Novel protein tyrosine phosphatase substrate-trapping double mutant and uses thereof

BACKGROUND OF THE INVENTION

[0003] Sequencing of the human and other genomes has radically changed the ways in which we identify and characterize genes. Typically, database searches that are based on structural similarities can assign a gene product to an established protein family. Although members of protein families often share a common mechanism of action, the cellular processes in which they are involved can be both highly specialized and fundamentally important.

[0004] Protein tyrosine phosphatases (PTPs), enzymes which have conserved catalytic domains but are involved in controlling a broad constellation of cellular processes (1-3), provide a striking example of conserved structures associated with functional diversity. The hallmark that defines the PTP superfamily is the active site amino acid sequence (H/V)C(X)5R(S/T), also referred to as the PTP signature motif, within the catalytic domain. To date, analysis of the nearly completed human genome has revealed 112 predicted human PTPs (4). Therefore, it is relatively easy to attribute a general role to a PTP gene product based upon structural homologies. However, determination of the exact physiological function of a PTP requires a tedious and protracted effort. Identification of the cellular substrates of individual PTPs will help to elucidate the biological functions of individual PTPs. A major challenge, though, is the development of technologies for rapid substrate identification that can be applied to the entire PTP family.

[0005] Detailed mechanistic studies have shown that PTPs utilize a common mechanism for phosphomonoester hydrolysis (5). See FIG. 1. The PTPs employ the active site cysteine (e.g., Cys215 in PTP1B) as the attacking nucleophile, thereby forming a thiophosphoryl enzyme intermediate (“E-P”) (6, 7). The E-P formation is assisted by a conserved aspartic acid (e.g., Asp181 in PTP1B), functioning as a general acid, to neutralize the build-up of a negative charge on the leaving group (8, 9). For the hydrolysis of E-P, Asp181, previously functioning as a general acid in E-P formation, acts as a general base, abstracting a proton from the attacking water (10, 11). This enhances the rate of E-P hydrolysis, thereby regenerating the active enzyme. The PTPs further accelerate the formation and hydrolysis of E-P by preferentially binding the pentacoordinated transition states with the guanidinium side chain of the active site arginine residue (e.g., Arg221 in PTP1B) (12, 13).

[0006] Because of the transient nature of the enzyme•substrate complex, it has been difficult to isolate substrates with wild-type PTPs. Based upon insights from mechanistic studies, two types of “substrate-trapping” mutant PTPs have been developed. In the first, the active site Cys residue is replaced by a Ser (14-16); in the second, the general acid Asp residue is substituted by an Ala (17, 18; see U.S. Pat. Nos. 5,912,138 and 5,951,979). These mutants retain the ability to bind substrates; however, because they are either unable to carry out substrate dephosphorylation (the Cys-to-Ser mutant) or severely impaired in carrying out substrate dephosphorylation (the Asp-to-Ala mutant), capture of the PTP enzyme•substrate complex becomes possible.

[0007] The substrate-trapping mutant PTPs have been used as affinity reagents to isolate and identify physiological substrates for various PTPs. Nevertheless, to date, only a limited number of PTP substrates have been identified by the substrate-trapping approach, and these have been mostly abundant proteins. For example, the adapter protein p130 has been found to be the target of several PTPs, including PTP1B (19), PTP-PEST (17), the Yersinia PTP (20), PTPα (21), LAR (22), and SAP (23). The fact that only a few proteins have been identified as PTP substrates is surprising, given the large number of protein tyrosine kinases and phosphotyrosine-(pTyr-) containing proteins in the cell. One possible explanation may be that the affinity of the available trapping mutants is not sufficiently high, such that only heavily populated phosphoproteins can be isolated. Accordingly, in view of the foregoing, there exists a need to create an improved PTP substrate-trapping mutant, with a higher affinity, that will enable the identification of less abundant substrates.

SUMMARY OF THE INVENTION

[0008] The inventors describe herein the design and characterization of a novel protein tyrosine phosphatase 1B (PTP1B) substrate-trapping double mutant (PTP1B/D181A/Q262A) that displays 6- and 28-fold higher affinity than the existing D181A and C215S mutants, respectively. Because both Asp181 and Gln262 are invariant among PTPs, this double mutant should be appropriate for substrate identification for all members of the PTP family. Identification and characterization of specific PTP/substrate interactions will permit the association of functions with individual PTPs, and implicate specific PTPs to specific signaling pathways.

[0009] Accordingly, the present invention provides protein tyrosine phosphatases (PTP) in which the invariant aspartate residue and the invariant glutamine residue are each replaced with a replacement amino acid residue, wherein the replacement residues together cause a reduction in catalytic rate (kcat) of the enzyme and an increase in substrate-binding affinity (Kd) of the enzyme.

[0010] The present invention also provides methods for identifying a substrate of PTPs by (a) contacting a candidate substrate with at least one PTP in which the invariant aspartate residue and the invariant glutamine residue are each replaced with a replacement amino acid residue, wherein the replacement residues together cause a reduction in catalytic rate (kcat) of the enzyme and an increase in substrate-binding affinity (Kd) of the enzyme; and (b) assessing the ability of the candidate agent to bind to the PTP.

[0011] Further provided are kits for identifying a substrate of a PTP, comprising: (a) at least one PTP in which the invariant aspartate residue and the invariant glutamine residue are each replaced with a replacement amino acid residue, wherein the replacement residues together cause a reduction in catalytic rate (kcat) of the enzyme and an increase in substrate-binding affinity (Kd) of the enzyme; and (b) reagents suitable for detecting binding of the PTP to a candidate substrate.

[0012] Finally, the present invention provides methods for identifying an agent that alters interaction between a PTP and a substrate of the PTP by (a) identifying a substrate of a PTP in accordance with the previously described method; (b) contacting the PTP and the substrate, in the presence of a candidate agent; and (c) assessing the ability of the candidate agent to inhibit PTP-substrate interaction.

[0013] Additional objects of the present invention will be apparent in view of the description which follows.

BRIEF DESCRIPTION OF THE FIGURES

[0014]
FIG. 1 illustrates a chemical mechanism for the PTP1B-catalyzed dephosphorylation reaction.

[0015]
FIG. 2 depicts an amino acid sequence alignment of 32 human PTPs, two yeast PTPs, and the Yersinia PTP YOP51, surrounding Gln262 in PTP1B. Conserved residues are shown in bold.

[0016]
FIG. 3 portrays calorimetric isothermal titration for the reaction of peptide Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2 with PTP1B/D181A/Q262A. The upper graph depicts raw data for thirty 8-μl injections of the peptide (0.5 mM stock) into the isothermal cell containing 32 μM PTP1B/D181A/Q262A, at 4-min intervals and 25° C. Both the protein and the peptide were in 50 mM 3,3-dimethylglutarate buffer (I=0.15 M, pH 7.0) containing 1 mM DTT. The lower graph depicts an integration curve showing experimental points that were obtained by integration of the above peaks as plotted against the molar ratio of the peptide to PTP1B/D181A/Q262A within the reaction cell. The solid line corresponds to the best fit to the data according to the nonlinear, least-squares regression algorithm, ORIGIN (29).

[0017]
FIG. 4 sets forth Western blots (WB) that show the level of tyrosine phosphorylation in the epidermal growth factor receptor (EGFR) upon expression of the wild-type and mutant PTP1Bs (4 μg plasmid/60 mm dish) in COS1 cells. Samples (50 μg total protein) were taken from the lysates of COS1 cells transfected with 4 μg of pJ3H plasmids expressing HA-tagged wild-type PTP1B, D181A, C215S, D181A/Q262A, or D181A/C215S. These samples were loaded into each lane for SDS-PAGE electrophoresis, and transferred onto a nitrocellulose membrane. The proteins on the membrane were first immunoblotted with anti-pTyr HRP conjugate (upper panel). The membrane was then stripped and re-probed with anti-hemagglutinin (anti-HA) HRP conjugate (lower panel).

[0018]
FIG. 5 illustrates immunoprecipitation (IP) and Western blots (WB) of the phospho-EGF receptor, and other less abundant PTP1B substrates, by the PTP1B substrate-trapping mutants. Wild-type PTP1B, D181A, D181A/Q262A, D181A/C215S, C215S, or blank vector (pJ3H) was used to transfect COS1 cells (4 μg plasmid/60 mm dish). The cells were maintained at 37° C. for 44-48 h. Twenty minutes before harvesting the cells, 20 ng/ml EGF was added to each dish. The PTP1B proteins were precipitated from the cell lysates using anti-hemagglutinin (anti-HA) antibody-conjugated agarose beads from the cell lysates (500 μg total protein) at 4° C. overnight. The beads were washed 3 times with the lysis buffer, mixed with 2×SDS sample buffer, and then boiled for 5 min. The immunocomplexes were resolved by SDS-PAGE, and transferred onto a nitrocellulose membrane. The membrane was probed with anti-pTyr HRP-conjugated antibody (lower panel). The membrane was then stripped and reblotted with anti-EGFR rabbit polyclonal antibody (middle panel). Thereafter, the membrane was further stripped, and re-probed with anti-HA HRP conjugate (upper panel).

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides mutant protein tyrosine phosphatases (PTP) having reduced or no catalytic activity relative to the wild-type PTP. The term “mutant” is used herein to refer to a gene or its gene product which exhibits at least one modification in its sequence or its functional properties as compared with the wild-type gene or its gene product. In contrast, the term “wild-type” refers to the characteristic genotype (or phenotype) for a particular gene (or its gene product), as found most frequently in its natural source (e.g., in a natural population). A wild-type animal, for example, expresses a functional PTP. Because the substrate-trapping PTP mutant of the present invention retains the ability to bind tyrosine phosphorylated substrates of the wild-type PTP, it is useful in binding, or trapping, one or more substrates of the PTP.

[0020] In the mutant PTPs of the present invention, the invariant aspartate residue is replaced with a replacement amino acid residue, and the invariant glutamine residue is replaced with a replacement amino acid residue. As used herein, the term “invariant residue” refers to an amino acid residue that is conserved within a particular family of proteins or enzymes. For example, in PTP1B, both Asp181 and Gln262 are invariant among other members of the PTP family. The invariant aspartate and glutamine residues may be identified in any other PTP by aligning the PTP nucleotide or amino acid sequence with that of PTP1B or another PTP for which the locations of the invariant aspartate and glutamine residues is known (see, e.g., FIG. 2). The replacement amino acids in the PTP enzyme of the present invention may be any amino acids that naturally occur. Examples of replacement amino acid residues include, without limitation, alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Preferably, the replacement amino acid residue is one with an aliphatic R group, e.g., isoleucine, valine or leucine. In the most preferred embodiments, the invariant aspartate and glutamine residues are replaced with alanine residues (see Experimental Details).

[0021] Examples of methods that may be employed in the synthesis of the PTP, and a mutant version of this sequence containing the replacement amino acid residues of the present invention, include, but are not limited to, solid-phase peptide synthesis, solution-method peptide synthesis, and synthesis using any of the commercially-available peptide synthesizers. The double mutant PTP of the present invention may contain coupling agents and protecting groups, which are used in the synthesis of protein sequences, and which are well-known to one of skill in the art.

[0022] The double mutant PTP of the present invention also may be produced from a PTP-encoding deoxyribonucleic acid (DNA) that has been mutated using methods known to one of skill in the art. These methods of DNA mutation include, but are not limited to, chemical mutagenesis, disruption (e.g., by allelic exchange), illegitimate recombination, PCR-mediated mutagenesis, signature-tagged mutagenesis, site-directed mutagenesis, targeted gene disruption, and transposon mutagenesis. Preferably, the method of mutation of the present invention is PCR-mediated mutagenesis.

[0023] The replacement amino acid residues in the PTPs of the present invention collectively bring about a reduction in the PTP's catalytic activity relative to the catalytic activity of the wild-type PTP. Specifically, the replacement residues together cause a reduction in catalytic rate (kcat) of the enzyme. In some embodiments of the present invention, the kcat of the PTP is reduced by at least 100-fold. In other embodiments of the present invention, the kd of the PTP is reduced by at least 1,000-fold.

[0024] The mutant PTPs of the present invention, which contains the replacement amino acid residues, do not lose their ability to bind tyrosine phosphorylated substrates of the wild-type PTP. In fact, the replacement amino acid residues in the PTPs of the present invention collectively bring about an increase in the PTP's binding ability relative to the binding ability of the wild-type PTP enzyme. Specifically, the replacement residues together cause an increase in substrate-binding affinity (Kd) of the enzyme. In some embodiments of the present invention, the Kd of the PTP is increased by at least 10-fold. In other embodiments of the present invention, the Kd of the PTP enzyme is increased by at least 30-fold.

[0025] The PTPs of the present invention may be any member of the PTP family, either prokaryotic (e.g., YOP51) or eukaryotic (e.g., from yeast or mammals, including humans), now known or later discovered. Examples of PTPs include, without limitation, any of the PTPs listed in FIG. 2. Preferred PTPs include PTP1B, PTP-PEST, PTPγ, TcPTP, DEP-1, PTPμ, LAR, CD45, PTPH1, PTPα, SHP1, SHP2, PTP-MEG1, PTPβ, HePTP, SAP-1, and YOP51. In particularly preferred embodiments, the PTP is PTP1B. Unless otherwise indicated, “PTP1B” includes both a PTP1B protein and a “PTP1B analogue”. A “PTP1B analogue” is a functional variant of the PTP1B peptide, having PTP1B biological activity, that has 60% or greater (preferably, 70% or greater) amino-acid-sequence homology with the PTP1B protein, as well as a fragment of the PTB1B protein having PTP1B biological activity. As further used herein, the term “PTP1B biological activity” refers to the activity of a protein or peptide that hydrolyzes phosphomonoesters, as described herein. Additionally, unless otherwise indicated, “protein” shall include a protein, protein domain, polypeptide, or peptide.

[0026] In some embodiments of the invention, the PTP is PTP1B, and the invariant aspartate residue is located at position 181 of the PTP1B amino acid sequence. In other embodiments, the PTP is PTP1B, and the invariant glutamine residue is located at position 262 of the PTP1B amino acid sequence. In further embodiments of the present invention, the PTP is PTP1B, the invariant aspartate residue is located at position 181 of the PTP1B amino acid sequence, and the invariant glutamine residue is located at position 262 of the PTP1B amino acid sequence. As described herein, this PTP1B substrate-trapping double mutant, PTP1B/D181A/Q262A, displays 6- and 28-fold higher affinity than the existing D181A and C215S mutants, respectively. See Experimental Details.

[0027] The present invention is also directed to methods for identifying a substrate of a PTP. The methods comprise the steps of: (a) contacting a candidate substrate with at least one PTP in which the invariant aspartate residue and the invariant glutamine residue are each replaced with a replacement amino acid residue, wherein the replacement residues together cause a reduction in catalytic rate (kcat) of the enzyme and an increase in substrate-binding affinity (Kd) of the enzyme; and (b) assessing the ability of the candidate agent to bind to the PTP. Also provided is a substrate identified by the method of the present invention.

[0028] In these methods, the PTP may be any member of the PTP family, as previously discussed in relation to the mutants of the invention.

[0029] In these methods, the invariant aspartate residue of the PTP is replaced with a replacement amino acid residue, and the invariant glutamine residue is replaced with a replacement amino acid residue. The replacement amino acids in the PTP may be any amino acids that naturally occur, as previously discussed.

[0030] In some embodiments of these methods, the invariant aspartate residue is replaced with an alanine residue. In other embodiments, the invariant aspartate residue is replaced with an alanine residue. In preferred embodiments, the invariant aspartate residue is replaced with an alanine residue, and the invariant glutamine residue is replaced with an alanine residue.

[0031] In these methods, the replacement amino acid residues in the PTP collectively bring about a reduction in the PTP's catalytic activity relative to the catalytic activity of the wild-type PTP. Specifically, the replacement residues together cause a reduction in catalytic rate (kcat) of the enzyme. In some embodiments of these methods, the kcat of the PTP is reduced by at least 100-fold. In other embodiments, the kcat of the PTP is reduced by at least 1,000-fold. Moreover, the replacement amino acid residues in the PTP collectively bring about an increase in the PTP's binding ability relative to the binding ability of the wild-type PTP. Specifically, the replacement residues together cause an increase in substrate-binding affinity (Kd) of the enzyme. In some embodiments of the present invention, the Kd of the PTP is increased by at least 10-fold. In other embodiments of the present invention, the Kd of the PTP is increased by at least 30-fold.

[0032] In some embodiments of these methods, the PTP is PTP1B, and the invariant aspartate residue is located at position 181 of the PTP1B amino acid sequence. In other embodiments, the PTP of the present invention is PTP1B, and the invariant glutamine residue is located at position 262 of the PTP1B amino acid sequence. In further embodiments, the PTP is PTP1B, the invariant aspartate residue is located at position 181 of the PTP1B amino acid sequence, and the invariant glutamine residue is located at position 262 of the PTP1B amino acid sequence. This PTP1B substrate-trapping double mutant may be referred to as PTP1B/D181A/Q262A.

[0033] In these methods, the substrate of the PTP can be any substrate, particularly a protein, that binds to, associates with, or interacts with the PTP. For example, the substrate of the PTP may be a tyrosine phosphorylated protein. Examples of tyrosine phosphorylated proteins that may be suitable substrates for a PTP include, without limitation, p130cas, the EGF receptor, p210bcr-abl, c-Src kinase, the insulin receptor, p160, p120, p70, and p60.

[0034] In these methods, a candidate substrate is contacted with at least one substrate-trapping PTP in which the invariant aspartate residue and the invariant glutamine residue are each replaced with a replacement amino acid residue. To effect contact, the candidate substrate may be combined with the substrate-trapping PTP complex between the candidate substrate and the substrate-trapping PTP, thereby producing a combination. For example, contacting may be effected in vitro, under conditions suitable for binding of the candidate substrate to the substrate-trapping PTP enzyme, for a period of time sufficient to permit binding of the candidate substrate to the substrate-trapping PTP. Appropriate binding conditions (e.g., temperature, pH, and salt concentration) are readily determinable by the skilled artisan.

[0035] After the candidate substrate has been combined with, or contacted with, the substrate-trapping PTP, an assessment may be made of the ability of the candidate substrate to bind to the PTP. For example, it may be determined whether or not a complex between the candidate substrate and the substrate-trapping PTP is present in the enzyme/substrate combination. In these embodiments, the presence of a complex in the combination between the candidate substrate and the substrate-trapping PTP is indicative that the candidate substrate is a substrate of the PTP.

[0036] The present invention is also directed to kits for identifying a substrate of a PTP. The kits comprise the following: (a) at least one of the previously described PTP mutants in which the invariant aspartate residue and the invariant glutamine residue are each replaced with a replacement amino acid residue; and (b) one or more ancillary reagents suitable for use in detecting binding of the PTP enzyme to a candidate substrate. The kits optionally may further comprise a container and/or instructions for utilizing the mutant and the reagent(s) to identify a PTP substrate.

[0037] In the kits of the present invention, the PTP may be any member of the PTP family, as previously discussed. Also, the candidate substrate of the PTP may be any substrate, e.g., a protein or oligopeptide, that binds to, associates with, or interacts with the PTP. For example, the substrate of the PTP may be a tyrosine phosphorylated protein. Examples of tyrosine phosphorylated proteins that may be suitable substrates for a PTP include, without limitation, p130cas, the EGF receptor, p210bcr-abl, c-Src kinase, the insulin receptor, p160, p120, p70, and p60.

[0038] In these kits, the ancillary reagent or reagents capable of detecting binding of the PTP to the candidate substrate (e.g., by detecting the presence or absence of an enzyme-substrate complex) are preferably secondary antibodies that bind selectively to one or the other of the enzyme and substrate, and which are further linked, either through a covalent linkage or by a noncovalent linkage, to a reporter molecule, including, without limitation, an enzyme, a fluorescent molecule, a light-emitting molecule, or a radioactive molecule. If the agent if located on a cell surface, antibody binding to the cell may result in cell agglutination. In such a case, the reagent capable of detecting the resulting complex will not be necessary.

[0039] The double mutant PTP of the present invention may be useful for reducing the activity of a substrate of a PTP. Accordingly, the present invention further provides a method for reducing in vivo the activity of a substrate of a PTP in a subject, comprising administering to the subject a mutant PTP in which the invariant aspartate residue is replaced with a replacement amino acid residue and the invariant glutamine residue is replaced with a replacement amino acid residue, and which forms a complex with the substrate. The subject is preferably a mammal (e.g., a human; a domestic animal; or a commercial animal, including a cow, a dog, a mouse, a monkey, a pig, and a rat), and is most preferably a human. The double mutant PTP is administered to the subject in an amount effective to reduce the activity of the substrate of the PTP. This amount may be readily determined by the skilled artisan.

[0040] The previously described double mutant PTPs of the present invention are suitable for reducing the activity of a substrate of a PTP, particularly a tyrosine phosphorylated protein, because it is capable of binding the phosphorylated protein without dephosphorylating the protein. As such, the double mutant PTP will form a complex with the substrate, thereby reducing its downstream effects.

[0041] The double mutant PTP of the present invention may be administered to a human or animal subject by known procedures, including, without limitation, nasal administration, oral administration, parenteral administration (e.g., epidural, epifascial, intracapsular, intracutaneous, intradermal, intramuscular, intraorbital, intraperitoneal [particularly in the case of localized regional therapies], intrasternal, intravascular, intravenous, parenchymatous, and subcutaneous administration), sublingual administration, transdermal administration, and administration by osmotic pump. Preferably, the PTP is administered nasally or orally.

[0042] For oral administration, a formulation of the PTP may be presented in solid or liquid preparations, e.g., capsules, tablets, powders, granules, dispersions, solutions, and suspensions. Such preparations are well known in the art, as are other oral-dosage forms not listed here. The formulation may have conventional additives, such as lactose, mannitol, corn starch, or potato starch. The formulation also may be presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins. Additionally, the formulation may be presented with disintegrators, such as corn starch, potato starch, or sodium carboxymethylcellulose. The formulations also may be presented with dibasic calcium phosphate anhydrous or sodium starch glycolate. Finally, the formulation may be presented with lubricants, such as talc or magnesium stearate.

[0043] For parenteral administration, a formulation of the PTP may be combined with a sterile aqueous solution that is preferably isotonic with the blood of the subject. Such a formulation may be prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile. The formulation may be presented in unit or multi-dose containers, such as sealed ampules or vials. The formulation may be delivered by any mode of injection, including, without limitation, epidural, epifascial, intracapsular, intracutaneous, intradermal, intramuscular, intraorbital, intraperitoneal (particularly in the case of localized regional therapies), intrastemal, intravascular, intravenous, parenchymatous, or subcutaneous.

[0044] For transdermal administration, a formulation of the PTP may be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the like, which increase the permeability of the skin to the enzyme, and permit the enzyme to penetrate through the skin and into the bloodstream. The composition of the enhancer and the enzyme also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which may be dissolved in a solvent, such as methylene chloride, evaporated to the desired viscosity, and then applied to backing material to provide a patch. For nasal administration, aerosol, nasal-mist, or nasal-spray a formulation of the PTP may be prepared in accordance with standard procedures known in the art for the preparation of nasal sprays.

[0045] A formulation of the PTP also may be released or delivered from an osmotic mini-pump or other time-release device. The release rate from an elementary osmotic mini-pump may be modulated with a microporous, fast-response gel disposed in the release orifice. An osmotic mini-pump would be useful for controlling release, or targeting delivery, of the enzyme.

[0046] In accordance with the methods of the present invention, the PTP may be administered to a subject by introducing to the subject the protein itself, or by introducing to the subject DNA encoding the enzyme, in a manner permitting expression of the protein. The enzyme of the present invention may be introduced to the subject by known techniques used for the introduction of drugs, including, for example, injection and transfusion. For non-invasive introduction of the PTP of the present invention, micro-encapsulated preparations, such as liposomes, also may be used. Liposomal vesicles may be prepared by various methods known in the art, and liposome compositions may be prepared using any one of a variety of conventional techniques for liposome preparation known to those skilled in the art.

[0047] DNA encoding the PTP may be introduced to the subject using conventional procedures known in the art, including, without limitation, electroporation, DEAE dextran transfection, calcium phosphate transfection, lipofection, monocationic liposome fusion, polycationic liposome fusion, protoplast fusion, creation of an in vivo electrical field, DNA-coated microprojectile bombardment, injection with recombinant replication-defective viruses, homologous recombination, in vivo gene therapy, ex vivo gene therapy, viral vectors, and naked DNA transfer, or any combination thereof. Recombinant viral vectors suitable for gene therapy include, but are not limited to, vectors derived from the genomes of viruses such as a retrovirus, HSV, adenovirus, adeno-associated virus, Semiliki Forest virus, cytomegalovirus, and vaccinia virus.

[0048] The present invention is further directed to methods for identifying an agent that alters interaction between a PTP and a substrate of the PTP. The methods, comprising the steps of: (a) identifying a substrate of a PTP using a method described herein; (b) contacting the PTP and the substrate, in the presence of a candidate agent; and (c) assessing the ability of the candidate agent to inhibit PTP-substrate interaction. Also provided is an agent identified in accordance with this method. An agent identified by the method of the present invention may be an antagonist, such as an agent that inhibits or decreases activity of a PTP. Additionally, an agent identified by the method of the present invention may be an agonist, such as an agent that enhances or increases PTP activity. An agent that is capable of altering interaction between a PTP and a substrate thereof also may be an agent that interacts with the PTP.

[0049] As used herein, an “agent” shall include a protein, polypeptide, peptide, nucleic acid (including DNA or RNA), antibody, Fab fragment, F(ab′)2 fragment, molecule, compound, antibiotic, drug, and any combinations thereof. Moreover, an agent may be either natural or synthetic, and exogenous or endogenous. A Fab fragment is a univalent antigen-binding fragment of an antibody, which is produced by papain digestion. An F(ab′)2 fragment is a divalent antigen-binding fragment of an antibody, which is produced by pepsin digestion. As used herein, the antibody of the present invention may be polyclonal or monoclonal, and may be produced by techniques well known to those skilled in the art. Polyclonal antibody, for example, may be produced by immunizing a mouse, rabbit, or rat with purified protein. Monoclonal antibody may then be produced by removing the spleen from the immunized mouse, and fusing the spleen cells with myeloma cells to form a hybridoma which, when grown in culture, will produce a monoclonal antibody. The antibody of the present invention also includes a humanized antibody, made in accordance with procedures known in the art.

[0050] According to the method of the present invention, an agent that alters (inhibits or enhances) interaction between a PTP and a substrate of the PTP may be identified by: identifying a substrate of a PTP using a method described herein; contacting the PTP and the substrate in the presence of a candidate agent; and assessing the ability of the candidate agent to inhibit PTP-substrate interaction. A substrate of a PTP may be identified in accordance with the above-described method for identifying a substrate of a PTP, which comprises the steps of: (a) contacting a candidate substrate with at least one PTP in which the invariant aspartate residue and the invariant glutamine residue are each replaced with a replacement amino acid residue, wherein the replacement residues together cause a reduction in catalytic rate (kcat) of the enzyme and an increase in substrate-binding affinity (Kd) of the enzyme; and (b) assessing the ability of the candidate agent to bind to the PTP.

[0051] In accordance with the method of the present invention, a PTP may be contacted with a substrate that has been identified in the manner described above, in the presence of a candidate agent. To effect contact, the substrate may be combined with the substrate-trapping PTP, in the presence of a candidate agent, under conditions appropriate for binding or formation of a complex between the substrate and the substrate-trapping PTP, thereby producing a combination. For example, contacting may be effected in vitro, under conditions suitable for binding of the substrate to the substrate-trapping PTP enzyme, for a period of time sufficient to permit binding of the substrate to the substrate-trapping PTP enzyme. Appropriate binding conditions (e.g., temperature, pH, and salt concentration) may be readily determinable by the skilled artisan.

[0052] After the substrate has been combined with, or contacted with, the substrate-trapping PTP, in the presence of the candidate agent, an assessment may be made of the ability of the candidate agent to alter (inhibit or enhance) interaction or binding between the PTP and the substrate. For example, the amount of enzymatic activity (or extent of binding) in the combination may be determined in the presence of the candidate agent, under conditions suitable for the formation of an enzyme-substrate complex, and this may be compared with the amount of enzymatic activity (or extent of binding) in the combination in the absence of the candidate agent under conditions suitable for the formation of an enzyme-substrate complex. In this embodiment of the present invention, a difference in enzymatic activity (or extent of binding) in the combination in the presence of the candidate agent, as compared with enzymatic activity (or extent of binding) in the absence of the candidate agent, is indicative that the candidate agent alters (inhibits or enhances) interaction between a PTP and a substrate of the PTP or interacts with a PTP.

[0053] An agent that alters (inhibits or enhances) interaction between a PTP and a substrate of the PTP, or an agent that interacts with a PTP, may be identified using an in vitro assay, such as an ELISA. Moreover, a specific inhibitor of PTP enzyme/substrate interaction may be obtained by high-throughput screening of a small-molecule library using purified PTP. Additionally, an agent that alters interaction between a PTP and a substrate of the PTP, or an agent that interacts with a PTP, may be identified using an in vivo assay, such as a yeast two-hybrid system.

[0054] The present invention is described in the following Experimental Details section, which is set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.

EXPERIMENTAL DETAILS

[0055] 1. Introduction

[0056] As described above, detailed mechanistic studies have shown that members of the protein tyrosine phosphatase (PTP) family utilize a common mechanism for phosphomonoester hydrolysis (FIG. 1) (5). Although PTPs share a common mechanism of action (hydrolysis of phosphotyrosine), the cellular processes in which they are involved can be both highly specialized and fundamentally important. It is believed that identification of cellular PTP substrates will help elucidate the biological functions of individual PTPs.

[0057] To date, two types of substrate-trapping mutants have been used to isolate PTP substrates. In the first, the active site Cys residue is replaced by a Ser (e.g., PTP1B/C215S); in the second, the general acid Asp residue is substituted by an Ala (e.g., PTP1B/D181A). Unfortunately, only a limited number of substrates (usually cellular proteins that are relatively abundant) have been identified with these two mutants.

[0058] Based upon mechanistic considerations, the inventors created novel PTP double mutants with improved substrate-trapping properties. These double mutants are described herein. Kinetic and thermodynamic characterization of the newly-designed PTP1B mutants indicate that PTP1B/D181A/Q262A displays lower catalytic activity than does D181A. In addition, PTP1B/D181A/Q262A possesses substrate-binding affinity that is 6- or 28-fold higher than that of PTP1B/D181A or PTP1B/C215S, respectively. Moreover, in vivo substrate-trapping experiments indicate that the double mutant PTP1B/D181A/Q262A exhibits much higher affinity for a bona fide PTP1B substrate, the epidermal growth factor receptor (EGFR), than does either PTP1B/D181A or PTP1B/C215S. Finally, the double mutant PTP1B/D181A/Q262A also trapped several novel, less abundant PTP1B substrates that are missed by both D181A and C215S mutants.

[0059] Accordingly, the inventors' newly-developed and improved substrate-trapping double mutant can serve as a powerful affinity reagent to isolate and purify both high- and low-abundance protein substrates. Given that both Asp181 and Gln262 are invariant among the PTP family, it is expected that this improved substrate-trapping mutant would be appropriate for identifying substrates for all members of the PTP family.

[0060] 2. Materials and Methods

[0061] A. Materials

[0062] p-nitrophenyl phosphate (pNPP) was purchased from Fluka Co. Other chemicals were purchased from Fisher Co. Solutions were prepared using deionized and distilled water. The preparation of the nonhydrolyzable phosphotyrosine (pTyr) mimetic phosphonodifluoromethyl phenylalanine-(F2Pmp-) containing peptide, Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2, was previously described (24). Recombinant human epidermal growth factor (EGF) was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-hemagglutinin (anti-HA) epitope mouse monoclonal antibody IgG-horseradish peroxidase (HRP) conjugate, anti-phosphotyrosine antibody PY20 and PY99 horseradish peroxidase conjugated, anti-HA conjugated agarose for immunoprecipitation, rabbit anti-EGF receptor polyclonal antibody, and goat anti-rabbit IgG-HRP were all purchased from Santa Cruz Biotechnology.

[0063] B. Protein Expression and Purification

[0064] The catalytic domain of PTP1B (residues 1 to 321) was used for in vitro study. PTP1B mutants, C215S, D181A, and Q262A, were described previously (9, 25, 26). PTP1B double mutants, D181A/C215S and C215S/Q262A, were generated by PCR reactions, according to the standard procedure of the Quick-Change™ site-directed mutagenesis kit (Stratagene), using pT7-7/PTP1B/C215S as a template. PTP1B double mutants, D181A/C215E and D181A/Q262A, were generated by similar PCR procedures, using pT7-7/PTP1B/D181A as a template. The recombinant wild-type and mutant PTP1Bs were expressed in E. coli, and purified to homogeneity as described (25, 27). Protein concentration was determined from absorbance measurement at 280 nm, using an absorbance coefficient of 1.24 for 1 mg/ml PTP1B.

[0065] C. Determination of Kinetic Constants Using pNPP as a Substrate

[0066] PTP activity was assayed at 25° C. and pH 7.0, in a reaction mixture (0.2 ml) containing pNPP concentrations ranging from 0.2 to 5 Km. The following buffered solution was used for activity measurements: pH 7.0, 50 mM 3,3-dimethylglutarate, 1 mM EDTA; the ionic strength was 0.15 M, as adjusted by addition of NaCl. Initial rate measurements for the enzyme-catalyzed hydrolysis of pNPP were conducted as previously described (26). Michaelis-Menten kinetic parameters were determined from a direct fit of the data to the Michaelis-Menten equation, using the nonlinear regression program, KINETASYST (Intellikinetics, State College, Pa.).

[0067] D. Determination of Kinetic Constants Using pTyr-containing Peptides as Substrates

[0068] All assays were performed at 25° C. in buffer (pH 7.0, ionic strength =0.15 M) containing 50 mM 3,3-dimethylglutarate and 1 mM EDTA. A continuous spectrophotometric assay, previously described (28), was employed to determine kcat and Km for the pTyr-containing peptides. The dephosphorylation reaction was monitored by either an increase in absorbance at 282 nm, or an increase in fluorescence at 305 nm. Fluorometric and absorbance determinations were performed on a Perkin-Elmer LS50B fluorometer and a Perkin-Elmer Lambda 14 spectrophotometer, respectively. The instruments were equipped with a water-jacketed cell holder, permitting maintenance of the reaction mixture at the desired temperature (25° C.).

[0069] E. Isothermal Titration Calorimetry

[0070] All isothermal titration calorimetry experiments were performed using an MCS Isothermal Titration Calorimetry System from Microcal Inc. (Northampton, Mass.). Experiments at pH 7.0 were conducted at 25° C., in 50 mM 3,3-dimethylglutarate buffer containing 1 mM DTT. The ionic strength of the buffer was adjusted to 0.15 M by addition of NaCl. Protein concentration in the calorimeter cell was 27-85 μM, while the ligand concentration in the syringe was 0.37-1 mM.

[0071] The PTP1B samples used in the isothermal titration calorimetry experiments were dialyzed completely against buffer. High-concentration stock solutions were prepared for ligands with distilled water, and adjusted to pH 7.0. Stock was diluted at least 26-fold with 50 mM 3,3-dimethylglutarate buffer before titration. Protein dilution during titration was determined by titration of buffer into the protein solution. The heat of protein dilution was found to be negligible. The heat of ligand dilution was corrected by subtracting the average heat of injection after saturation.

[0072] The binding data were analyzed using ORIGIN software (29). Binding constant, K, and enthalpy change, ΔH, were used to calculate free energy change, ΔG, and entropy change, ΔS, according to equation [1]:

−RTLnK=ΔG=ΔH−TΔS
[1]

[0073] where R is the gas constant and T is the absolute temperature.

[0074] F. Mammalian Expression Plasmids

[0075] The hemagglutinin-tagged (HA-tagged) full-length human wild-type PTP1B in pJ3H expression vector was a gift from Dr. Chemoff (30). The HA-tagged PTP1B mutants, C215S, D181A, D181A/C215S, and D181A/Q262A, were generated by PCR reactions, according to the standard procedure of the Quick-Change™ site-directed mutagenesis kit (Stratagene), using pJ3H-HA-PTP1B as a template. All mutations were verified by DNA sequencing.

[0076] G. Cell Culture and Transient Transfection

[0077] Monkey kidney COSI cells (ATCC: CRL-1650)were maintained in Dulbecco's modified Eagle's minimum essential medium (DMEM), supplemented with 10% fetal bovine serum (Life Technologies, Inc.), penicillin (50 units/ml), streptomycin (50 μg/ml), and L-glutamine (2 mM) under a humidified atmosphere containing 5% CO2. In transient transfection experiments, COS1 cells were inoculated at a density of 2×105 cells/60-mm dish, and grown overnight in DMEM containing 10% fetal bovine serum. Transfection was performed using LipoTAXIR according to the manufacturer's recommendations (Stratagene, Cedar Creek, Tex.). Each transfection assay included 4 μg of purified DNA mixed with LipoTAXIR in a 1:5 DNA:lipid ratio per 60-mm dish of cells. Following transfection, the cells were exchanged to normal culture medium, and maintained for 44 to 48 h at 37° C. and 5% CO2. The efficiency of transfection, as assessed by 5-bromo-4-chloro-3-indolyl-β-D galactoside (X-Gal) staining of pCMV-β-gal-transfected (β-Gal staining kit, Invitrogen) COS cells, was 25%.

[0078] H. Cell Lysis, Immunoprecipitation, and Western Blotting

[0079] The transfected cells (treated with or without EGF) were lysed with “lysis buffer” containing 50 mM Tris-HC1 (pH 7.5), 5 mM EDTA, 150 mM NaCl, 10 mM sodium phosphate, 10 mM sodium fluoride, 5 mM iodoacetic acid, 1 mM benzamidine, 1% Triton X-100, 10 μg/ml leupeptin, and 5 μg/ml Aprotinin. Lysate protein concentration was estimated using Bio-Rad protein assay reagent (Bio-Rad, Hercules, Calif.).

[0080] For immunoprecipitation, 1 mg of cell lysate was immunoprecipitated overnight with 30 μl of agarose-conjugated anti-hemagglutinin antibody at 4° C. Immunocomplexes were washed four times with the cell lysis buffer, and boiled for 5 min in SDS-PAGE sample buffer. Proteins in the cell lysate were separated by 8% SDS-PAGE under reducing conditions, according to the method of Laemmli (31), followed by blotting onto nitrocellulose membranes (Protran, Schleicher & Schuell). The nitrocellulose membranes were blocked with 5% non-fat dry milk in Tris-buffered saline/Tween (TBST) buffer (20 mM Tris-HC1, 150 mM NaCl, 0.1% Tween-20; pH 7.6) for 1 h at 25° C.

[0081] Thereafter, blots were incubated overnight with primary antibodies diluted in TBST containing 5% non-fat dry milk at 4° C. The blots were then rinsed with TBST (3 times), for 5 min at 25° C., and incubated with the appropriate secondary antibody diluted in TBST containing 5% non-fat dry milk, for 1 h at 25° C. The blots were rinsed 3 more times with TBST for 5 min before detection by enhanced chemiluminescence (ECL) (Cell Signaling Technology). For re-probing, blots were stripped by incubating in 62.5 mM Tris-HC1, 2% SDS, and 100 mM 2-mercaptoethanol (pH 6.7) for 30 min at 50° C. Stripped blots were then rinsed extensively with TBST, and re-probed as described above.

[0082] 3. Results and Discussion

[0083] PTPs play a central role in controlling many diverse signal transduction pathways in cells. Although PTPs share a common catalytic mechanism (hydrolysis of phosphoamino acids), they have distinct (and often unique) biological functions in vivo. Genetics and biochemical studies indicate that PTPs are involved in a number of disease processes (3). However, despite the detailed understanding of PTP catalysis, the molecular basis for the diverse biological functions of PTPs is poorly understood. In part, this lack of understanding is a result of the paucity of information concerning the physiological substrates of most PTPs. Therefore, the identification of physiological substrates for individual PTPs remains one of the central goals in this field. One effective strategy for identifying and characterizing in vivo PTP substrates employs high affinity PTP substrate-trapping mutants.

[0084] Currently, two types of substrate-trapping mutants are being used to identify PTP substrates. In the first mutant, the active site Cys residue is replaced by a Ser, which leads to a complete loss of phosphatase activity (14-16, 32). In the second, the general acid Asp residue is substituted by an Ala (17, 18), which results in a decrease in kcat of several orders of magnitude (8, 18). So far, only relatively abundant cellular proteins have been identified with these two mutants. It is possible that the affinity for substrates of the existing trapping mutants is not high enough to enable the capture of less abundant proteins.

[0085] The goal of the present study is to create a mutant PTP with enhanced substrate-trapping properties, for use in PTP substrate identification. The inventors' strategy to achieve this goal is to exploit the common mechanism of PTP catalysis to create substrate-trapping mutants that can be applied to all PTP members. In doing so, the inventors decided to focus on invariant residues that are important for PTP catalysis.

[0086] There are two ways to improve a substrate-trapping mutant: (1) by lowering the dissociation constant (Kd) between the enzyme and the substrate; and (2) by further slowing down substrate turnover (kcat). Since PTP1B/D181A displays higher affinity for substrates than does PTP1B/C215S, both in vitro and in vivo (18, 24), the inventors sought to improve its potency by introducing into it a second site mutation. Because the general acid deficient mutant PTP1B/D181A still contains residual phosphatase activity, the inventors considered mutating additional invariant residues to further reduce its activity, and possibly to further increase its substrate-binding affinity. The criteria for selecting the second site mutations are as follows: (1) mutation of the second residue must lead either to a further decrease in activity or to an increase in affinity; and (2) mutation of the second residue must not alter substrate specificity. Thus, the chosen residues ideally should be located in or near the active-site pocket (i.e., pTyr binding site), and should perform universal catalytic functions.

[0087] In view of the above criteria, the active site Cys215 is an obvious choice because of its essential role in catalysis. Thus, substitutions of Cys215 in PTP1B/D181A should render the double mutant inactive. However, because the side chain of the active site Cys exists as a thiolate anion at physiological pH (33), the Cys-to-Ser mutation is not a simple substitution of OH for SH; rather, it is a replacement of the negatively charged thiolate anion with a neutral hydroxyl group. Indeed, the inventors have observed that the Cys-to-Ser mutation causes significant structural/dynamic perturbations in the active site (24, 32, 34, 35). Therefore, in addition to PTP1B/D181A/C215S, the inventors also prepared PTP1B/D181A/C215E in order to mimic the thiolate anion in the active site.

[0088] The next residue that the inventors chose was Gln262, which is invariant among all PTPs (FIG. 2). The crystal structure of PTP1B/C215S bound with Ac-DADEpYL-NH2 showed that the side chain of Gln262 is close to the phenyl ring of pTyr, and may define a portion of the rim for the pTyr binding pocket (37). Previous kinetic studies revealed that the kcat/Km and kcat for the PTP1B/Q262A-catalyzed hydrolysis of the epidermal growth factor receptor peptide, DADEpYLIPQQG (EGFR988-998), and phosphorylated lysozyme were reduced by 10- and 100-fold, respectively (18, 26). This suggests that Gln262 may play a role in both the formation of the thiophosphoryl enzyme intermediate (E-P) and the E-P hydrolysis step. Studies on the structural equivalents, Gln446 in the Yersinia PTP (36) and Gln262 in PTP1B (38), also suggest that the invariant Gln262 residue is important for the optimal positioning of the nucleophilic water molecule, which facilitates efficient E-P hydrolysis (FIG. 1). To determine whether mutation of Gln262 would improve existing substrate-trapping mutants, the inventors prepared PTP1B/C215S/Q262A and PTP1B/D181A/D262A.

[0089] A. Kinetic Characterization

[0090] All recombinant PTP1B proteins were expressed in Escherichia coli, and purified to near homogeneity, as judged by SDS polyacrylamide gel electrophoresis, using procedures described previously (25, 27) (data not shown). The kinetic parameters for the wild-type and mutant PTP1B-catalyzed hydrolysis of pNPP are summarized in Table 1.

1TABLE 1Kinetic Parameters for the Wild-type and MutantPTP1B-Catalyzed Hydrolysis of pNPPPTP1BKm(μM)kcat(s−1)kcat/Km(M−1 s−1)aWild-type2400 ± 200 9.8 ± 0.5(4.1 ± 0.4) × 103aD181A 80 ± 30(3.1 ± 0.3) × 10−2(3.9 ± 1.5) × 102bQ262A 58 ± 60.21 ± 0.01(3.6 ± 0.3) × 103aD181A/Q262A 2.4 ± 0.5(2.8 ± 0.1) × 10−3(1.2 ± 0.2) × 103cC215ScD181A/C215ScC215S/Q262AcD181A/C215EaMeasured at 25° C. and pH 7.0. bMeasured at 30° C. and pH 7.0 (26). cNo measurable activities were observed for C215S, C215S/D181A, C215S/Q262A, and D181A/C215E.

[0091] Table 2 lists the steady-state kinetic parameters for the wild-type and mutant PTP1Bs, using the EGFR988-998 peptide DADEpYLIPQQG as a substrate. As expected, no measurable activities were observed for C215S, D181A/C215S, C215S/Q262A, and D181A/C215E. For the D181A mutant, the kcat was 320-fold lower than that of the wild-type enzyme, while the Km value was 30-fold lower with pNPP as a substrate (Table 1). With the EGFR988-998 peptide as a substrate, the kcat value for D181A was 920-fold lower than that for PTP1B, while the Km value was 22-fold higher (Table 2). As shown previously, Q262A exhibited 38- and 7.4-fold decreases in Km for pNPP and the EGFR988-998 peptide, respectively (26). In addition, the kcat for the Q262A-catalyzed hydrolysis of pNPP and the EGFR988-998 peptide was reduced by 80- and 150-fold, respectively (26).

2TABLE 2Kinetic Parameters for the Wild-type and Mutant PTP1B-CatalyzedHydrolysis of the EGFR988-998 peptide, DADEpYLIPQQGPTP1BKm(μM)kcat(s−)kcat/Km(M−1 s−1)aWild-type 2.6 ± 0.3 30 ± 1(1.2 ± 0.1) × 1071D181A 56 ± 2 (6.5 ± 0.2) × 10−2(1.2 ± 0.1) × 103bQ262A0.35 ± 0.050.29 ± 0.002(8.3 ± 1.1) × 105aD181A/Q262A 51 ± 4 (2.7 ± 0.1) × 10−3(5.3 ± 0.5) × 101aMeasured at 25° C. and pH 7.0. bMeasured at 30° C. and pH 7.0 (26).

[0092] The substitution of Gln262 by an Ala in D181A resulted in a 33-fold decrease in Km, and an 11-fold decrease in kcat, with pNPP as a substrate (Table 1). Interestingly, with the EGFR988-998 peptide as a substrate, the kcat for D181A/Q262A was 24-fold lower than that for D181A, while the Km value for D181A/Q262A was similar to that for D181A (Table 2). Collectively, then, it appears that mutation at either Asp181 or Gln262 results in large reductions in kcat, irrespective of the substrates, and that the kcat for the double mutant is lower than those for the single mutants.

[0093] B. The Kinetic Parameter Km Is Not an Accurate Indicator of Substrate-Binding Affinity

[0094] The overall mechanism of the PTP-catalyzed reaction involves a number of steps that are represented schematically in Scheme 1, where ArOPO32−can be either aryl phosphates or pTyr-containing peptides/proteins. The reaction proceeds through a sequence involving substrate binding (substrate dissociation constant Ks=k−1/k1), which is then cleaved with phosphoryl transfer (k2) to the active site nucleophilic Cys residue (E-P formation). A subsequent general base-catalyzed reaction with water cleaves the phosphoenzyme intermediate E-P (k3), and the release of phosphate completes the catalytic cycle.

[0095] Applying the steady-state assumption to [E-P], it can be shown that kcat=[k2k3/(k2+k3)] and Km=[Ksk3/(k2+k3)]. Thus, the kcat term describes the rate-limiting step under saturating concentrations of substrate, and is mostly determined by the E-P hydrolysis step (k3) for the wild-type PTP1B (26, 39). The Km parameter is an apparent dissociation constant that can be treated as the overall dissociation constant of all enzyme-bound species (40). For example, when k2 is much greater than k3, the concentration of E-P is much greater than [E•ArOPO32−], so that E-P contributes more to Km than does E•ArOPO32−, and is the predominant enzyme-bound species. Thus, Km is not an accurate measure of substrate-binding affinity. Rather, its value is smaller than the substrate-dissociation constant Ks by a factor of k3/(k2+k3).

[0096] The dramatic decrease in Km for the Q262A mutant, which may be indicative of increased accumulation of the E-P intermediate in the Q262A-catalyzed reactions, is consistent with the observation that substitution of Gln262 by an Ala has a more severe effect on k3 than on k2 (26, 37). Interestingly, it appears that, for both D181A and the double mutant D181A/Q262A, the mutation may have a more negative effect on k3 in the pNPP reaction, resulting in a decrease in Km. In contrast, in the peptide-substrate reaction, the mutation may have a more deleterious effect on k2, leading to an increase in Km. Thus, it is difficult to predict the substrate-binding affinity of the double mutant (D181A/Q262A) based purely upon the apparent Km values.

[0097] C. D181A/Q262A Displays Higher Affinity to Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2 than Does D181A

[0098] To determine directly the substrate-binding affinity for substrates of the PTP1B double mutants, the inventors performed isothermal titration microcalorimetry (ITC) experiments. ITC allows a simultaneous determination of the binding constant (K), stoichiometry, and the enthalpy change (ΔH) associated with the binding of a ligand to a macromolecule (29). From these parameters, the Gibbs free energy of binding (ΔG) and the entropy change (ΔS) of binding can also be derived from the expression ΔG=−RTLnK=ΔH-TΔS. Because of the inherent hydrolytic activity, it has not been possible to study directly the binding interactions between a PTP and its substrate. In order to compare binding interactions between wild-type PTP1B and its various mutants (either active or inactive), the inventors have used peptides that contain a nonhydrolyzable pTyr analog, phosphonodifluoromethyl phenylalanine (F2Pmp), in the ITC experiments (24).

[0099] The F2Pmp-containing peptide, Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2, is an excellent non-hydrolyzable substrate analog that exhibits high affinity for PTP1B (24, 41, 42). Using ITC, the inventors have previously determined the dissociation constant (Kd) and thermodynamic parameters for the binding of Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2 to PTP1B and to the mutants, C215S and D181A (24). The inventors showed that the active site Cys215-to-Ser mutant PTP1B binds Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2 with the same affinity as the wild-type enzyme (Table 3). In addition, the inventors found that the general acid deficient mutant D181A binds the same ligand 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better substrate-trapping reagent than C215S (17, 18).

3TABLE 3Thermodynamic Parameters for the Binding ofPTP1B and Its Substrate-Trapping Mutants withAc-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2KdΔHTΔSΔGPTP1B(μM)(kcal mol−1)(kcal mol−1)(kcal mol−1)aWild-type0.24 ± 0.05 −3.9 ± 0.25.1 ± 0.2−9.0 ± 0.1aC215S0.19 ± 0.03−10.4 ± 0.2−1.2 ± 0.2 −9.2 ± 0.1aD181A0.04 ± 0.01 −6.5 ± 0.13.7 ± 0.1−10.2 ± 0.1 bD181A/Q262A0.0068 ± 0.0023 −4.5 ± 0.56.6 ± 0.2−11.1 ± 0.2 bD181A/C215E 1.3 ± 0.09−28.4 ± 0.3−20.4 ± 0.3 −8.0 ± 0.03All experiments were performed at 25° C. and pH 7.0. aData from (24). bData from this study.

[0100] To determine whether any one of the double mutants (D181A/Q262A, D181A/C215S, D181A/C215E, and C215S/Q262A) displayed improved binding properties over the D181A mutant, the inventors measured the Kdvalues of the double mutants for Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2, and the thermodynamic parameters associated with binding, using ITC under conditions (pH 7.0, ionic strength of 0.15 M, and 25° C.) identical to those employed for the wild-type PTP1B and for the C215S and D181A mutants (Table 3). A typical titration curve and binding isotherm for the binding of D181A/Q262A to Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2 is shown in FIG. 3. From curve-fitting of the binding isotherms, the stoichiometry for the binding of the peptide to D181A/Q262A was determined to be 1:1. The dissociation constant, Kd, was 6.8 nM, which is ˜30-fold lower than those of the wild-type PTP1B and the C215S mutant (Table 3). More importantly, D181A/Q262A also exhibited nearly 6-fold higher affinity for Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2 than did the D181A mutant.

[0101] The association process between D181A/Q262A and the F2Pmp-containing peptide is both enthalpically (ΔH=-4.5 kcal/mol) and entropically (TΔS=6.6 kcal/mol) favored, yielding a ΔG for binding of −11.1 kcal/mol at pH 7.0 and 25° C. The increased substrate-binding affinity for the D181A/Q262A mutant, as compared with that for the D181A mutant, appears to result from a larger increase in the TAS term, which more than offsets the smaller decrease in ΔH of binding (Table 3).

[0102] The inventors were not able to obtain Kd values for the D181A/C215S and C215S/Q262A mutants because the titration curves were rather complex. The observed titration curves either yielded a very low binding stoichiometry (˜0.1 for D181A/C215S), or displayed two peaks (C215S/Q262A) with one absorbing heat and the other releasing heat (data not shown). The inventors noted that, even for the C215S single mutant, the titration curve also displayed two peaks at a lower temperature (15° C.).

[0103] Because all of the mutants were purified to homogeneity (i.e., a single band in SDS-PAGE), these observations suggest that the C215S-containing mutants may exist in aqueous solution as an equilibrium mixture of more than one conformation. Indeed, the recently solved crystal structure of the apo PTP1B/C215S mutant showed that C215S can exist in a conformation that is different from that observed in the C215S/substrate complex (43). It is possible that removal of the negative charge from the thiolate group in Cys215 may lead to a disruption of the active-site conformation (24). However, the inventors were able to obtain the Kd and thermodynamic parameters for the binding of Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2 to D181A/C215E (Table 3). The affinity of D181A/C215E for the peptide (Kd=1.3 μM) was 7- and 32-fold lower than those of the C215S and D181A.mutants, respectively. Thus, substitution of Cys215 in PTP1B/D181A does not improve PTP1B's affinity for substrates. In contrast, substitution of Gln262 by an Ala in PTP1B/D181A generates a much-improved PTP1B substrate-trapping mutant, with a substrate-binding affinity 6-fold higher than that of PTP1B/D181A.

[0104] D. D181A/Q262A Is a Better Substrate-Trapping Mutant than D181A In Vivo

[0105] From the experiments described above, the inventors have shown that the D181A/Q262A mutant not only displays lower catalytic activity than the D181A mutant, but also exhibits higher substrate-binding affinity toward a peptide substrate analog. In the following, the inventors present evidence that D181A/Q262A is also a better substrate-trapping mutant in vivo.

[0106] To determine whether the double mutant D181A/Q262A also possesses higher affinity for physiological substrates of PTP1B, the inventors performed substrate-trapping experiments in COS1 cells. It has been shown previously that, in COS1 cells, the epidermal growth factor (EGF) receptor is the major substrate for PTP1B, and that the D181A mutant has a higher affinity for the EGF receptor than does the C215S mutant (18). Because EGF receptor is the major PTP1B substrate in COS1 cells, the inventors wanted to determine the effects of expression of PTP1B and its various forms of trapping mutants, in this cell type, on the level of EGF receptor tyrosine phosphorylation.

[0107] Accordingly, the inventors expressed the full-length, HA-tagged wild-type, D181A, C215S, D181A/Q262A, and D181A/C215S mutants of PTP1B in COS1 cells, and then immunoblotted the cell lysates with an anti-pTyr antibody (FIG. 4). The protein expression level of wild-type and mutant PTP1Bs was shown to be similar by Western blotting of the cell lysates using anti-HA antibodies (FIG. 4).

[0108] Consistent with earlier work (18), the inventors found that the level of pTyr in the EGF receptor in cells expressing wild-type PTP1B was comparable to that in the vector control. In addition, the inventors also noted that the tyrosine phosphorylation level of EGF receptor was higher in cells expressing the D181A, rather than the C215S, mutant (FIG. 4). These results serve as important controls to demonstrate that, as shown previously, D181A has higher affinity for the EGF receptor than does C215S, and that the trapping mutants bind to and protect substrates from dephosphorylation by endogenous PTPs.

[0109] The inventors next evaluated the ability of D181A/Q262A and D181A/C215S to promote accumulation of the pTyr-phosphorylated EGF receptors in COS1 cells. The inventors discovered that D181A/C215S displayed a comparable affinity to that of C215S (FIG. 4). In contrast, the inventors were satisfied to find that enhancement of EGF receptor phosphorylation was most pronounced in cells expressing D181A/Q262A, rather than any other, PTP1B mutants (FIG. 4).

[0110] Immunoblotting of the cell lysates with anti-pTyr antibodies showed that, indeed, the D181A/Q262A mutant was much better (more than 3-fold) than the D181A mutant in protecting the phosphorylated EGF receptors in COS1 cells (FIG. 4). These data suggest that D181A/Q262A displays the highest substrate-binding affinity among all mutant forms of PTP1B applied in the inventors' experiments, and that it can strongly protect the EGF receptor from dephosphorylation by the endogenous PTPs.

[0111] Finally, the inventors determined whether the binding interaction between the trapping mutants and the substrate would be stable enough to endure isolation procedures. The inventors expressed the wild-type and the mutant forms of PTP1B in COS1 cells. After the cells were treated with EGF (20 ng/ml for 20 min), the expressed PTP1B proteins were immunoprecipitated from the cell.lysates using anti-HA antibody-conjugated agarose. The associated substrates were visualized by anti-pTyr and anti-EGFR immunoblotting, and the amount of co-immunoprecipitated PTP1B was measured with HRP-conjugated anti-HA antibody.

[0112] As shown in FIG. 5, more than 3-fold greater quantities of tyrosine-phosphorylated EGF receptor were co-precipitated with D181A/Q262A than with the D181A mutant. In addition, the amount of EGF receptor protein associated with D181A/Q262A and D181A also corresponded to the level of pTyr in the EGF receptor, suggesting that only the phosphorylated EGF receptor bound to the trapping mutants (FIG. 5). Moreover, the D181A/Q262A double mutant also trapped several novel, less-abundant tyrosine phosphorylated proteins (e.g., P160, P120, P70, and P60; see FIG. 5) that were not visible in either of the D181A or C215S samples. Collectively, then, the inventors have demonstrated, from both substrate protection and substrate pull-down experiments, that D181A/Q262A is a substrate-trapping mutant superior to D181A.

[0113] 4. Summary

[0114] Based on insights from mechanistic studies, the inventors designed several mutant PTP1Bs in order to-create substrate-trapping mutants with improved properties over the existing mutants. Kinetic and thermodynamic characterization indicated that PTP1B/D181A/Q262A possesses further decreased catalytic activity and increased substrate-binding affinity than does PTP1B/D181A. These properties suggest that D181A/Q262A would serve as an improved substrate-trapping mutant, as compared with D181A. Indeed, in vivo substrate-trapping experiments indicated that this is the case: D181A/Q262A displays higher affinity for a bona fide PTP1B substrate, the EGF receptor, than does D181A. Further, D181A/Q262A also trapped several novel, less-abundant protein substrates that are missed by both D181A and C215S mutants. Thus, this newly-developed and improved substrate-trapping mutant can be used as a powerful affinity reagent to isolate and purify PTP1B physiological substrates.

[0115] Because of the conserved roles of Asp181 and Gln262 in PTP catalysis, and the proximity of these residues to the active site (pTyr pocket), it is unlikely that D181A/Q262A and D181A would exhibit substrate specificity different from that of the wild-type enzyme. Furthermore, given that both Asp181 and Gln262 are invariant among the PTP family, it is predicted this improved substrate-trapping mutant would be applicable to the identification of physiological substrates for all members of the PTP family.

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[0159] All publications mentioned hereinabove are hereby incorporated by reference in their entireties. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

Novel protein tyrosine phosphatase substrate-trapping double mutant and uses thereof

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