Arg Asn Pro Ser Gln Arg Ile Gly Gly Gly Pro Gly Asp Ala 320 325 330gct gat gtg cag aga cat ccc ttt ttc cgg cac atg aat tgg gac gac 1120Ala Asp Val Gln Arg His Pro Phe Phe Arg His Met Asn Trp Asp Asp 335 340 345ctt ctg gcc tgg cgt gtg gac ccc cct ttc agg ccc tgt ctg cag tca 1168Leu Leu Ala Trp Arg Val Asp Pro Pro Phe Arg Pro Cys Leu Gln Ser 350 355 360gag gag gac gtg agc cag ttt gat acc cgc ttc aca cgg cag acg ccg 1216Glu Glu Asp Val Ser Gln Phe Asp Thr Arg Phe Thr Arg Gln Thr Pro365 370 375 380gtg gac agt cct gat gac aca gcc ctc agc gag agt gcc aac cag gcc 1264Val Asp Ser Pro Asp Asp Thr Ala Leu Ser Glu Ser Ala Asn Gln Ala 385 390 395ttc ctg ggc ttc aca tac gtg gcg ccg tct gtc ctg gac agc atc aag 1312Phe Leu Gly Phe Thr Tyr Val Ala Pro Ser Val Leu Asp Ser Ile Lys 400 405 410gag ggc ttc tcc ttc cag ccc aag ctg cgc tca ccc agg cgc ctc aac 1360Glu Gly Phe Ser Phe Gln Pro Lys Leu Arg Ser Pro Arg Arg Leu Asn 415 420 425agt agc ccc cgg gtc ccc gtc agc ccc ctc aag ttc tcc cct ttt gag 1408Ser Ser Pro Arg Val Pro Val Ser Pro Leu Lys Phe Ser Pro Phe Glu 430 435 440ggg ttt cgg ccc agc ccc agc ctg ccg gag ccc acg gag cta cct cta 1456Gly Phe Arg Pro Ser Pro Ser Leu Pro Glu Pro Thr Glu Leu Pro Leu445 450 455 460cct cca ctc ctg cca ccg ccg ccg ccc tcg acc acc gcc cct ctc ccc 1504Pro Pro Leu Leu Pro Pro Pro Pro Pro Ser Thr Thr Ala Pro Leu Pro 465 470 475atc cgt ccc ccc tca ggg acc aag aag tcc aag agg ggc cgt ggg cgt 1552Ile Arg Pro Pro Ser Gly Thr Lys Lys Ser Lys Arg Gly Arg Gly Arg 480 485 490cca ggg cgc taggaagccg ggtgggggtg agggtagccc ttgagccctg 1601Pro Gly Arg 495tccctgcggc tgtgagagca gcaggaccct gggccagttc cagagacctg ggggtgtgtc 1661tgggggtggg gtgtgagtgc gtatgaaagt gtgtgtctgc tggggcagct gtgcccctga 1721atcatgggca cggagggccg cccgccacac cccgcgctca actgctcccg tggaagatta 1781aagggctgaa tcatgaaaaa aaaaaaaaaa aaaaa 1816 2 495 PRT Homo sapiens p70(beta) S6 Kinase protein 2Met Ala Arg Gly Arg Arg Ala Arg Gly Ala Gly Ala Ala Met Ala Ala 1 5 10 15Val Phe Asp Leu Asp Leu Glu Thr Glu Glu Gly Ser Glu Gly Glu Gly 20 25 30Glu Pro Glu Leu Ser Pro Ala Asp Ala Cys Pro Leu Ala Glu Leu Arg 35 40 45Ala Ala Gly Leu Glu Pro Val Gly His Tyr Glu Glu Val Glu Leu Thr 50 55 60Glu Thr Ser Val Asn Val Gly Pro Glu Arg Ile Gly Pro His Cys Phe 65 70 75 80Glu Leu Leu Arg Val Leu Gly Lys Gly Gly Tyr Gly Lys Val Phe Gln 85 90 95Val Arg Lys Val Gln Gly Thr Asn Leu Gly Lys Ile Tyr Ala Met Lys 100 105 110Val Leu Arg Lys Ala Lys Ile Val Arg Asn Ala Lys Asp Thr Ala His 115 120 125Thr Arg Ala Glu Arg Asn Ile Leu Glu Ser Val Lys His Pro Phe Ile 130 135 140Val Glu Leu Ala Tyr Ala Phe Gln Thr Gly Gly Lys Leu Tyr Leu Ile145 150 155 160Leu Glu Cys Leu Ser Gly Gly Glu Leu Phe Thr His Leu Glu Arg Glu 165 170 175Gly Ile Phe Leu Glu Asp Thr Ala Cys Phe Tyr Leu Ala Glu Ile Thr 180 185 190Leu Ala Leu Gly His Leu His Ser Gln Gly Ile Ile Tyr Arg Asp Leu 195 200 205Lys Pro Glu Asn Ile Met Leu Ser Ser Gln Gly His Ile Lys Leu Thr 210 215 220Asp Phe Gly Leu Cys Lys Glu Ser Ile His Glu Gly Ala Val Thr His225 230 235 240Thr Phe Cys Gly Thr Ile Glu Tyr Met Ala Pro Glu Ile Leu Val Arg 245 250 255Ser Gly His Asn Arg Ala Val Asp Trp Trp Ser Leu Gly Ala Leu Met 260 265 270Tyr Asp Met Leu Thr Gly Ser Pro Pro Phe Thr Ala Glu Asn Arg Lys 275 280 285Lys Thr Met Asp Lys Ile Ile Arg Gly Lys Leu Ala Leu Pro Pro Tyr 290 295 300Leu Thr Pro Asp Ala Arg Asp Leu Val Lys Lys Phe Leu Lys Arg Asn305 310 315 320Pro Ser Gln Arg Ile Gly Gly Gly Pro Gly Asp Ala Ala Asp Val Gln 325 330 335Arg His Pro Phe Phe Arg His Met Asn Trp Asp Asp Leu Leu Ala Trp 340 345 350Arg Val Asp Pro Pro Phe Arg Pro Cys Leu Gln Ser Glu Glu Asp Val 355 360 365Ser Gln Phe Asp Thr Arg Phe Thr Arg Gln Thr Pro Val Asp Ser Pro 370 375 380Asp Asp Thr Ala Leu Ser Glu Ser Ala Asn Gln Ala Phe Leu Gly Phe385 390 395 400Thr Tyr Val Ala Pro Ser Val Leu Asp Ser Ile Lys Glu Gly Phe Ser 405 410 415Phe Gln Pro Lys Leu Arg Ser Pro Arg Arg Leu Asn Ser Ser Pro Arg 420 425 430Val Pro Val Ser Pro Leu Lys Phe Ser Pro Phe Glu Gly Phe Arg Pro 435 440 445Ser Pro Ser Leu Pro Glu Pro Thr Glu Leu Pro Leu Pro Pro Leu Leu 450 455 460Pro Pro Pro Pro Pro Ser Thr Thr Ala Pro Leu Pro Ile Arg Pro Pro465 470 475 480Ser Gly Thr Lys Lys Ser Lys Arg Gly Arg Gly Arg Pro Gly Arg 485 490 495 3 2346 DNA Homo sapiens CDS (28)..(1602) p70(alpha) S6 Kinase gene 3gcacgaggct gcggcgggtc cgggccc atg agg cga cga agg agg cgg gac ggc 54 Met Arg Arg Arg Arg Arg Arg Asp Gly 1 5ttt tac cca gcc ccg gac ttc cga gac agg gaa gct gag gac atg gca 102Phe Tyr Pro Ala Pro Asp Phe Arg Asp Arg Glu Ala Glu Asp Met Ala 10 15 20 25gga gtg ttt gac ata gac ctg gac cag cca gag gac gcg ggc tct gag 150Gly Val Phe Asp Ile Asp Leu Asp Gln Pro Glu Asp Ala Gly Ser Glu 30 35 40gat gag ctg gag gag ggg ggt cag tta aat gaa agc atg gac cat ggg 198Asp Glu Leu Glu Glu Gly Gly Gln Leu Asn Glu Ser Met Asp His Gly 45 50 55gga gtt gga cca tat gaa ctt ggc atg gaa cat tgt gag aaa ttt gaa 246Gly Val Gly Pro Tyr Glu Leu Gly Met Glu His Cys Glu Lys Phe Glu 60 65 70atc tca gaa act agt gtg aac aga ggg cca gaa aaa atc aga cca gaa 294Ile Ser Glu Thr Ser Val Asn Arg Gly Pro Glu Lys Ile Arg Pro Glu 75 80 85tgt ttt gag cta ctt cgg gta ctt ggt aaa ggg ggc tat gga aag gtt 342Cys Phe Glu Leu Leu Arg Val Leu Gly Lys Gly Gly Tyr Gly Lys Val 90 95 100 105ttt caa gta cga aaa gta aca gga gca aat act ggg aaa ata ttt gcc 390Phe Gln Val Arg Lys Val Thr Gly Ala Asn Thr Gly Lys Ile Phe Ala 110 115 120atg aag gtg ctt aaa aag gca atg ata gta aga aat gct aaa gat aca 438Met Lys Val Leu Lys Lys Ala Met Ile Val Arg Asn Ala Lys Asp Thr 125 130 135gct cat aca aaa gca gaa cgg aat att ctg gag gaa gta aag cat ccc 486Ala His Thr Lys Ala Glu Arg Asn Ile Leu Glu Glu Val Lys His Pro 140 145 150ttc atc gtg gat tta att tat gcc ttt cag act ggt gga aaa ctc tac 534Phe Ile Val Asp Leu Ile Tyr Ala Phe Gln Thr Gly Gly Lys Leu Tyr 155 160 165ctc atc ctt gag tat ctc agt gga gga gaa cta ttt atg cag tta gaa 582Leu Ile Leu Glu Tyr Leu Ser Gly Gly Glu Leu Phe Met Gln Leu Glu170 175 180 185aga gag gga ata ttt atg gaa gac act gcc tgc ttt tac ttg gca gaa 630Arg Glu Gly Ile Phe Met Glu Asp Thr Ala Cys Phe Tyr Leu Ala Glu 190 195 200atc tcc atg gct ttg ggg cat tta cat caa aag ggg atc atc tac aga 678Ile Ser Met Ala Leu Gly His Leu His Gln Lys Gly Ile Ile Tyr Arg 205 210 215gac ctg aag ccg gag aat atc atg ctt aat cac caa ggt cat gtg aaa 726Asp Leu Lys Pro Glu Asn Ile Met Leu Asn His Gln Gly His Val Lys 220 225 230cta aca gac ttt gga cta tgc aaa gaa tct att cat gat gga aca gtc 774Leu Thr Asp Phe Gly Leu Cys Lys Glu Ser Ile His Asp Gly Thr Val 235 240 245aca cac aca ttt tgt gga aca ata gaa tac atg gcc cct gaa atc ttg 822Thr His Thr Phe Cys Gly Thr Ile Glu Tyr Met Ala Pro Glu Ile Leu250 255 260 265atg aga agt ggc cac aat cgt gct gtg gat tgg tgg agt ttg gga gca 870Met Arg Ser Gly His Asn Arg Ala Val Asp Trp Trp Ser Leu Gly Ala 270 275 280tta atg tat gac atg ctg act gga gca ccc cca ttc act ggg gag aat 918Leu Met Tyr Asp Met Leu Thr Gly Ala Pro Pro Phe Thr Gly Glu Asn 285 290 295aga aag aaa aca att gac aaa atc ctc aaa tgt aaa ctc aat ttg cct 966Arg Lys Lys Thr Ile Asp Lys Ile Leu Lys Cys Lys Leu Asn Leu Pro 300 305 310ccc tac ctc aca caa gaa gcc aga gat ctg ctt aaa aag ctg ctg aaa 1014Pro Tyr Leu Thr Gln Glu Ala Arg Asp Leu Leu Lys Lys Leu Leu Lys 315 320 325aga aat gct gct tct cgt ctg gga gct ggt cct ggg gac gct gga gaa 1062Arg Asn Ala Ala Ser Arg Leu Gly Ala Gly Pro Gly Asp Ala Gly Glu330 335 340 345gtt caa gct cat cca ttc ttt aga cac att aac tgg gaa gaa ctt ctg 1110Val Gln Ala His Pro Phe Phe Arg His Ile Asn Trp Glu Glu Leu Leu 350 355 360gct cga aag gtg gag ccc ccc ttt aaa cct ctg ttg caa tct gaa gag 1158Ala Arg Lys Val Glu Pro Pro Phe Lys Pro Leu Leu Gln Ser Glu Glu 365 370 375gat gta agt cag ttt gat tcc aag ttt aca cgt cag aca cct gtc gac 1206Asp Val Ser Gln Phe Asp Ser Lys Phe Thr Arg Gln Thr Pro Val Asp 380 385 390agc cca gat gac tca act ctc agt gaa agt gcc aat cag gtc ttt ctg 1254Ser Pro Asp Asp Ser Thr Leu Ser Glu Ser Ala Asn Gln Val Phe Leu 395 400 405ggt ttt aca tat gtg gct cca tct gta ctt gaa agt gtg aaa gaa aag 1302Gly Phe Thr Tyr Val Ala Pro Ser Val Leu Glu Ser Val Lys Glu Lys410 415 420 425ttt tcc ttt gaa cca aaa atc cga tca cct cga aga ttt att ggc agc 1350Phe Ser Phe Glu Pro Lys Ile Arg Ser Pro Arg Arg Phe Ile Gly Ser 430 435 440cca cga aca cct gtc agc cca gtc aaa ttt tct cct ggg gat ttc tgg 1398Pro Arg Thr Pro Val Ser Pro Val Lys Phe Ser Pro Gly Asp Phe Trp 445 450 455gga aga ggt gct tcg gcc agc aca gca aat cct cag aca cct gtg gaa 1446Gly Arg Gly Ala Ser Ala Ser Thr Ala Asn Pro Gln Thr Pro Val Glu 460 465 470tac cca atg gaa aca agt ggc ata gag cag atg gat gtg aca atg agt 1494Tyr Pro Met Glu Thr Ser Gly Ile Glu Gln Met Asp Val Thr Met Ser 475 480 485ggg gaa gca tcg gca cca ctt cca ata cga cag ccg aac tct ggg cca 1542Gly Glu Ala Ser Ala Pro Leu Pro Ile Arg Gln Pro Asn Ser Gly Pro490 495 500 505tac aaa aaa caa gct ttt ccc atg atc tcc aaa cgg cca gag cac ctg 1590Tyr Lys Lys Gln Ala Phe Pro Met Ile Ser Lys Arg Pro Glu His Leu 510 515 520cgt atg aat cta tgacagagca atgcttttaa tgaatttaag gcaaaaaggt 1642Arg Met Asn Leu 525ggagagggag atgtgtgagc atcctgcaag gtgaaacaag actcaaaatg acagtttcag 1702agagtcaatg tcattacata gaacacttcg gacacaggaa aaataaacgt ggattttaaa 1762aaatcaatca atggtgcaaa aaaaaactta aagcaaaata gtattgctga actcttaggc 1822acatcaatta attgattcct cgcgacatct ttctcaacct tatcaaggat tttcatgttg 1882atgactcgaa actgacagta ttaagggtag gatgttgctc tgaatcactg tgagtctgat 1942gtgtgaagaa gggtatcctt tcattaggca agtacaaatt gcctataata cttgcaacta 2002aggacaaatt agcatgcaag cttggtcaaa cttttcccag gcaaaatggg aaggcaaaga 2062caaaagaaac ttaccaattg atgttttacg tgcaaacaac ctgaatcttt tttttatata 2122aatatatatt tttcaaatag atttttgatt cagctcatta tgaaaaacat cccaaacttt 2182aaaatgcgaa attattggtt ggtgtgaaga aagccagaca acttctgttt cttctcttgg 2242tgaaataata aaatgcaaat gaatcattgt taacacagct gtggctcgtt tgagggattg 2302gggtggacct ggggtttatt ttcagtaacc cagctgcgga gcct 2346 4 525 PRT Homo sapiens p70(alpha) S6 Kinase protein 4Met Arg Arg Arg Arg Arg Arg Asp Gly Phe Tyr Pro Ala Pro Asp Phe 1 5 10 15Arg Asp Arg Glu Ala Glu Asp Met Ala Gly Val Phe Asp Ile Asp Leu 20 25 30Asp Gln Pro Glu Asp Ala Gly Ser Glu Asp Glu Leu Glu Glu Gly Gly 35 40 45Gln Leu Asn Glu Ser Met Asp His Gly Gly Val Gly Pro Tyr Glu Leu 50 55 60Gly Met Glu His Cys Glu Lys Phe Glu Ile Ser Glu Thr Ser Val Asn 65 70 75 80Arg Gly Pro Glu Lys Ile Arg Pro Glu Cys Phe Glu Leu Leu Arg Val 85 90 95Leu Gly Lys Gly Gly Tyr Gly Lys Val Phe Gln Val Arg Lys Val Thr 100 105 110Gly Ala Asn Thr Gly Lys Ile Phe Ala Met Lys Val Leu Lys Lys Ala 115 120 125Met Ile Val Arg Asn Ala Lys Asp Thr Ala His Thr Lys Ala Glu Arg 130 135 140Asn Ile Leu Glu Glu Val Lys His Pro Phe Ile Val Asp Leu Ile Tyr145 150 155 160Ala Phe Gln Thr Gly Gly Lys Leu Tyr Leu Ile Leu Glu Tyr Leu Ser 165 170 175Gly Gly Glu Leu Phe Met Gln Leu Glu Arg Glu Gly Ile Phe Met Glu 180 185 190Asp Thr Ala Cys Phe Tyr Leu Ala Glu Ile Ser Met Ala Leu Gly His 195 200 205Leu His Gln Lys Gly Ile Ile Tyr Arg Asp Leu Lys Pro Glu Asn Ile 210 215 220Met Leu Asn His Gln Gly His Val Lys Leu Thr Asp Phe Gly Leu Cys225 230 235 240Lys Glu Ser Ile His Asp Gly Thr Val Thr His Thr Phe Cys Gly Thr 245 250 255Ile Glu Tyr Met Ala Pro Glu Ile Leu Met Arg Ser Gly His Asn Arg 260 265 270Ala Val Asp Trp Trp Ser Leu Gly Ala Leu Met Tyr Asp Met Leu Thr 275 280 285Gly Ala Pro Pro Phe Thr Gly Glu Asn Arg Lys Lys Thr Ile Asp Lys 290 295 300Ile Leu Lys Cys Lys Leu Asn Leu Pro Pro Tyr Leu Thr Gln Glu Ala305 310 315 320Arg Asp Leu Leu Lys Lys Leu Leu Lys Arg Asn Ala Ala Ser Arg Leu 325 330 335Gly Ala Gly Pro Gly Asp Ala Gly Glu Val Gln Ala His Pro Phe Phe 340 345 350Arg His Ile Asn Trp Glu Glu Leu Leu Ala Arg Lys Val Glu Pro Pro 355 360 365Phe Lys Pro Leu Leu Gln Ser Glu Glu Asp Val Ser Gln Phe Asp Ser 370 375 380Lys Phe Thr Arg Gln Thr Pro Val Asp Ser Pro Asp Asp Ser Thr Leu385 390 395 400Ser Glu Ser Ala Asn Gln Val Phe Leu Gly Phe Thr Tyr Val Ala Pro 405 410 415Ser Val Leu Glu Ser Val Lys Glu Lys Phe Ser Phe Glu Pro Lys Ile 420 425 430Arg Ser Pro Arg Arg Phe Ile Gly Ser Pro Arg Thr Pro Val Ser Pro 435 440 445Val Lys Phe Ser Pro Gly Asp Phe Trp Gly Arg Gly Ala Ser Ala Ser 450 455 460Thr Ala Asn Pro Gln Thr Pro Val Glu Tyr Pro Met Glu Thr Ser Gly465 470 475 480Ile Glu Gln Met Asp Val Thr Met Ser Gly Glu Ala Ser Ala Pro Leu 485 490 495Pro Ile Arg Gln Pro Asn Ser Gly Pro Tyr Lys Lys Gln Ala Phe Pro 500 505 510Met Ile Ser Lys Arg Pro Glu His Leu Arg Met Asn Leu 515 520 525 5 31 PRT Homo sapiens Sequence derived from C-terminus of ribosomal S6 protein 5Lys Glu Ala Lys Glu Lys Arg Gln Glu Gln Ile Ala Arg Arg Arg Leu 1 5 10 15Ser Ser Leu Arg Ala Ser Thr Ser Lys Ser Glu Ser Ser Gln Lys 20 25 30 6 19 PRT Homo sapiens Sequence derived from C-terminus of ribosomal S6 protein 6Arg Arg Arg Leu Ser Ser Leu Arg Ala Ser Thr Ser Lys Ser Glu Ser 1 5 10 15Ser Gln Lys 7 10 PRT Artificial Sequence Description of Artificial Sequence Synthetic protease target substrate for p70(alpha) S6 Kinase isoforms 7Lys Lys Arg Asn Arg Thr Leu Ser Val Ala 1 5 10 8 18 PRT Artificial Sequence Description of Artificial Sequence Synthetic protease target substrate for p70(beta) S6 Kinase isoforms 8Arg Arg Leu Ser Ser Leu Arg Ala Ser Thr Ser Lys Ser Glu Ser Ser 1 5 10 15Gln Lys

Claims

1. An isolated nucleic acid molecule selected from the group consisting of:(a) an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or the complete complement thereof; (b) an isolated nucleic acid molecule having at least 95% nucleotide sequence identity with the entire contiguous open reading frame of SEQ ID NO: 1 and encoding a protein capable of phosphorylating ribosomal S6 protein; (c) an isolated nucleic acid molecule which hybridizes to the nucleotide sequence of SEQ ID NO: 1 or the complete complement thereof under conditions which employ 0.1×SSC at 68° C. and which encodes a protein capable of phosphorylating ribosomal S6 protein; and (d) an isolated nucleic acid molecule which encodes a protein comprising the amino acid sequence of SEQ ID NO: 2.

2. An isolated nucleic acid molecule which encodes a fragment of a protein comprising the amino acid sequence of SEQ ID NO: 2 wherein the fragment is capable of phosphorylating ribosomal S6 protein.

3. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises nucleotides 77-1561 of SEQ ID NO: 1.

4. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule consists of nucleotides 77-1561 of SEQ ID NO: 1.

5. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule consists of nucleotides 77-1564 of SEQ ID NO: 1.

6. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises nucleotides 116-1561 of SEQ ID NO: 1.

7. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule consists of nucleotides 116-1561 of SEQ ID NO: 1.

8. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule consists of nucleotides 116-1564 of SEQ ID NO: 1.

9. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule contains a nucleotide substitution at a position corresponding to nucleotides 1277, 1278 or 1279 of SEQ ID NO: 1.

10. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes a protein comprising an aspartic acid substitution for threonine at amino acid 401 of SEQ ID NO: 2.

11. The isolated nucleic acid molecule of any one of claims 1 and 2-10, wherein the nucleic acid molecule is operably linked to one or more expression control elements.

12. A vector comprising the isolated nucleic acid molecule of any one of claims 1 and 2-10.

13. A host cell transformed to contain the nucleic acid molecule of any one of claims 1 and 2-10.

14. A host cell comprising the vector of claim 12.

15. The host cell of claim 13, wherein said host cell is selected from the group consisting of prokaryotic hosts and eukaryotic hosts.

16. A method for producing a protein comprising the step of culturing a host cell of claim 13 under conditions in which the protein encoded by the nucleic acid molecule is expressed.

17. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule in (b) has at least 97% nucleotide sequence identity with the entire contiguous open reading frame of SEQ ID NO: 1.

18. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule in (b) has at least 98% nucleotide sequence identity with the entire contiguous open reading frame of SEQ ID NO: 1.

19. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule in (b) has at least 99% nucleotide sequence identity with the entire contiguous open reading frame of SEQ ID NO: 1.

20. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule hybridizes to the nucleotide sequence of SEQ ID NO: 1 or the complete complement thereof under conditions which employ 0.1×SSC at 68° C.