CA2375145A1 - Substrate trapping protein tyrosine phosphatases - Google Patents

Abstract

Compositions and methods are provided pertaining to novel substrate trapping mutant protein tyrosine phosphatases (PTPs) that are catalytically impaired but which retain the ability to bind phosphotyrosine-containing protein substrate(s), and that are further modified by the replacement of at least o ne tyrosine residue with an amino acid that cannot be phophorylated. Uses of su ch PTPs for identification of PTP substrates, and of agents that alter PTP- substrate interactions are disclosed, as are methods of altering PTP activities.

Description

SUBSTRATE TRAPPING PROTEIN TYROSINE PHOSPHATASES
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Continuation-in-Part ofU.S. Application Serial No.
09/144,925, filed September 1, 1998, which is a Divisional of U.S. Application No. 08/685,992, filed July 25, 1996. This application also claims the benefit of U.S.
Provisional Application No. 60/137,319 (attorney docket number CSHL99-06P), filed June 3, 1999. The teachings of these applications are incorporated herein by reference in their entirety STATEMENT OF GOVERNMENT INTEREST
Work described herein was supported by government funding under research grant CA 53840 awarded by the National Institutes ofHealth. The government may have certain rights in this invention.
TECHNICAL FIELD
The present invention relates generally to compositions and methods useful for treating conditions associated with defects in cellular biochemical pathways such as those controlling cell proliferation, cell differentiation and/or cell survival. The invention is more particularly related to substrate trapping mutants of protein tyrosine phosphatase polypeptides, and variants thereof. The present invention is also related to the use of such polypeptides to identify antibodies and other agents, including small molecules, that modulate biological signal transduction and cellular biochemical pathways.

WO 00/75339 '' PCT/US00/14211 B~CKGROLT1D OF THE INVE~T1DN
Reversible protein tyrosine phosphorylation. coordinated by the action of protein tyrosine kinases (PTKs) that phosphorylate certain tyrosine residues in polypeptides. and protein tyrosine phosphatases (PTPs) that dephosphorylate certain phosphotyrosine residues, is a kev met:~hanism in regulating manv cellular activities. It is becoming apparent that the diversity and complexity of the PTPs and P'TKs are comparable. and that PTPs are equally important in delivering both positive and negative signals for proper function of cellular machinery. Regulated tyrosine phosphorylation contributes to specific pathways for biological signal transduction.
including those associated with cell division, proliferation and differentiation. Defects and/or malfunctions in these pathways may underlie certain disease conditions for which effective means for intervention remain elusive, including for example.
malignancy. autoimmune disorders, diabetes. obesity and infection.
The protein tyrosine phosphatase (PTP) family of enzymes consists of 1 ~ more than X00 structurally diverse proteins that have in common the highly conserved 250 amino acid PTP catalytic domain. but which display considerable variation in their non-catalytic se~nents (Charbonneau and Tonks. 1992 Annu. Rev. Cell Biol.
8:=~6~
493; Tonks. 1993 Semin. Cell Biol. x:373-X53). This structural diversity presumably reflects the diversity of physiological roles of individual PTP family members. which in certain cases have been demonstrated to have specific functions in ~owth.
development and differentiation (Desai et al.. 1996 Cell 8~:~99-609; Kishihara et al..
1993 C~ll ~-~:1-~S-1~6; Perlcins et al.. 1992 Cell ~0:'??5-236; Pingel and Thomas. 1989 Cell .18:10»-106; Schultz et x1..1993 Cell -3:1~~-1~5~).
a.~lthoush recent studies have also generated considerable information regarding the structure. expression and regulation of PTPs. the nature of the tyrosine phosphorylated substrates throw which the PTPs exert their elects remains to be determined. Studies with a limited number of synthetic phosphopeptide substrates have demonstrated some differences in the substrate selectivities of different PTPs (Cho et al.. ; 99 ~ Protein Sc: l. ': 97 -98-~: Dec:~ert et al.. 199: Eur. J. Biochem.
?~ I :67~-681 ~.
.-W alvses of PTP-mediated dephosphoryiation of PTP substrates suggesmhat catalytic activity may be favored by the presence of certain amino acid residues at specific positions in the substrate polypeptide relative to the phosphorylated tyrosine residue (Ruzzene 'et al.. 1993 Eur. J. Biochem. ?ll:'?89-?9~; Zhang et al.. 1994 Biochemistry 33:??85-~~90). Thus. although the phvsiolo~ical relevance of the substrates used in these studies is unclear. PTPs display a certain level of substrate selectivity in vitro.
The PTP family of enzymes contains a common evolutionarily conserved seu~nent of approximately 250 amino acids known as the PTP catalytic domain. Within this conserved domain is a unique signature sequence motif.
[I/V]HC~G~[S/T~G SEQ ID N0:36, that is invariant among all PTPs. The cysteine residue in this motif is invariant in members of the family and is known to be essential for catalysis of the phosphoryrosine dephosphorylation reaction. It functions as a nucleopbl1e to attack the phosphate moiety present on a phosphotyrosine residue of the incoming substrate. If the cysteine residue is altered by site-directed mutagenesis to serine (e.g.. in cysteine-to-serine or 1 ~ ''CS"mutants) or alanine (e.g.. cysteine-to-alanine or 'CA'~ mutants). the resulting PTP
is catalvtically attenuated but retains the ability to complex with. or bind.
its substrate, at least in vitro. Such mutants can be made, for example, using the PTP family member MKP-1 (Sun et al.. 1993 Cell i.i:48?--X93), as well as other PTPs. However, although these CS mutants can in 'eneral bind erTectively to phosphoryrosyl substrates in vitro to ?0 form stable enzyme-substrate complexes. in many cases such complexes cannot be isolated in vivo, for example when both the mutant PTP and the phosphoryrosyl protein substrate are present together within a cell. Thus. the CS mutants are of limited usefulness and cannot be employed to isolate all combinations of PTPs and substrates.
Currently. desirable Goals for determining the molecular mechanisms that Govern PTP-mediated cellular events include. inter alia. determination of PTP
interacting molecules. substrates and binding parmers. and identification of agents that relate PTP activities. In some situations. however. current approaches may lead to an understandina_ of certain aspects of the regulation of tyrosine phosphorylation by PTPs.
but still may not provide strategies to control specific tyrosine phosphorylation and,~or 30 dephosphorylation events Within a cell.

Accordingly. there is a need in the art for an improved ability to regulate phosphotvrosine signaling, including regulation of PTPs. ~n increased understanding of PTP re_ulation may facilitate the development of methods for modulating the activity of proteins involved in phosphoryrosine signaling pathways, and for treating conditions associated with such pathways. The present invention fulfills these needs and further provides other related advantages.
SUMMARY OF THE ITJVENTION
The present invention provides hove! substrate trapping mutant or altered forms of mammalian PTPs, also referred to as substrate trapping PTPs (ST-PTPs), which bind (trap) one or more substrates of the PTP. Binding of the ST-PTP to a PTP
substrate results in the formation of a complex that can be readily observed.
and. if desired. isolated_ and characterized. These mutant forms of PTPs have attenuated catalytic activity (lack catalytic activity or have reduced catalytic activity) relative to the wild type PTP, but retain the ability to bind tyrosine phosphorylated substrates) of the I~ wild type PTP. ST-PTPs are useful, for example, to determine the fine substrate specificity of one or more PTPs.
It is an aspect of the invention to provide a substrate trapping mutant protein tyrosine phosphatase in which the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause si~ificant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than I per minute: and at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated. In certain embodiments at least one wildtype tyrosine residue is replaced with an amino acid that is alanine.
cysteine_ aspartic acid. ~lutamine. glutamic acid. phenylalanine. ~lycine.
histidine.
?5 isoleucine. lysine. leucine. methionine, aspara:~ne. proline. arginine, valine or tryptophan. In certain other embodiments at least one tyrosine residue that is replaced is located in a protein tyrosine phosphatase catalytic domain. In certain embodiments at least one t'-rosine residue that is replaced is located in a protein tyrosine phosphatase active site. and in certain other embodiments at least one tyrosine residue is replaced with phenylalanine. In certain other embodiments at least one tyrosine residue that is replaced is a protein tyrosine phosphatase conserved residue. and in certain further embodiments the conserved residue corresponds to tyrosine at amino acid position 676 in human PTPH1. In certain embodiments at least one tyrosine residue is replaced with an amino acid that stabilizes a comple:c comprising the protein tyrosine phosphatase and at least one substrate molecule. In certain embodiments the substrate trapping mutt comprises a mutated PTPH1, and in certain embodiments the substrate trapping mutant comprises a mutated protein tyrosine phosphatase that is PTP 1 B. PTP-PEST, PTP~!, uLKP-I, DEP-1. PTP~. PTPXl, PTPX10, SHP?, PTP-P~, PTP-~IEGI, LC-PTP, TC-PTP, CD45, LAR or PTPHl. In certain embodiments the substrate trapping mutant comprises a mutated PTP-PEST phosphatase in which the amino acid at position 231 is replaced with a serine residue.
It is another aspect of the present invention to provide a method of identifying a tyrosine phosphorylated protein which is a substrate of a protein tyrosine phosphatase, comprising the steps of combining a sample comprising at least one tyrosine phosphorylated protein with at least one substrate trapping mutant protein tyrosine phosphatase. in which (i) the wildtype protein tyrosine phosphatase catalync domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than I per minute, and (ii) at least one wildtvpe tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated_ under conditions and for a time sufficient to permit formation of a comple:c between the tyrosine phosphorylated protein and the substrate napping mutt protein tyrosine phosphatase; and determining the presence or absence of a comple:c comprising the tyrosine phosphorylated protein ?5 and the substrate trapping mutant protein tyrosine phosphatase. wherein the presence of the comple:c indicates that the tyrosine phosphoryiated protein is a substrate of the protein tyrosine phosphatase with which it forms a comple:c. In certain embodiments the substrate trapping mutant comprises a mutated protein tyrosine phosphatase that is PTP1B: PTP-PEST. PTP~!. VIKP-1_ DEP-I. PTPu. PTPYI. PTPX10. SHP'_'. PTP-PEZ.
;0 PTP-VlEGI. LC-PTP. TC-PTP. CD~~. L~R or PTPH1. In certain embodiments the sample comprises a cell that e:cpresses the tyrosine phosphorylated protein.
and in certain further embodimenu the cell has been transfected with at 1 east one nucleic acid molecule encoding the substrate. In certain other embodiments at least one substrate trapping mutant protein tyrosine phosphatase is e:cpressed by a cell. and in certain ~ further embodimenu the cell has been transfected with at least one nucleic acid molecule encoding the substrate trapping mutant protein tyrosine phosphatase.
In certain other embodiments the sample comprises a cell that e:cpresses (l) the tyrosine phosphoryiated protein which is a substrate of the protein tyrosine phosphatase, and (ii) the substrate trapping mutant protein tyrosine phosphatase. In certain other embodimenu the cell has been transfected with (l) at least one nucleic acid encoding the substrate. and (ii) at least one nucleic acid encoding the substrate trapping mutant protein tyrosine phosphatase. In certain other embodimenu the sample comprises a cell lysate containing at least one tyrosine phosphorylated protein. and in certain funkier embodimenu the cell lysate is derived from a cell transfected with at least one nucleic 1 ~ acid encoding the tyrosine phosphorylated protein. In certain other further embodiments the cell lysate is derived from a cell ttansfected with at least one nucleic acid encoding a protein tyrosine kinase. In certain other embodimenu at least one substrate trapping mutant protein tyrosine phosphatase is present within a cell lysate.
and in certain further embodimenu the cell lysate is derived from a cell transfected with at least one nucleic acid encoding the substrate trapping mutant protein tyrosine phosphatase. In other embodiments. the tyrosine phosphoryiated protein is ~JCP.
p130'u. the EGF receptor. p~10 bcr:abl. VIAP lanase. Shc (Tiganis et al., 1998 :~lol.
Cell. Biol. 18:162?-16~~) or the insulin receptor.
Turnips to another aspect_ the present invention provides a method of ~5 identifying an anent which alters the interaction between a protein tyrosine phosphatase and a tyrosine phosphoryiated protein which is a substrate of the protein tyrosine phosphatase. comprising contacting in the absence and in the presence of a candidate aQent_ a protein tyrosine phosphatase and a tyrosine phosphorylated protein which is a substrate of the protein tyrosine phosphatase under conditions and for a time sufficient ~0 for detectable dephosphon~lation of the substrate to occur. wherein the tyrosine phosphorylated protein which is a substrate of the protein tyrosine phosphatase is identified by (I) combining a sample comprising at least one tyrosine phosphorylated protein with at least one substrate trapping mutant protein tyrosine phosphatase. in which (l) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute, and (ii) at least one wildtype tyrosine residue is, replaced with an amino acid that is not capable of being phosphorylated. under conditions and for a time sufficient to permit formation of a complex between the tyrosine phosphorylated protein and the substrate trapping mutant protein tyrosine phosphatase: and (2) determining the presence or absence of a complex comprising the tyrosine phosphorylated protein and the substrate trapping mutant protein tyrosine phosphatase. wherein the presence of the complex indicates that the tyrosine phosphorylated protein is a substrate of the protein tyrosine phosphatase with which it forms a complex: and comparing the level of 1~ dephosphorylation of the substrate in the absence of the agent to the level of dephosphorylation of the substrate in the presence of the agent wherein a difference in the level of substrate dephosphorylation indicates the agent alters the interaction between the protein tyrosine phospharase and the substrate.
In another aspect the present invention provides a method of identifying an agent which alters the interaction between a protein tyrosine phosphatase and a tyrosine phosphorylated protein which is a substrate of the protein tyrosine phosphatase, comprising contacting in the absence and in the presence of a candidate agent.
a protein tyrosine phosphatase and a tyrosine phosphorylated protein which is a substrate of the protein tyrosine phosphatase under conditions and for a time su~cient to permit formation of a complex between the tyrosine phosphorylated protein and the substrate trappinmutant protein tyrosine phosphatase. wherein the substrate trapping mutant protein tyrosine phosphatase comprises a mutated protein tyrosine phosphatase in which (l) the wildtype protein tyrosine phosphatase catalvric domain invariant aspartate residue is replaced with an amino acid which does not cause si~ificant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than I per minute.

and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that ~s not capable of being phosphorylated: and comparing the level of complex formation in the absence of the agent to the level of complex formation in the presence of the agent.
wherein a difference in the level of complex formation indicates the agent alters the interaction between the protein tyrosine phosphatase and the substrate.
In another aspect the invention provides a method of reducing the activity of a tyrosine phosphorylated protein. comprising administering to a subject a substrate trapping mutant of a protein tyrosine phosphatase in which (i) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause si~ificant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than I per minute. and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated. whereby interaction of the substrate trapping mutant protein tyrosine phosphatase with the tyrosine phosphorylated protein reduces the activity of the tyrosine phosphorylated protein. In certain embodiments the tyrosine phosphorylated protein is VCP: p130°S, the EGF receptor. p210 bcr:abl. VtAP kinase.
Shc (Tiganis et al.. 1998 Llol. Cell. Biol. 18:1622-I6~-~) or the insulin receptor. In certain other embodiments the protein tyrosine phosphatase is PTP1B, PTP-PEST, PTPI, VLKP-1.
DEP-1. PTPu. PTPX1, PTP'Y10. SHP'_'. PTP-PEZ. PTP-VIEG1, LC-PTP, TC-PTP.
CD~~, LAR or PTPHl.
In still another aspect the invention provides a method of reducing a transtomzing effect of at least one oncoQene associated with p130'~
phosphorylation comprising administering to a mammal capable of expressing p130'u a substrate trapping mutant of PTP-PEST in which (i) the wildtvpe protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than I per minute. and (ii1 at least one wildrype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated:
whereby the substrate trapping mutant interacts with p130"~ to reduce the transforming effect of at least one oncogene associated with p130"S phosphorylation. In certain embodiments the oncogene is v-cric, v-src or c-Ha ras.
Turning to another aspect, the present invention provides a method of reducing formation of signaling complexes associated with p 130°x, comprising administering to a mammal capable of e:cpressing pI~O"~ a substrate trapping mutant of PTP-PEST in which (l) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute. and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated: whereby the substrate trapping mutant interacts with p130'~ to reduce the formation of signaling comple:ces associated with p130'~.
The present invention provides. in another aspect. a method of reducing cytotoxic effects associated with protein tyrosine phosphatase administration or overeYpression_ comprising administering to a mammal a substrate trapping mutant of a protein tyrosine phosphatase in which (l) the wildtvpe protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute. and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated.
'Earning now to another aspect of the invention. there is provided an isolated nucleic acid molecule encoding a substrate trapping mutant protein tyrosine phosphatase in which the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause si~ificant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute: and in which at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphoryiated. In certain embodiments the invention provides an antisense oligonucieotide comprising at Least 1 ~
consecutive nucleotides complementary to the nucleic acid molecule encoding a substrate trapping .i0 mutant protein tyrosine phosphatase. as just described.

It is another aspect of the invention to provide a fusion protein comprising a poiypeptide sequence fused to a substrate trapping mutant protein tyrosine phosphatase in which the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause ~ significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute: and in which at least one wildtype protein tyrosine phosphazase tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated. In certain embodiments the polypeptide is an enzyme or a variant or fragment thereof. In some embodiments the polypeptide sequence fused to a substrate 10 trapping mutant protein tyrosine phosphatase is cieavable by a protease. In certain other embodiments the polypeptide sequence is an affinity tag polypeptide having affinity for a lisand.
In still another aspect. the Present invention provides a recombinant expression construct comprising at least one promoter operably linked to a nucleic acid 1 ~ encoding a substrate trapping mutant protein tyrosine phosphatase in which wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than I per minute: and in which at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated. In certain embodiments the promoter is a regulated promoter.
and in certain other embodiments the substrate trapping mutant protein tyrosine phosphatase is expressed as a fusion protein with a polypeptide product of a second nucleic acid sequence. In certain further embodiments the polypeptide product of the second nucleic acid sequence is an enzyme- In certain other embodiments the expression construct is a 5 recombinant viral expression construct In certain other embodiments the present invention provides a host cell comprising a recombinant expression construct according to those just described. In certain embodiments the host cell is a prokaryotic cell and in certain zmbodiments the host cell is a eukaryotic cell.
The vresent invention provides. in another aspect. a method of producing ~0 a recombinant substrate trapping mutant protein tyrosine phosphatase.
comprisin°_-culturing a host cell comprising a recombinant e:cpression construct comprising at least one promoter operably linked to a nucleic acid sequence encoding a substrate trapping mutant protein tyrosine phosphatase in which the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does ? not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute; and in which at least one wildtype protein tyrosine phosphatase tyrosine residue is replaced with an amino acid that is not capable of being phosphoryiated. In ceztmn embodiments the promoter is a regulated promoter. In certain other embodiments the invention provides a method of producing a recombinant substrate trapping mutant protein tyrosine phosphatase, comprising culturing a host cell infected with the recombinant viral e:cpression construct described above.
The present invention. in another aspect provides a pharmaceutical composition comprising a substrate trapping mutant protein tyrosine phosphatase in which the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate 1 ~ residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute;
and in which at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated, in combination with a pharmaceutically acceptable carrier or diluent.
In yet another aspect the invention provides a pharmaceutical composition comprising an agent that interacts with a substrate trapping mutant protein tyrosine phosphatase in which the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat ~5 to less than 1 per minute: and in which at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphoryiated. in combination with a pharmaceutically acceptable carrier or diluent. In certain other embodiments the invention provides a kit for identifying a tyrosine phosphorylated protein substrate of a protein tyrosine phosphatase comprisin= at least one substrate trapping mutant protein 30 tyrosine phosphatase in which ~i1 the wildtype protein tyrosine phosphatase catalytic 1?
domain invariant aspartate residue is replaced tenth ~ ~o mid which does not cause sisnincant alteration of the Km of the ezrzvrne 'nut which results in a reduction in Kcat to less than I pe: minute, and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphoryiated; and ancillary reagents ~ suitable for use in detecting the presence or absence of a complex between the protein tyrosine phosphatase and a tyrosine phosphorylated protein.
These and other aspects of the present invention will become apparent upon reference to the following deed d~~Ption and attached drawings. All references (including websites) disclosed herein are hereby incorporated by reference in their entireties as if each was incorporated individually.
BRIEF DESCRIPTION OF THE DR.~WII'TGS
Figures lA-lE show a multiple amino acid sepuence alignment of the paralytic domains of various PTPs_ the positions of amino acid residues of PTP1B that interact with substrate are indicated wtth small ~'owh~, and the residue numbering 1~ at the bottom of the alignment corresponds to that for PTP1B. Figs. lA-lE
show a taultiple sequence alignment of the catalytic domains of PTPs (SEQ ID NOS:1-35).
Cvtosoiic eukarvotic PTPs and domain 1 of RPTPs are combined into one soup:
domains 2 of R.PTPs are in a second group and the Yersinia PTP is in a third.
Invanant residues shared among all three gzoups are shown in lower case. Invariant and highly conserved residues within a group are shown is italics and bold, respectively.
Within the Yersinia PTP sequence, residues that are either invariant or highly conserved between the cvtosolic and RPTP domain sc~ ~ m i~~ ~d bold., respectively.
Figure ? shows the Vmax. Kcat and Km of various PTPIB mutants toward RCVLL (reduc=d and carboayamidomethvlated and maleylated lysozyme).
Figure ~ presents phase contrast micrographs that show inhioition of stable NIH.",~T~ cell lines overe:cpressing PTPH1 (-. induced:
uninduc~d~.
Figure ~ presents gzowZh ctmres (mean values from triplicate plating) that show YTowzh inhibition of stable ~~ cell lines overe~pressing PTPHI .

l~
Figure ~ shows inhibition of cell cycle progression by PTPH1 overexpression at indicated time a$er release from hydroxyurea block. by irnmunoblot analysis using antibodies specific for HA epitope tag (PTPH1) or cyclin (~.
induced; -, uninduced).
Figure 6 shows identification of pp97/VCP as a PTPH1 substrate ~n vitro by anti-phosphotyrosine immunoblot analysis of 293 cell lysate proteins trapped by substrate trapping mutant PTPHl(D811A).
Figure 7 shows the amino acid sequence of pp97/V CP (nebi database accession number Z140~) [SEQ ~ NO='~2].
Figure 8 shows identification of pp97/VCP as a PTPH1 substrate in vivo by immunoblot analysis of ?93 cellular proteins trapped by and co-immunoprecipitated with substrate trapping mutant PTPH1(Y676FID811 ~).
Figure 9 shows localization of VCP tyrosine residues reco~ized by PTPH1 to the C-terminal region of VCP.
1~ Figure 10 shows dephosphorylation of VCP in stable NIH3T3 cell lines expressing wildtype PTPH 1.
Figure 11 shows overall profile of tyrosine phosphorylated proteins in stable ~T3 cell lines e:cpressing wildtype PTPH1.
DEVILED DESCRIPTION OF Tl~ INV~ON
The present invention is directed to novel substrate trapping mutant protein tyrosine phosphatases (PTPs) derived from a PTP that has been mutated such that the PTP catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause si~ificant alteration of the Vtichaelis-Vlenten constant (Km) of the enzyme but which results in a reduction of the catalytic rate constant (Kcat), and 25 that has further been mutated by replacement of at least one tyrosine residue with an amino acid that is not capable of being phosphorylated. The invention is based. in part.
on the unexpected tinciing that under certain conditions n vivo. a PTP enzyme may itself undergo tyrosine phosphorylation in a manner that can alter interactions between the PTP and other molecules. including PTP substrates. As defined herein. a phosphatase is a member of the PTP family if it contains the si~ature motif [I/V]HCX.aG~[S/T]G (SEQ ID X0:36). Dual specificity PTPs, i.e., PTPs which dephosphoryiate both phosphorylated tyrosine and phosphorylated serine or threonine, are also suitable for use in the invention. Appropriate PTPs include. but are not limited to, PTP1B, PTP-PEST, PTP~I, VIKP-1, DEP-1, PTPu_ PTPY1, PTPY10, SHP?, PTP
PEZ_ PTP-~G1, LC-PTP, TC-PTP, CD~~, L~1R and PTPH1.
As noted above, substrate trapping mutant PTPs are derived from wildtype PTPs that have been mutated such that the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute; and at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated. In this regard. amino acid sequence analysis of known PTPs reveals the presence of twenty seven invariant residues within the PTP pt'imaN structure (Barford et al., 1994 Science 1~ 263:1397-1404; Jia et al., 199 Science ?68:174-178), including an aspartate residue in the catalytic domain that is invariant among PTP family members. When the amino acid sequences of multiple PTP family members are ali~ed (see, for instance.
Figure lA-E; see also, e.g.. Barford et al., 1995 Varure Struct. Biol. 2:1043), this invariant aspartate residue may be readily identified in the catalytic domain region of each PTP
sequence at a corresponding position relative to the PTP signature sequence motif [UV]HC~G~YR~S/T]G (SEQ ID NO:36), which is invariant among all PTPs (see.
e.o . W098/0471'_'; Flint et al.. 1997 Proc. Vat. .cad Sci. 94:1680 and references cited therein). However. the e.~cact amino acid sequence position numbers of catalytic domain invariant aspartate residues may be different from one PTP to another, due to sequence ?5 shifts that may be imposed to ma.Yimize aliment of the various PTP
sequences (see.
e.o . Barford et al.. 199 Nature Stn~ct. Biol. ?:1043 for an alignment of various PTP
sequenced.
In particular. portions of two PTP polypeptide sequences are regarded as "correspondina~~ amino acid sequences_ regions. fra_ments or the like. based on a convention of numbering one PTP sequence according to amino acid position number.

1~
and then aligning the sequence to be compared in a manner that ma.~cimizes the number of amino acids that match or that are conserved residues. for e:cample. that remain polar (e.g.. D. E, K. R. H. S. T. N, Q), hydrophobic (e.o , ~. P, V. L. I. Vt, F. W.
'~ or neutral (e.~.. C. G) residues at each position. Similarly, a DNA sequence encoding a candidate PTP that is to be mutated as provided herein. or a portion. region, fragment or the like.
may correspond to a known wildtype PTP-encoding DNA sequence according to a convention for numbering nucleic acid sequence positions in the known wildtype PTP
DNA sequence, whereby the candidate PTP DNA sequence is aligned with the known PTP DNA such that at least 70%, preferably at least 80% and more preferably at least 90% of the nucleotides in a given sequence of at least 20 consecutive nucleotides of a sequence are identical. In certain preferred embodiments. a candidate PTP DNA
sequence is heater than 95°'o identical to a corresponding known PTP
DNA sequence.
In certain particularly preferred embodiments, a portion, region or fragment of a candidate PTP DNA sequence is identical to a corresponding known PTP DNA
1 ~ sequence. As is well known in the art. an individual whose DNA contains no irregularities (e.o , a common or prevalent form) in a particular gene responsible for a given trait may be said to possess a wildtype genetic complement (genotype) for that gene, while the presence of irregularities known as mutations in the DNA for the gene.
for example. substitutions, insertions or deletions of one or more nucleotides. indicates a mutated or mutant genotype.
As noted above, in certain embodiments of the present invention there is provided a substrate trapping mutant PTP in which catalytic domain invariant aspartate and at least one tyrosine residue are replaced. as provided herein.
Identification of the catalytic domain invariant aspartate residue in PTP sequences other than those disclosed in Barford et al. ( 1990 may be achieved by comparing sequences using computer alsorithms well known to those having ordinary skill in the art. such as GENEWORKS.
Alit or the BLAST algorithm (.-~ltschul. J. :Llol. Biol. '19:~~~-~6~. 1991:
Henikotf and Henikot~ Pror. W t1. :cad Sri. L.S~ a9:1091~-10919. 199'?. which is available at the ~iCBI website (http::/mywincbi.nlm.niiz.govicgi-bin/BL.~ST).

Certain embodiments of the invention pertain in part to novel PTPs in which the invariant aspartate residue is replaced with an amino acid which does not cause sisnificant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min'). These PTPs retain the ability to form a complex with. or bind to. their tyrosine phosphorylated substrates. but are catalytically attenuated (l. e.. a substrate trapping mutant PTP retains a similar Km to that of the corresponding wildrype PTP, but has a Vmax which is reduced by a factor of at least 10Z-10' relative to the wildtype enzyme, depending on the activity of the wildtype enzyme relative to a Kcat of less than 1 mini'). This attenuation includes catalytic activity which is either reduced or abolished relative to the wildtype PTP.
For example, the invariant aspartate residue can be changed or mutated to an alanine.
valine. leucine, isoleucine: proline. phenylalanine. tryptophan. methionine. glycine. serine.
threonine, cysteine, tyrosine. aspan~;n_e, ~utamine. lysine, ar~nine or histidine.
The preferred substrate trapping mutant PTPs described herein, in which 1 ~ the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min'), and in which at least one tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated, may further comprise other mutations. In particularly preferred embodiments. such additional mutations relate to substitutions. insertions or deletions (most preferably substitutions) that assist in stabilizing the PT'Plsubstrate complex. For example, mutation of the serine,%threonine residue in the si~ati~re motif to an alanine residue (S/T~ ~
mutant) may change the rate-determining step of the PTP-mediated substrate dephosphorylation reaction. For the unmodified PTP, formation of the transition state may be rate-?5 limiting, whereas in the case of the S~T~:~ mutant the breakdown of the transition state may become rate-Limiting, thereby stabilizing the PTP/substrate complex.
Such mutations may be valuably combined with the replacement of the PTP catalytic domain invariant aspartate residue and the replacement of PTP tyrosine as provided herein. for example_ with re?ard to stabilizing the PTP-substrate complex and facilitating its .0 isolation. :~s another e:cample. substitution of any one or more other amino acids present in the wildtype PTP that are capable of being phosphoryiated as provided herein (e.g.. serine. threonine, tyrosine) with an amino acid that is not capable of being phosphorylated may be desirable, with regard to the stability of a PTP-substrate complex.
As noted above, the present invention provides substrate trapping mutant PTPs in which catalytic domain invariant aspartate and at least one tyrosine residue are replaced, wherein the tyrosine is replaced with an amino acid that is not capable of being phosphorylated. The amino acid that is not capable of being phosphorylated may.
in prefezred embodiments, be alanine, cysteine, aspartic acid, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine. leucine, methionine, asparagine, proline. ar~inine. valise or trvptophan. The desirability of the tyrosine replacement derives from the surprising observation that under certain conditions in vivo.
a PTP
enzyme may itself underjo tyrosine phosphorylation in a manner that can alter interactions between the PTP and other molecules. including PTP substrates.
PTP
substrates include any naturally or non-naturally tyrosine-phosphorylated peptide, polypeptide or protein that can specifically bind to and/or be dephosphorylated by a PTP as provided herein. Thus, replacement of a tyrosine residue found in the wildtype amino acid sequence of a particular PTP with another amino acid as provided herein stabilizes a complex formed'ov the subject invention substrate trapping mutant P'?'P and a PTP substrate when the amount of complex that is present and/or the affinity of the mutant PTP for the substrate increases_ relative to comple:c formation using a PTP in which the tyrosine residue is not replaced As noted above, the present invention exploits the substrate trapping mutant PTPs described herein to provide a method of identifying a tyrosine ?5 phosphorylated protein that is a substrate of a wildtype PTP. According to this aspect of the invention_ a sample comprisins at least one tyrosine phosphorylated protein is combined with at least one substrate trapping mutant PTP as provided herein.
and the presence or absence of a complex comprising the substrate and the mutant PTP
is determined. T'ne binding inte:action between a PTP and a PTP substrate may result in ;0 the formation of a complex. which refers to the affinity interaction of the PTP and the is PTP substrate. A complex may include a sigria,ling complex. which refers to any complex that by virtue of its formation. its stable association and/or its dissociation directly or indirectly provides a biological signal. Such signals may include.
for example by way of illustration and not limitation. intracellular and/or intercellular events that lead to molecular binding. covalent or non-covalent modification of molecular structure, gene expression. genetic recombination. ?enetic integration, nucleic acid synthesis or subcellular particle assembly. and may also include endocvtic.
phagocvtic. nucleolytic, proteolvtic. lipolvtic. hydrolytic. catalytic. or other regulatory events.
Determination of the presence of a stable complex between a PTP and a PTP substrate refers to the use of any methodology known in the art for demonstrating an intermolecular interaction between a PTP and a PTP substrate according to the present disclosure. Such methodologies may include. by way of illustration and not limitation. co-purification, co-precipitation, co-immunoprecipitation, radiometric or 16 ffuorimetric assays, western immunoblot analyses. amity capture including atf~ty techniques such as solid-phase ligand-counteriigand sorbent techniques, affinity' chromato~aphy and surface affinity plasmon resonance, and the like. For these and other useful affnity techniques, see. for example. Scopes. R.K., Protein Purification:
Principles and Practice. 1987. Springer-Veriag. NY: Weir. D.M., Handbook of Erperimental Immunology, 1986, Blackwell Scientific. Boston; and Hermanson.
G.T. et al.. Immobili=ed .-lffrniry Ligand Techniques, 1992. Academic Press, Inc., California:
which are hereby incorporated by reference in their entireties, for details regarding techniques for isolating and chatacteriang complexes. including affinity techniques. A
PTP may interact with a PTP substrate via specific binding if the PTP binds the substrate with a Ka of heater than or eq~ to bout 10'~ M-l. preferably of 'eater than or equal to about 10= 1~L-l, more preferably of greater than or equal to about 106 Vl-I
and still more preferably of Greater than or equal to about 10 % VI-~ to 10~ M-' . .Wfinities of binding partners such as a PTP and a PTl' substrate can be readily determined using conventional techniques. for ~:campie those desc:ibed by Scatchard et al.. .-inn. V. Y.
:0 .cad Sci. ~ 1:660 ~19~9).

Without wishing to be bound by theory, it is contemplated that phosphorylated tyrosine residues that are part of a PTP molecule itself may influence the interaction between the PTP molecule and PTP substrate molecules. which include tyrosine phosphorylated proteins that a PTP may bind and/or dephosphorylate.
:according to this non-limiting theory. a conserved tyrosine residue present in a PTP
primary structure may be a receptor for transfer of a phosphate soup from the highly reactive thiophosphaze intermediate that may be formed between the invariant cysteine residue found in the simature motif that resides in the active site of the PTP
catalytic domain (as described above) and the phosphate soup present in the form of phosphotyrosine on the PTP substrate phosphoproteirL Thus. although a conserved tyrosine residue in a PTP active site may facilitate intermolecular orientation of the PTP
relative to its substrate by providing a hydrophobic interaction with the substrate phosphotyrosine, and may further act as a phosphate acceptor. the invention is not so Limited.
1 ~ As described above. the present invention provides a mutated PTP in which at least one tyrosine residue is replaced with an amino acid that cannot be phosphorylated. Preferably the tyrosine residue is located in the PTP
catalytic domain, which refers to the approximately 250 amino acid region that is highly conserved among the various PTPs. as noted above (see also, e.o., Barford_ 1998 ~rrn.
Rev.
~0 Biophys. Biomol. Struct. ~7:1~3; Jia_ 1997 Biochem. Cell Biol. 75:17; Van Vactor et al..
1998 Curr. Opin Genet. bevel. 8:112) Vlore preferably. the tyrosine residue is located in a PTP active site. which refers to the region within the PTP catalytic domain that contains the PTP si~ature motif and which also includes those amino acids that form the PTP binding site pocket or "cradle' for substrate binding and dephosphorylation.
25 further inciudinQ the invariant aspastate-containing loop (when present) and adjacent peptide backbone sequences that contribute to substrate reco~ition and catalysis (see.
e.g.. Jia_ 1997. In a most preferred embodiment. the tyrosine residue is replaced with phenvlalanine. and in another most preferred embodiment. the tyrosine residue is a conserved residue that corresponds to the tyrosine situated at position 676 in the amino ;0 acid sequence of human PTPH1. and which also corresponds to the amino acid residue at position ~6 in the PTP-1B sequence shown in Figure 1. In other preferred embodiments, the tyrosine residue is a PTP conserved residue. which includes tyrosine residues that are present at corresponding positions within two or more PTP
amino acid sequences relative to the position of the signature motif. In other preferred 5 embodiments. the tyrosine residue is replaced with an amino acid that stabilizes a comple:c formed by the PTP and at least one substrate molecule. as provided herein.
~s noted above. PTPs that may be useful according to the present invention include any PTP which has an invariant aspartate residue in a corresponding position in the catalytic domain. and a tyrosine residue. By way of illustration and not 10 limitation, in certain preferred embodiments of the present invention, the substrate trapping mutant PTP has at least one tyrosine residue found in the corresponding wildtype sequence replaced with phenwlalanine. In certain particularly preferred embodiments. the PTP is PTPH1 having the invariant aspartate replaced by alanine and the tyrosine at position 676 replaced by phenylalanine. PTPH1(Y676F/D811A). In 1 ~ certain other embodiments. the PTP is a mutated PTP-PEST phosphatase in which the cysteine found in the corresponding wildtype sequence is replaced with serine and at Least one wildtype tyrosine residue is replaced with an amino acid that cannot be phosphorylated. It should be recognized. however. that mutant PTPs other than those specifically desr:ibed herein can readily be made by ali~ing the amino acid sequence ?0 of a PTP catalytic domain with the amino acid sequence of PTPs that are described herein (including those provided by the cited references), identifying the catalytic domain invariant aspartate residue and at least one tyrosine residue. and changing these residues. for e:cample by site-directed mutagenesis of DNA encoding the PTP.
Vfodification of DNA may be performed by a variety of methods.
'_'S inciudin= site-specinc or site-,iirected mutagenesis of DNA sncodina the PTP and the use of DNA amplification methods using primers to introduce and amplify alterations in the DN -~ template. such as PCR splicing by overlap extension (SOE). Site-directed mutagenesis is t~~picallv effected using a phaQe vector that has single- and doubie stranded forms. such as Vt 13 phage vectors. which are well-~now-n and commercially ~0 available. Other suitable vectors that contain a single-stranded phaae origin of replication may be used (see. e.o . Veira et al.. .Meth. En..-ymol. 1 ~ :3.
1987). In General, site-directed mutagenesis is performed by preparing a single-stranded vector that encodes the protein of interest (e.o . a member of the PTP family). :fin oligonucieotide primer that contains the desired mutation within a re°_-ion of homology to the DNA in the single-stranded vector is annealed to the vector followed by addition of a DNA
polymetase, such as E toll DNA polymerase I (Klenow fragment), which uses the double stranded region as a primer to produce a hetezoduplex in which one strand encodes the altered sequence and the other the original sequence. Additional disclosure relating to site-directed mutagenesis may be found, for example, in Kunkel et GI.
10. (ll~Ierhods in Er~ymol. 1 ~~:367, 1987); and in U.S. Patent Nos. ~.~ 18.84 and 4,737,462. The heteroduplex is introduced into appropriate bacterial cells.
and clones that include the desired mutation are selected. The resulting altered DNA
molecules may be expressed recombinantly in appropriate host cells to produce the modified protein.
1~ Specific substitutions of individual amino acids through introduction of site-directed mutations are well-known and may be made according to methodologies with which those having ordinary skill in the art will be familiar. The effects on catalytic activity of the resulting mutant PTP may be determined empirically merely by testing the resulting modified protein for the preservation of the Km and reduction of 20 Kcat to less than 1 per minute as provided herein and as previously disclosed (e.g., W098l04712; Flint et al.. 1997 Proc. iVat. .cad Sci. 94:1680). The effects on the ability to tyrosine phosphorylate the resulting mutant PTP molecule can also be determined empirically merely by testing such a mutant for the presence of phosphotyrosine. as also provided herein. for example. following exposure of the mutant to conditions in vitro or in vivo where it may acs as a PTK acceptor.
Although the specific examples of PTP mutants described below are DA
(aspartate to alanine) mutants. YF (tyrosine to phenylalanine) mutants. CS
mutants and combinations thereof, it will be understood that the subject invention substrate trapping mutant PTPs are not limited to these amino acid substitutions. The invariant aspartate 30 residue can be changed. for example by site-directed mutaaenesis. to anv amino acid that does not cause si~ificant alteration of the Kl-n of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min'). For example.
the invariant aspartate residue can be changed or mutated to an alanine. valine_ leucine_ isoleucine: proline. phenylalanine, tryptophan, methionine, glycine. serine.
threonine, S cysteine. tyrosine. asparagine. ~lutamine_ lysine. ar~nine or histidine, or other natural or non-natural amino acids known in the art inciudina derivatives. variants and the like.
Similarly. substitution of at least one tyrosine residue may be with any amino acid that is not capable of being phosphorylated (i.e.. stable. covalent modification of an amino acid side chain at a hydroxyl with a phosP~e pup), for example alanine, cysteine_ aspartic acid. giutamine, gluutamic acid, phenyialanine. ~ycine, histidine, isoleucine, lysine, leucine_ methionine. asparagine, proline, arQinine, valine or tryptophan_ or other natural or non-natural amino acids known in the art inciudiny derivatives.
variants and the like.
The nucleic acids of the present invention may be in the form of R.hIA or in the form of DNA. which DNA includes cDNA. aenomic DNA, and synthetic DNA.
The DNA may be double-stranded or single-stranded. and if single stranded may be the coding strand or non-coding (anti-sense) strand. A nucleic acid molecule encoding a substrate trapping mutant PTP in which the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does ?0 not cause si~ificant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute. and in which at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated. may be identical to the coding sequence known in the art for any even PTP. as described above. or may be a different coding sequence. which. as a result of the redundancy or de~eneracv of the genetic code. encodes the same PTP.
The present invention fiirther relates to variants of the herein described nucleic acids which encode fragznents_ analogs and derivatives of a PTP
polypeptide_ includinz a mutated PTP such as a substrate trappins mutant PTP. T'ne variants of the nucleic acids eacodins PTPs may be naturally occuring allelic variants of the nucleic ;0 acids or non-naturally occurring variants. As is known in the art. an allelic variant is an alternate form of a nucleic acid sequence which may have at least one of a substitution, a deletion or an addition of one or more nucleotides. any of which does not substantially alter the function of the encoded PTP polvpeptide.
Equivalent DNA constructs that encode various additions or substitutions of amino acid residues or sequences. or deletions of terminal or internal residues or sequences not needed for biological activity are also encompassed by the invention. For example, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids. preventing formation of incorrect intramolecular disulfide bridges upon renaturation. Other equivalents can be prepared by modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KE:~ protease activity is present. EP ~ 1?.91-~ discloses the use of site-specific mutagenesis to inactivate KEG prot~e processing sites in a protein. KEG
protease processing sites are inactivated by deleting, adding or substituting residues to alter :~rg-Arg, Arg-Lys, and Lys-__.~rg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEY' cleavage.
and conversion of erg-Lys or Lys-erg to Lvs-Lys represents a conservative and preferred approach to inactivatin°_- KE.~? sites.
The present invention further relates to PTP polypeptides including substrate trapping mutant PTPs. and in particular to methods for producing recombinant pTP polypeptides by culturing host cells containing PTP expression constructs.
and to isolated recombinant PTP polypeptides. The polvpeptides and nucleic acids of the present invention are preferably provided is an isolated form. and in certain preferred embodiments are purified to homogeneity. The terms "fra~nent.~ ''derivative'' and -=~~og" when referring to PTP poiypeptides or fusion proteins. including substrate trapping mutant PTPs. refers to any PTP polypeptide or fusion protein that retains essentially the same biological function or activity as such polypeptide. Thus-an analog includes a proprotein which c :n be activated by cleavage of the proprotein portion to produce an active PTP polypeptide. The polypeptides of the present ?4 invention may be recombinant polvpeptides or synthetic polypeptides. and are preferably recombinant polvpeptides.
;~ fira~ent derivative or analog of a PTP polvpeptide or fusion protein.
including substrate trapping mutant PTPs. may be (l) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and such substituted amino acid residue may or may not be one encoded by the genetic code. or (ii) one in which one or more of the amino acid residues includes a substituent soup. or (iii) one in which the PTP polypeptide is fused with another compound. such as a compound to increase the half life of the polvpeptide (e.o , polyethylene glycol), or (iv) one in which additional amino acids are fused to the PTP polypeptide. including amino acids that are employed for purification of the PTP polypeptide or a proprotein sequence. Such fragments.
derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
1~ The polypeptides of the present invention include PTP polypeptides and fusion proteins having amino acid sequences that are identical or similar to PTP
sequences known in the art. For e:cample by way of illustration and not limitation- the human PTP polypeptides (including substrate trapping mutant PTPs) referred to below in the E.~camples are contemplated for use according to the instant invention.
as are polypeptides having at least 70% similarity (preferably 70% identity), more preferably 90°,'° similarity (more preferably 90% identity) and still more preferably 95% similarity (still more preferably 9~°,% identity) to the polypeptides described in references cited herein and in the E-'tamPles and to portions of such polypeptides. wherein such portions of a PTP polypeptide generally contain at least 30 amino acids and more preferably at 5 least ~ 0 amino acids.
As knowm in the art "similarity" between two polypeptides is determined by comparins the amino acid sequence and conserved amino acid substitutes thereto of the polvpeptide -to the sequence of a second polvpeptide (e.o . using GE~1EWORKS.
:~li~ or the BL:~ST algorithm. as described abovel. Fra~nents or portions of the ;0 polypeptides of the present invention may be employed for producing the corresponding ?5 full-length poiypeptide by peptide synthesis; therefore. the fragments may be employed as intermediates for producing the full-length polvpeptides. Fragments or portions of the nucleic acids of the present invention may be used to synthesize full-length nucleic acids of the present invennon.
The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For e:cample, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated.
but the same nucleic acid or polypeptide, separated from some or all of the co-e:cisting materials in the natural system, is isolated. Such nucleic acid could be part of a vector and/or such nucleic acid or polvpeptide could be part of a composition. and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. The term "gene' means the se~nent of DNA involved in producing a polypeptide chain: it includes regions preceding and following the coding region "leader and trailer' as well as intervening sequences (introns) between individual 1~ coding segments (e:cons).
As described herein. the invention provides a fusion protein comprising a polvpeptide fused to a substrate trapping mutant PTP in which the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but ?p which results in a reduction in Kcal to less than 1 pe: minute, and in which at least one wildtype protein tyrosine phosphatase tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated. Such PTP fusion proteins are encoded by nucleic acids have the substrate trapping mutant PTP coding sequence fused in frame to an additional coding sequence to provide for e:cpression of a PTP polypeptide sequence fused to an additional functional or non-functional polvpeptide sequence that permits.
for e:campie by way of illustration and not Limitation. detection. isolation and/or purification of the PTP fusion protein. Such PTP fusion proteins may permit detection.
isolation and/or purification of the PTP fusion protein by protein-protein ai~nity. metal affinity or charge atFnitv-based polypeptide purincation. or by specific protease cleavage of a fusion protein containing a fusion sequence that is cleavable by a protease such that the PTP polypeptide is separable from the fusion protein.
Thus. PTP fusion proteins may comprise affinity tag polypeptide sequences. which :efers to polypeptides or peptides added to PTP to facilitate detection and isolation of the PTP via a specific affinity interaction with a ligand.
The ligand may be any molecule. receptor, counterreceptor, antibody or the like with which the a~niry tag may interact through a specific binding interaction as provided herein. Such peptides include. for e:cample, poly-His or the antigenic identification peptides described in U.S. Patent No. ~,011.9I? and in Hopp et al., (1988 BiolTechnolo~
6:1204), or the ~'RESST" epitope tag (Invitrogen, Carisbad, CA). The affinity sequene~ may be a he:ca-histidine tag as supplied. for e:cample, by a pBAD~His (Invitrogen) or a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host or. for e:cample, the af~lnity sequence may be a hemagglutinin (HA) tag when a mammalian host e.o., COS-7 cells, is used.
1 ~ The HA tag corresponds to an antibody defined epitope derived from the influenza hemagglutinin protein (Wilson et al.. 198- Cell 3 i :767).
PTP fusion proteins may further comprise immunoglobulin constant region polypeptides added to PTP to facilitate detection, isolation andlor localization of PTP: The immunoglobulin constant region polvpeptide preferably is fused to the C-terminus of a PTP polypeptide. General preparation of fusion proteins comprising heterologous polvpeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described_ e.o , by Ashkenazi et al. (PV:~S
v;Srl 88: I0~35. 1991) and Byre et al. (Nature 3-~-1:677, 1990). A gene fusion encoding the PTP:Fc fusion protein is inserted into an appropriate e:cpression vector. In certain ?5 embodiments of the invention. PTP:Fc fusion proteins may be allowed to assemble much like antibody molecules. whereupon interchain disulfide bonds form between Fc polvpeptides. yielding dimeric PTP fusion proteins.
. PTP fusion proteins having Specific binding affinities for pre-selected antigens by virtue of fusion polypeptides comprising immunoglobulin V-re?ion domains encoded by DNA sequences -linked in-frame to sequences encoding PTP
are ''7 also within the scope of the invention. including variants and fragments thereof as provided herein. General strategies for the construction of fusion proteins having immunoglobulin V-region fusion polvpeptides are disclosed. for example, in EP
0318664; U.S. 6,132,406; U.S. 6,091,6 I3; and U.S. ~.-X76.786.
6 The nucleic acid of the present invention may also encode a fusion protein comprising a PTP polypeptide fused to other polypeptides having desirable affinity properties. for example an enzyme such as glutathione-S-transferase.
As another example, PTP fusion proteins may also comprise a PTP polypeptide fused to a Staphylococcus aurezrs protein A polypeptide; protein :~ encoding nucleic acids and their use in constructing fusion proteins having affinity for immunoglobulin constant regions are disclosed generally, for example, in U.S. Patent 5.100,788. Other useful affinity polypetides for construction of PTP fusion proteins may include streptavidin fusion proteins, as disclosed. for example. in WO 89/03422; U.S. ~,-X89>>28;
U.S.
6,672,691; WO 93/24631; U.S. 6,168,049; U.S. 6?72:~5~ and elsewhere, and avidin fusion proteins (see, e.j . EP 611,747). r1s provided herein and in the cited references, PTP polypeptide sequences. including substrate trapping mutant PTPs, may be fused to fusion polypeptide sequences that may be full length fusion polypeptides and that may alternatively be variants or fragments thereof.
The present invention also contemplates PTP fusion proteins that contain polypeptide sequences that direct the fusion protein to the cell nucleus. to reside in the lumen of the endoplasmic reticulum (ER), to be secreted from a cell via the classical ER-Golgi secretory pathway (see. e.o . von Heijne, J. ~Llembrane Biol. 11 ~
:195-201, 1990), to be incorporated into the plasma membrane, to associate with a specific evtoplasmic component including the cvtoplasmic domain of a transmembrane cell ?5 surface receptor or to be directed to a particular subcellular location by any of a variety of known intracellular protein sorring mechanisms with which those skilled in the art will be familiar (See. e.o.. Rothman. .Vature X72:66-63. 1994. :~drani et al..
1998 .I.
Biol: C~rem. '_'73:10 17. and references cited therein.). Accordingly. these and related embodiments are ;.ncompassed by the instant compositions and methods directed to ~s targeting a polypeptide of interest to a predefined intracellular. membrane or extracellular localization.
The present invention also relates to vectors and to constructs that include nucleic acids of the present invention. and in particular to "recombinant expression constructs' that include any nucleic acids encoding PTP
polypeptides according to the invention as provided above; to host cells which are genetically engineered with vectors and/or constructs of the invention and to the production of PTP
polypeptides and fusion proteins of the invention. or fragments or variants thereof. by recombinant techniques. PTP proteins can be expressed in mammalian cells.
yeast bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RIVAs derived from the DNA constructs of the present invention. appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described. for example. by Sambrook, et al.; :Llolecular Cloning: <~ Laboratory Vlarruah Second Edition. Cold Spring Harbor.
New York, (1989).
Generally. recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g.. the ampicillin resistance gene of E. coli gad S cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor. acid phosphatase, or heat shock proteins_ among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences.
Optionally. the heterologous sequence can encode a fusion protein including an N-terminal ?5 identification peptide imparting desired characteristics. e.o .
stabilization or simplified purification of expressed recombinant product.
L: seful expression constructs for bacterial use are constructed by inserting into an expression vector a structural DNh sequence encoding a desired protein toge~.her with suitable translation initiation and termination sisals in operable ~0 reading phase with a functional promoter. The construct may comprise one or more ?9 phenotypic selectable markers and an origin of replication to ensure maintenance of the vector construct and. if desirable. to provide amplification within the host.
Suitable prokaryotic hosts for transformation include E. coll. Bacillus subrilis, Salmonella typhimurium and various species within the genera Pseudomonas. Streptomyces.
and Staphylococcus. although others may also be employed as a matter of choice.
:any other plasmid or vector may be used as long as they are replicable and viable in the host As a representative but nonlimiting example. useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR~2? (ATCC 3701'x. Such commercial vectors include. for e,t~ple, p~?~;_; (pharmacia Fine Chemicals. Uppsala. Sweden) and GEM1 (Promega Biotec, Madison. Wisconsin. USA). These PBR:p~ ~~backbone'' sections are combined with an appropnate promoter and the structural sequence to be expressed.
1 j Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density. the selected promoter, if it is a regulated promoter as provided herein, is induced by appropriate means (e.g.. temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically hariested by centrifugation, disrupted by physical or chemical means, and the resulting c:ude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication. mechanical disruption- or use of cell lysing agents; such methods are well how to those skilled in the art.
Thus. for example. the nucleic acids of the invention as provided herein may be included in anv one of a variety of expression vector constructs as a recombinant expression construct for expressing a PTP polypeptide. Such vectors and constructs include chromosomal. nonchromosomal and synthetic DNA sequences. e-,g..
derivatives of SV -10: bacterial plasmids: phage DN.-~: baculovirus: vesst plasmids:
vectors derived from combinations of plasmids and phage DNA.. viral DNA. such as ;0 vaccinia. adenovirus. fowl pox virus. and pseudorabies. However. any other vector may be used for preparation of a recombinant expression construct as long as it is replicable and viable in the host.
The appropriate DNA sequences) may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate 5 restriction endonuciease sites) by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification. far enzymatic reactions involving DNA ligase. DNA polymezase, restriction endonucleases and the like.
and various separation techniques are those known and commonly employed by those skilled is the art. A number of standard techniques are described, for example. in IO Ausubel et al. (1993 Current Protocols in Llolecular Biology, Greene Publ.
P.ssoc. Inc.
& John Wiiey 3t Sons. Inc., Boston. ~); S~brook et al. (1989 :Ltolecular Cloning, Second Ed., Cold Spring Harbor Laboratory. Piainview. iV~; Maniatis et al.
(1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, ~; and elsewhere.
The DNA sequence in the expression vector is operatively linked to at l~ least one appropriate expression control sequences (e.g.. a promoter or a regulated promoter) to direct mRNA synthesis. Representative e.~camples of such expression control sequences include LTR or SV.~O promoter. the E cvli lac or trp. the phage lambda. P~ promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. Promoter regions can be selected from 20 _any desired gene using C~-~T (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK? i2-8 and pCVi7.
Particular named bacterial promoters include lack lacZ. T~, T7, apt, lambda P~, P:. ~d nP' Eukaryotic promoters include CVIV immediate early. HSV thymidine kinase, early and late SV40, LTRs from retrovirus. and mouse metallothionein-I. Selecnon of the appropriate vector and promoter is well within the level of ordinary skill in the art, ~d preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter or regulated promoter operably linked to a nucleic acid encoding a PTP polvpeptide is described herein.
:~s noted'above. in certain embodiments the vector may be a viral vector ;0 such as a retroviral vector. For example. retroviruses from which the retroviral plasmid ;l vectors may be derived include. but are not limited to, Moloney Marine Leukemia Virus. spleen necrosis virus. retroviruses such as Rous Sarcoma Virus. Harvey Sarcoma virus. avian leukosis virus. gibbon ape leukemia virus. human immunodeficiency virus.
adenovirus. Myeioproliferative Sarcoma V leas, and mammary tumor virus.
The viral vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV=10 promoter, and the human cytomegalovirus (CAN) promoter described in Miller. et al..
Biotechniques % :980-990 ( 1989), or any other promoter (e.o , cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III. and (3-actin promoters). Other viral promoters which may be employed include. but are not limited to, adenovitus promoters. thymidine kinase (TK) promoters. and B 19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachinss contained herein. and may be from among either regulated promoters or promoters as described above.
1 ~ The retroviral plasmid vector is employed to transduce pac.~caging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to. the PE~O1, P.A l 17. v~-2, yr-AUI. PAl?, T19-1~Y, VT-19-17-H2, wCRE, y~CRIP. GP+E-86. GPlenvAml'_'. and D~~1 cell lines as described in Miller. F~uman Gene Tnerapv, 1:~-l~ (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include. but are not limited to, eiectroporation. the use of liposomes, and calcium phosphate precipitation. In one alternative. the retroviral plasmid vector may be encapsulated into a liposome. or coupled to a lipid. and then administered to a host.
The producer cell line Qenerates infectious retroviral vector particles which include the nucleic acid sequences) encoding the PTP polypeptides or fusion proteins. Such retroviral vector particles then may be employed- to transduce eukarvotic cells. either in vitro or in vivo. The transduced eukaryotic cells will e~cpress the nucleic acid sequencet s1 encoding the PTP polypeptide or fusion protein.
Eukaryotic cells which may be transduced include. but are not limited to.
embryonic stem cells, embryonic carcinoma cells. as well as hematopoietic stem cells, hepatocytes.
fibroblasu. myoblasu, keratinocytes. endothelial cells. bronchial epithelial cells and various other culture-adapted cell lines.
As another example of an embodiment of the invention in which a viral vector is used to prepare the recombinant PTP expression construct in one preferred embodiment. host cells transduced by a recombinant viral construct directing the expression of PTP polypeptides or fusion proteins may produce viral particles containing expressed PTP polvpeptides or fusion proteins that are derived from portions of a host cell membrane incorporated by the viral particles during viral budding. In another preferred embodiment, PTP encoding nucleic acid sequences are cloned into a baculovirus shuttle vector, which is then recombined with a baculovirus to generate a recombinant baculovirus expression constrict that is used to infect. for example. S~
host cells. as described in Baculovirus Expression Protocols. ~l~lethods in .Llolecular Biology Vol. 39. Christopher D. Richardson, Editor, Human Press, Totowa, NJ, 1995;
piwnica Worms, ''E.Ypression of Proteins in Insect Cells Using Baculoviral Vectors;' Section II in Chapter 16 in: Short Protocols in :Llolecular Biology. 2"d Ed., Ausubel et al., eds.. John Wilev & Sons. New York_ New York, 199?, pages 16-32 to 16-~8.
In another aspect the present invention relates to host cells containing the above described recombinant PTP expression consmicu. Host cells are genetically ?0 engineered (transduced, transformed or transfected) with the vectors and/or expression constructs of this invention which may be. for example, a cloning vector. a shuttle vector or an expression construct The vector or construct may be, for e:cample. in the form of a plasmid. a viral particle. a phage. etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating ~5 promoters. selecting transformanu or amplifying particular genes such as genes encoding PTP polypeptides or PTP fusion proteins. The culture conditions for particular host cells selected for zxpression. such as temperature. pH and the like. will be readily apparent ~o the ordinarily skilled artisan.
The host cell can be a higher eukaryotic cell. such as a mammalian ce! 1.
~0 or a lower eukar<<otic cell. such as a yeast cell. or the host cell can be a prokaryotic cell.

>;
such as a bacterial cell. Representative examples of appropriate host cells according to the present invention include, but need not be limited to, bacterial cells, such as E. coli.
Streptomyces. Salmonella typhimurium; fungal cells. such as yeast: insect cells. such as Drosophila S2 and Spodoptera SfP; animal cells. such as CHO> COS or ?9S cells;
adenoviruses; plant cells. or any suitable cell already adapted to in vitro propagadon or so established de novo. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
Various mammalian cell culture systems can also be employed to express recombinant protein. The invention is therefore directed in part to a method of producing a recombinant substrate trapping mutant protein tyrosine phosphatase. by culturing a host cell comprising a recombinant expression construct that comprises at least one promote: operably linked to a nucleic acid sequence encoding a substrate trapping mutant protein tyrosine phosphatase in which the wildtype protein tyrosine phosphatase catalytic domain invariant aspattate residue is replaced with an amino acid 1 ~ which does not cause si~ificant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute. and in which at least one wildtype protein tyrosine phosphatase tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated. In certain embodiments, the promoter may be a regulated promoter as provided he:ein. for example a tetracylc:ne-repressible promoter.
In certain embodiments the recombinant expression construct is a recombinant viral expression construct as provided herein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts. described by Gluzman, Cell ?3:175 (1981), and other cell lines capable of e.~cpressing a compatible vector. for example, the C1?7, 3T3. CHO. HeLa and BHK cell lines. Mammalian expression vectors will comprise an ?5 origin of replication. a suitable promoter and enhancer. and also any necessary ribosome binding sites. polvadenvlation site. splice donor and acceptor sites.
transcriptional termination sequences. 'and ~' l3aukin Q nontranscribed sequences. for example as desc:ibed he:ein regarding the preparation of PTP zxpression constructs. DVS
sequences derived from the SV:~O splice. and polyadenylation sites may be used to ;0 provide the required nontranscribed genetic dements. Introduction of the construct into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar. including but not limited to. for eaampie, calcium phosphate transfection. DE.aE-De:ctran mediated transfection. or eiectroporation (Davis et aL.
1986 Basic _Llerhods in :Llolecular Biolo~)-Identification of nucleic acid molecules for use as antisense agents.
which includes antisense oligonucleotides and ribozymes specific for nucleic acid sequences encoding PTPs (including subsu'ate ~ppmg mutant PTPs) or variants or dents thereof and of DNA oligonucleotides encoding PTP genes (including substrate trapping mutant PTPs) for targeted delivery for genetic therapy.
involve methods well known in the art- For e:cample, the desirable properties. lengths and other characteristics of such oligonucieotides are well known. In certain preferred embodiments such an antisense oligonucleotide comprises at least 1 ~
consecutive nucleotides complementary to an isolated nucleic acid molecule encoding a substrate trapping mutant PTP as provided herein. Antisense oligonucleotides are typically designed to resist degradation by endogenous nucieolytic enzymes by using such linkages as: phosphorothioate. methylphosphonate, sulfone, sulfate, ketyl.
phosphorodithioate, phosphorattudate, phosphate esters. and other such linkages (see.
e.g., Agrwal et al., Tetrehedron Lett. ?8:559-X542 (1987); Miller et al., .I.
:gym. Chem.
Soc: 93:6657-6665 (1971): Stec et al.. Tetrehedron Lett. ?6:?191-2194 (1985);
Moody ZO et al., ~Vucl. .-Icidr Res. I ?:769-~78~ ( 1989); Uzaanski et al., ~Vucl.
.=lcids Res. ( 1989);
Letsinger et al., Tetrahedron X0:137-1=~S (1984); Eckstein. dnnu. Rev.
Biochem.
5:367-:~02 (1985); Eckstein- Trends Biol. Sci. I-x:97-100 (1989); Stein In:
Oligodeoryrrucleotides. ,~ntisense Inhibitors of Gene Expression. Cohere, Ed.
Macmillan Press, London. pp. 97-117 (1989); Japer et al.. Biochemistry 2; :7237-7246 (1988)).
Antisense nucleotides are oligonucleotides that bind in a sequence-specific manner to nucleic acids_ such as mR:~t~ or DNA. When bound to mR'~A
that has complementary sequences. antisense prevents translation of the mR.'~1A
(see. e.,;..
U.S. Patent ~lo. p_168,0>; to ~ltman et al.: U.S. Patent Rio. 5.190.951 to Inouve. U.S_ Patent ~o. 5.1>j.917 to Burch: LT.S. Patent Vo. 5.087.617 to Smith and Clusel et al.
30 (1990 .Vucl. .~c:dr Res. 5!:5-10=-~-X11- which describes dumbbell antisense ;5 oligonucleotides). Triplex molecules refer to single DNA strands that bind duplex DNA forming a colinear triplex molecule, thereby preventing transcription (see, e.,;..
U.S: Patent No. x.176.996 to Hogan et al., which describes methods for making synthetic oligonucleotides that bind to target sites on duplex DNA).
According to this embodiment of the invention, particularly useful antisense nucleotides and triple:c molecules are molecules that are complementary to or bind the sense strand of DNA or mRNA that encodes a PTP polypeptide (including substrate trapping mutant PTPs), such that inhibition of translation of mR:'~1A encoding the PTP polypeptide is erected.
A ribozyme is an RNA molecule that specifically cleaves RNA
substrates, such as mR'VA. resulting m specific inhibition or interference with cellular gene expression. There are at least five known classes of ribozymes involved in the cleavage andlor ligation of R.~A chains. Ribozymes can be targeted to any R:~iA
transcript and can catalytically cleave such transcripts (see, e.g., U.S.
Patent No.
S.?72,262; U.S. Patent No. ~.1=x.019; and U.S. Patent Nos. ~,168,05~, x,180,818, 5,116,742 and ~.093,?46 to Cech et al.). According to certain embodiments of the invention. any such PTP (including substrate trapptag mutant PTP) mR.:~'~-specific ribozyme, or a nucleic acid encoding such a ribozyr.:e. may be delivered to a host cell to effect inhibition of PTP gene expression. Ribozymes. and the like may therefore be delivered to the host cells by DNA encoding the ribozyme linked to a eukaryotic promoter. such as a eukaryotic viral promoter, such that upon introduction into the nucleus. the ribozyme will be directly transcribed.
The expressed recombinant PTP polypeptides or fusion proteins (including substrate trapping mutant PTPs) may be useful in intact host cells;
in intact '_'S organelles such as cell membranes. intracellular vesicles or other cellular organelles: or in disrupted cell preparations including but not limited to cell homogenates or lysates.
microsomes. uni- and muitilamellar membrane vesicles or other preparations.
Alternatively. expressed recombinant PTP polypeptides or fusion proteins can be recovered and purified from recombinant cell cultures by methods including ammonium ~0 sulfate or ethanol precipitation. acid extraction. anion or canon exchange chromatography. phosphocellulose chromatography, hydrophobic interaction chromato°raphy, affinity chromato~aphy, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used- as necessary. in completing configuration of the mature protein. Finally. high performance liquid chromatography (HPLC) can be employed for final purification steps.
Turning to another aspect of the invention, there is provided a method of identifying a tryosine phosphorylated protein which is a substrate of a PTP. A
''sample"
as used herein refers to a biological sample containing at least one tyrosine phosphorylated protein. and may be provided by obtaining a blood sample.
biopsy specimen. tissue e:cplant. organ culture or any other tissue or cell preparation from a subject or a biological source. A sample may further refer to a tissue or cell preparation in which the morphological integrity or physical state has been disrupted. for example, by dissection. dissociation. solubilization, fractionation- homogenization.
biochemical or chemical earaction, pulverization, lyophilization, sonication or any other means for 1~ processing a sample derived from a subject or biological source. In certain preferred embodiments, the sample is a cell lysate, and in certain particularly preferred embodiments the lysate is a detergent solubilized cell lysate from which insoluble components have been removed according to standard cell biology techniques.
The subject or biological source may be a human or non-human animal. a primar'-' cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromosomally inte~ated or episomal recombinant nucleic acid sequences. immortalized or immortalizable cell lines. somatic cell hybrid cell lines, differentiated or differentiatable cell lines. transformed cell lines and the like.
Optionally. in certain situations it may be desirable to treat cells in a biological sample with pervanadate as described herein. to enrich the sample in tyrosine phosphorylated proteins. Other means may also be employed to et~ect an increase in the population of tyrosine phosphorylated proteins present in the sample. including the use of a subject or biological source that is a cell line that has been transfected with at least one gene encoding a protein tyrosine lCinases. Additionally or alternatively. protein tyrosine ~0 phosphoryiation may be stimulated in subject or biological source cells using any one or more of a variety of well known methods and compositions known in the art to stimulate protein tyrosine kinase activity. These stimuli may include. without limitation. exposure of cells to cytokines. ~owth factors. hormones, peptides.
small molecule mediators or other agents that induce PTK-mediated protein tyrosine phosphorylation. Such agents may include, for example, interleukins, interferons.
human ~owth hormone. insulin and fibroblast ~owth factor (FGF), as well as other agents with which those having ordinary skill in the art will be familiar.
According to the subject invention. a sample comprising at least one tyrosine phosphorylated protein is combined with at least one substrate trapping mutant IO PTP as provided herein. under conditions and for a time sufficient to permit formation of a complex between the tyrosine phosphorylated protein and the substrate trapping mutant PTP. Suitable conditions for formation of such complexes are known in the art and can be readily determined based on teachings provided herein. including solution conditions and methods for detecting the presence of a complex. Next the presence or absence of a complex comprising the tyrosine phosphorylated protein and the substrate trapping mutant PTP is determined. wherein the presence of the complex indicates that the tyrosine phosphorylated protein is a substrate of the PTP with which it forms a complex.
Substrate trapping mutant PTPs that associate in complexes with tyrosine phosphorylated protein substrates may be identified by any of a variety of techniques laiown in the art for demonstrating an intermolecular interaction between a PTP and a PTP substrate as described above. for example, co-purification. co preoipitation. co-immunoprecipitation_ radiometric or fluorimetric assays, western immunoblot analyses. amity capture including affinity techniques such as solid-phase ~5 li~and-counteriisand sorbent techniques. amity chromatography and surface affinity plasmon resonance. and the like (see. e.o . LT.S. Patent ~Io. ~.3~2.660).
Determination of the presence of a PTP!substrate complex may employ antibodies. including monoclonal. polvcional. chimeric and single-.:hain antibodies. and the like.
that specifically bind'to the PTP or the tyrosine phosphorylated protein substrate.
Labeled ;0 PTPs and/or Labeled tyrosine phosphoryiated substrates can also be used to detect the presence of a comple:c. The PTP or phosphorylated protein can be labeled by covalently or non-covalently attaching a suitable reporter molecule or moiety.
for e:cample any of various enzymes. fluorescent materials, luminescent materials and radioactive materials. E:camples of suitable enzymes include. but are not limited to, horseradish pero:cidase, biotin. alkaline phosphatase, ~-~alactosidase and aceryicholinesterase. E:camples of suitable fluorescent materials include. but are not limited to, umbeiliferone. fluorescein, ffuorescein isothiocyanate. rhodamine.
dichlorotriazinylamine lluorescein. dansyl chloride and phycoerythrin.
Appropriate luminescent materials include luminol, and suitable radioactive materials include radioactive phosphorus [''P], iodine ['''~I or'3'I] or tritium [3H].
Using such approaches, representative comple:ces of PTP 1 B with p210 bcr:abl. of PTP-PEST with p 130". of TC-PTP with Shc (e.,;.. Tiganis et al..
1998 .Llol.
Cell. Biol. 18:1622-I6~~) and of PTPH1 with pp97/VCP may be readily identified by western immunoblot analysis as described below. These associations may be observed.
1 S for e.~cample, in lysates from several cell lines and in transfected cells. indicating that p210 bcr:abl, pi30'~, Shc and VCP represent major physiologically relevant substrates for PTP1B, PTP-PEST. TC-PTP and PTPH1, respectively. The compositions and methods of the present invention. which may be used. as e:cemplified herein.
to identify specific tyrosine phosphoryiated substrates for PTPIB. PTP-PEST and PTPHI, are generally applicable to any member of the PTP family, including but not limited to TC-PTP, PTP,j, MKP-I. DEP-I. PTP~. SHP?, PTP-PEZ, PTP-v~Gl. LC-PTP, CD45, L.4R and PTPY10.
In certain embodiments of this aspect of the invention. the sample may comprise a cell that naturally e:cpresses the tyrosine phosphorylated protein that is a ''S PTP substrate. while in certain other embodiments the sample may comprise a cell that has been transfected ~.vith one or more nucleic acid molecules encoding the substrate protein. For e:cample: the sample may comprise a cell or population of cells that has been transfected with a nucleic acid library such as a cDNA library that contains at least one nucleic acid molecule encoding a substrate protein. Any tyrosine phosphorylated protein is suitable as a potential substrate in the present invention.
Tyrosine phosphorylated proteins are well known in the art. Specific examples of appropriate substrates include. without limitation. p 1 ~0'u, pp97/VCP. the EGF receptor.
p210 bcr:abl. ~fAP kinase. Shc and the insulin receptor. Of particular interest are tyrosine phosphorylated proteins that have been implicated in a mammalian disease or disorder.
According to the present invention_ substrates may include full length tyrosine phosphoryiated proteins and polypeptides as well as fra~nents (e.o , portions), derivatives or analogs thereof that can be phosphorylated at a tyrosine residue. Such fragments. derivatives and analogs include any PTP substrate polypeptide that retains at least the biological function of interacting with a PTP as provided herein.
for example by forming a complex with a PTP. :~ fragment. derivative or analog of a PTP
substrate polypeptide: including substrates that are fusion proteins, may be (l) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in 1~ which one or more of the amino acid residues includes a substituent soup, or (iii) one in which the substrate polvpeptide is fusers with another compound. such as a compound to increase the half life of the polypeptide (e.g.. polyethylene glycol) or a detectable moiety such as a reporter molecule. or (iv) one in which additional amino acids are fused to the substrate polypeptide. including amino acids that are employed for purification of the substrate polypeptide or a proprotein sequence. Such fragments.
derivatives and analogs are deemed to be within the scope of those skilled in the art.
The subject invention also contemplates certain embodiments wherein the substrate trapping mutant PTP that is combined with the sample) is a mutant PTP
that is expressed by a cell, including embodiments wherein the cell has been transfected with one or more nucleic acid molecules encoding the mutant PTP. Thus. the method of identifying a tyrosine phosphory lated protein which is a substrate of a PTP may include in certain embodiments combining a sample comprising a tyrosine phosphorylated protein with a mutant PTP wherein the sample comprises a c~11 e:cpressing either or both of the tyrosine phosphorylated protein and the mutant PTP.

.~0 Optionally. the cell may be transfected with nucleic acids encoding either or both of the tyrosine phosphorylated protein and the mutant PTP.
In another aspect_ the invention provides methods of identifying an agent that alters the interaction between a PTP and a tyrosine phosphorylated protein that is a substrate of the PTP. through the use of screening assays that detect the ability of a candidate agent to alter (l. e.. increase or decrease) such interaction. The interaction between the PTP and its substrate may be determined enzymatically, for e:cample by detecting catalytic substrate dephosphorylation. Alternatively. the interaction between the PTP (including a substrate trapping mutant PTP) and its substrate may be determined as a binding interaction. and in preferred embodiments such interaction is manifested as detection of a comple:c formed by PTP-substrate binding, according to criteria described herein. Agents identified according to these methods may be aaonists (e.g., agents that enhance or increase the activity of the wildrype PTP) or antagonists (e.o , agents that inhibit or decrease the activity of the wildrype PTP) of PTP activity.
1 ~ Agents may be identified from among naturally occurring or non-naturally occurring compounds. including synthetic small molecules as described below.
In certain embodimenu. wherein the screening assay is directed to PTP
catalytic activity. the tyrosine phosphorylated protein that is a substrate of the PTP can be identined as described above. which method features the use of a novel substrate trapping mutant PTP as disclosed herein. Accordingly, a PTP and a tyrosine phosphoryiated substrate are combined in the absence and in the presence of a candidate agent where the substrate has first been identified as described above using a substrate trapping mutant PTP. The PTP and the substrate are combined under conditions permissive for the detectable dephosphorylation of the subsnate to occur.
?S Anv suitable method may be used to detect phosphoprotein dephosphorylation: such methods are well known in the art and include. without limitation. detection of substrate catalysis by one or more of. e.o .
radiometric.
tluorimetric. densitometric. spectrophotometric. chromatoQraphic_ eiec:rophoretic.
colorimetric or biometric assays. T'ne level of dephosphorylatiun of the substrate in the absence of the agent is compared to the level ut' dephosphorylation of the substrate in =~ 1 the presence of the agent, such that a difference in the level of substrate dephosphorylation (e.g., a statistically significant increase or decrease) indicates the anent alters the interaction between the protein tyrosine phosphatase and the substrate.
For instance. an znzymatic activity assay utilizing a wildtype PTP can be carried out in the absence and presence of a candidate agent. Enzymatic activity assays known in the art include. for e:cample, PTP activity assays using a tyrosine phosphorylated 'zP-labeled substrate as described in Flint et al. ( 1993 E
L1B0 f IZ:I937-19~. A decrease in the PTP enzymatic acnnty tn the presence of the candidate agent indicates that the agent inhibits the interaction between the PTP and its substrate. Conversely, an increase in PTP enzymatic acnvity tn the presence of the agent indicates that the agent enhances the interaction between the PTP and its substrate.
In certain other embodiments, wherein the screening assay is directed to identifying an agent capable of altering a substrate trapping mutant PTP-substrate 1 ~ binding interaction.. the substrate trapping mutant PTP (as described herein) and a tyrosine phosphoryiated substrate are combined in the absence and in the presence of a candidate agent under conditions and for a time su~cient to permit formation of a comple:c between the tyrosine phosphoryiated protein and the substrate trapping mutant PTP. thereby producing a combination. The formation of a comple:c comprising the tyrosine phosphorylated protein and the substrate trapping mutant protein tyrosine phosphatase in the combination is ne:ct determined (as also provided herein), wherein a difference between the level of comple:c formation (e.g.. a statistically significant difference) in the absence and in the presence of the agent indicates that the agent alters (i.e.. increases or decreases) the interaction between the protein tyrosine phosphatase ?5 and the substrate. Altemativelv. a competitive bindins assay can be carried out utilizing the substrate trapping mutant'PTP in the absence and presence of a candidate agent.
Competitive binding assays known in the art include. for e~cample. LT.S.
Patent Rio. ~.~ ~=.660. which desc:ibes methods suitable for use according to these embodiments of the present invention. ~ decrease in the e:ctent of PTP-substrate ~0 binding in the presence of the anent to be tested indicates that the went inhibits the 4'' interaction between the PTP and iu substrate. Conversely, an increase in the extent of binding in the presence of the agent to be tested indicates that the agent enhances the interaction between the PTP and iu substrate.
Candidate menu for use in a method of screening for an agent that alters the interaction between a PTP and its tyrosine phosphorylated protein substrate according to the present invention may be provided as "libraries'' or collections of compounds. compositions or molecules. Candidate agents that may interact with one or more PTPs (including agents that interact with a substrate trapping mutant PTP
as provided herein) may include members of phosphotvrosyl peptide libraries as described in Songyang et al. (1995 Vatz~re 373:>>6->j9; 1993 Cell 7?:767-778) that bind to the PTP. Peptides identified from such peptide libraries can then be assessed to determine whether tyrosine phosphorylated proteins containing these peptides e:cist in nature.
Alternatively, libraries of candidate molecules to be screened may typically include compounds known in the art as °small molecules" and having molecular weighu less 1~ than 10' daltons. preferably less than 10' daltons and still more preferably less than 10' daltons. For e:cample, members of a library of test compounds can be administered to a plurality of samples, each containing at least one substrate trapping mutant PTP and at least one tyrosine phosphorylated protein that is a substrate of the PTP as provided herein, and then assayed for their ability to enhance cr inhibit mutant PTP
binding to ZO the substrate. Compounds so identified as capable of altering PTP-substrate interaction (e.g.. binding andlor substrate phosphotyrosine dephosphorylation) are valuable for therapeutic and/or dia~ostic purposes, since they permit treatment and/or detection of diseases associated with PTP activity. Such compounds are also valuable in research directed to molecular si~aling mechanisms that involve PTPs, and to refinements in 25 the discovery and development of future compounds e:chibiting heater specificity.
Candidate agents further may be provided as members of a combinatorial library. which o_ refe:ablv includes synthetic agents prepared according to a plurality of predetermined chemical reactions performed in a plurality of reaction vessels.
For e:cample. various startins compounds may be prepared employing one or more of solid-30 phase synthesis. recorded random mi.~c methodologies and recorded reaction split techniques that permit a liven constituent to traceably undergo a plurality of permutations and/or combinations of reaction conditions. The resulting products comprise a library that can be screened followed by iterative selection and synthesis procedures. such as a synthetic combinatorial library of peptides (see e.,;..
PCT~'LrS9Ii0869~. PCTIUS9110~666. which are hereby incorporated by reference in their entireties) or other compositions that may include small molecules as provided herein (see e.o , PCTlLJS94/085~2. EP 0'rl~ U.S. 5.798,035. U.S. 5,789,172, U.S.
5,751,629, which are hereby incorporated by reference in their entireties).
Those having ordinary skill in the art will appreciate that a diverse assortment of such libraries may be prepared according to established procedures, and tested using substrate trapping mutant PTPs according to the present disclosure.
The invention also pertains to a method of reducing the activity of a tyrosine phosphorylated protein. comprising administering to a subject a substrate trapping mutant PTP in which (i) the wildtype PTP catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 miri') (e.o . an alanine residue), and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated.
whereby interaction of the substrate trapping mutant protein tyrosine phosphatase with the tyrosine phosphorylated protein reduces the activity of the tyrosine phosphorylated protein. In certain preferred embodiments. the tyrosine phosphorylated protein is VCP, p130"~, the EGF receptor. p210 ber:abl. ~ Vie- Shc or the insulin receptor. In certain other preferred embodiments. the protein tyrosine phosphatase is PTP 1 B, PTP
PEST. PTP;~, VIKP-l. DEP-1, PTPu. PTP.'C1. PTPYIO. SHP''. PTP-PEZ. PTP-VtEGl.
35 LC-PTP, TC-PTP, CD45. L~R or PTPHI.
Without wishing to be bound by theory. such a mutant PTP may reduce the activity of the corresponding wildtype PTP by forming a complex with the tyrosine phosphorylated protein substrate of the wildtype PTP. thereby rendering the substrate unavailable for catalytic deahosphorylation by the wildrype enzyme. The substrate trapping mutant PTP thus binds to the phosphoprotein substrate without dephosphorylating it (or catalyzing dephosphorylation at a neatly reduced rate). thereby blocking the activity of the dephosphorylated protein substrate and reducing its downstream effects. :~s used herein. "reducing" includes both reduction and complete abolishment of one or more activities or functions of the phosphorylated protein substrate.
In one aspect of the method of reducing the activity of a tyrosine phosphoryiated protein. a method is provided for reducing the transforming effects of at least one oncogene associated with phosphorylation of p130'~, a substrate of PTP-PEST. The method Generally comprises administering to a subject a substrate trapping mutant PTP-PEST in which the wildtvpe PTP catalytic domain invariant aspartate residue is replaced with an alanine residue, and in which at least one wildtvpe tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated.
Whereas wildtype PTP-PEST binds and dephosphorylates the substrate p130"5, thereby negatively regulating this substrate's downstream biological effects, the subject 1~ invention substrate trapping PTP-PEST mutants bind but cannot dephosphorylate p 1;0"~ (or do so at a ~eatiy reduced rate). According to the non-limiting theory disclosed above. the substrate is thus sequestered in the complex with the substrate trapping PTP-PEST and cannot e.~cert its downstream effects. In certain embodiments of this method, the oncogene may be one of v-crk, v-5re or c-Ha ras.
Similarly. the invention relates to a method of reducing the formation of signaling complexes associated with p130"', particularly those signaling complexes which induce mitogenic pathways, comprising administering to a mammal substrate trapping mutant PTP-PEST as provided above. The PTP binds to andlor dephosphorylates p1~0'~. thereby negatively regulating the downsue3m effects of ~5 p130'_- and reducing the formation of signaling complexes associated with p1~0"s. ~s another example. in certain embodiments the invention relates to regulation of the cell cycle by the PTPHl substrate pp97/VCP. wherein a substrate trapping mutant as provided herein ( l. e.. a double mutant that is caralytically attenuated and in which a wildtype tyrosine has been replacedl can alter the interaction between PTPHI
and VCP.

~s provided herein. the substrate trapping mutant PTPs of the present invention may be useful in virtually any situation where biological regulation involving PTP-regulated signal transduction is involved, for example. in place of. or in addition to, a corresponding wildtvpe PTP. T'ne advantages of such utility of the subject invention lie in the ability of a substrate trapping mutant PTP to mimic the function of its corresponding wildtype enzyme. e.o - to impair the biological signaling activity of a tyrosine phosphorylated substraxe subsequent to dephosphorylation mediated by wildtype PTP, without inducing the harmful cytotoxic effects commonly observed when wildtype PTP is administered and/or overexpressed. Thus. the invention also pertains to a method of reducing the cytotoxic effects associated with administration or overexpression of wild type PTPs. For example. CS mutants of VIKP-1 have been shown to have the same functional effect as wild type VIKP-I without induction of potentially harmful side effects. Thus. PTPs described herein. in which the wildtype PTP catalytic domain invariant aspartate residue is replaced with an amino acid which 1 ~ does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min') (e.g., an alanine residue), and in which at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated. can in many situations be substituted for a counterpart wildtype enzyme, where such a counterpart wildtype enzyme can specifically interact with the same substrate as the mutant PTP.
The substrate trapping mutant PTPs described herein may also be used therapeutically to alter (l. e.. increase or decrease) the activity of a tyrosine phosphorylated protein, such as by a gene therapy method in which a nucleic acid. for example. a recombinant expression construct as described above, encoding a substrate trapping mutant PTP (or a functional portion thereof) which retains the ability to bind to its tyrosine phosphorylated substrate. is introduced into a subject and is expressed. The mutant PTP replaces. either partially or totally. a corresponding host PTP
enzyme that is normally produced in the subject or may compete with the host PTP for binding to the substrate. For example. where a specinc tyrosine phosphorylated protein substrate may ~0 be implicated in a particular disease or disorder. at least one PTP capable of dephosphorylating the suspect substrate may be identified. A corresponding substrate trapping mutant PTP can be administered either directly or by gene therapy.
using the compositions and methods described herein. Such a mutant PTP may sequester the tyrosine phosphorylated substrate, thereby inhibiting or reducing the substrates role in the disease process. In a preferred embodiment, the substrate trapping mutant PTP of the present disclosure is administered in place of a corresponding wildtype enzyme. tn order to reduce the cytotoxic effects associated with overe:cpression of the wild type enzyme. Procedures for geae therapy are known in the art (see, e.g., U.S.
Patent No.
5,399.36) and can be modified by known methods known in order. to express the I O subject invention substrate trapping mutant PTPs.
The methods of the present invention are specifically e:cemplified herein with respect to the phosphatases PTPHl. PTPIB and PTP-PEST: however. it is understood that the invention is not limited to these specific PTPs but is applicable to all members of the PTP family. In order to identify potential substrates of PTPHI, I~ PTPIB and PTP-PEST. mutant (i.e., altered or substrate trapping) forms of PTPH1, PTP I B and PTP-PEST are generated as described herein that are catalytically attenuated but that retain the ability to bind substrates.
In certain embodiments. the invention relates in part to PTP 1 B(D 13 I r1), in which the aspartate residue at position 131 of wildtype PTPIB is repiaeed with 20 alanine. and in which fiuther a PTP tyrosine residue may optionally be replaced with a non-phosphorylatable residue. In certain other embodiments the invention relates to the phosphatase PTP-PEST(D 199A) and in certain other embodiments to PTP
PEST(C231 S), which in either case may further have a PTP tyrosine residue optionally replaced with a non-phosphorylatable residue. In particularly preferred embodiments 25 the invention relates to PTPH1(Y676F~'D8I I~).
As noted above. in certain embodiments the invention relates . to a substrate trappins mutant PTP-PEST. PTP-PEST is an 86 kDa cvtosolic PTP
(Chartist et al.. 199 Biochem. .I 308:~'_'~--~~~: den Hertog et al., 1992 Biochem.
Biophys. Res.
Commzrn. 18-~:1~'-Il-1==~9: Take!cawa et al.. 199? Biocnem. Biopnys. Res.
Commun. 189'.
30 1='_'~-1'_'30: Yang et al.. 1993 J. Biol. Chem. ?68:66=''-6623: Yang et a1._ l99? J. Biol.

~7 Chem. Z68: I 7 660) which is e:~pressed ubiquitously in mammalian tissues (Yi et al..
1991 Blood -8:?''2?-??~8), and which e:chibits hi°h specific activity when assayed in vitro using artificial tyrosine phosphorylated substrates (Garton and Tonks, 199 E:LIBO
.I. 13:3763-3771). PTP-PEST is subject to regulation via phosphorylation of Ser39 in vitro and in vivo. This modification is catalyzed by both protein kinase C
(PKC) and protein kinase A (PK~), and results in reduced PTP-PEST enzyme activity due to an increase in the Km for the dephosphorylation reaction catalyzed by this PTP
(Garton and Tonks, 1994 E:LIBO ,I. 13:3763-3771). Additional intracellular regulatory mechanisms may include PTP-PEST-mediated dephosphorylation of one or more cytosolic substrates of tyrosine kinases.
As disclosed herein and described in the Examples. the substrate specificities of PTP 1 B and of PTPHl may be characterized by methods that relate to PTP catalytic and/or binding interactions with substrate. e.o .
dephosphorylation and substrate trapping in vitro and in vivo. PTP1B (see. e.o.. Barford et al..
1994 Science 263:1397; Jia et al., 1996 Science ?68:1750 and PTPH1 (see. e.o . U.S. Patent Nos.
6.696,911 and 6,863,781) are well known in the art. The substrate trapping methods provided herein are generally applicable to any PTP by virtue of the invariant PTP
catalytic domain aspartate residue and the frequency of tyrosine in PTP amino acid sequences. and should therefore prove useful in delineating the substrate preferences of other PTP family members. In particular. the use of mutant catalvtically impaired PTPs to trap. and thereby isolate. potential substrates permits the identification of physiologically important substrates for individual PTPs, leading to improved understanding of the roles of these enzymes in revelation of cellular processes_ Furthermore. replacement of PTP tyrosine residues with amino acids that cannot be 26 phosphorylated provides substrate trapping mutant PTPs that are not impaired in their ability to interact with tyrosine phosphorylated protein substrates.
The present invention also pertains to pharmaceutical compositions comprising a substrate trapping mutant PTP in which (l) the wildtype PTP
catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause ~0 si~incant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 miri') (e.o , an alanine residue); and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphoryiated (e.,;., not seine or threonine_ nor any other naturally occurring or non-natuallv occurring amino that may be phosphorylated). The PTP of the preseat invention may therefore be formulated with a physiologically acceptable medium such as, for e:cample, a pharmaceutically acceptable carrier or diluent, to prepare a pharmaceutical composition.
For administration to a patient one or more polypeptides (including substrate trapping mutant PTPs), nucleic acid molecules (including recombinant e:cpression constructs encoding substrate trapping mutant PTPs) and/or modulating agents (including agents that interact with a PTP and/or a substrate trapping mutant PTP) are generally formulated as a pharmaceutical composition. :~
pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension or emulsion.
which additionally comprises a physiologically acceptable carrier (i.e., a non-toxic 1 ~ material that does not interfere with the activity of the active ingredient). S uch compositions may be in the form of a solid, liquid or gas (aerosol).
Alternatively, compositions of the present invention may be formulated as a lyophilizate or compounds may be encapsulated within liposomes using well known technology.
Pharmaceutical compositions within the scope of the present invention may also contain other components. which may be biologically active or inactive. Such components include. but are not limited to. buffers (e.o , neutral buffered saline or phosphate buffered saline), carbohydrates (e.o , glucose. tnannose, sucrose or de:ctrans), mannitol.
proteins. polypeptides or amino acids such as glycine_ antio~cidants, cheiating agent such as EDT: or glutathione. stabilizers. dyes. flavoring agents. and suspending agents and/or preservatives.
Any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of the present invention. Carriers for therapeutic use are well known. and are described_ for e:cample. in Remingtons Pharmacezuical Sciences. Vtack Publishing Co. (A.R. Gennaro ed. 1980. In <leneral.
the type of carrier is selected based on the mode of administration.
Pharmaceutical compositions may be formulated for any appropriate manner of administration.
including, for example, topical. oral. nasal. intraocular, intrathecal, rectal, vaginal, sublingual or parenteral administration- including subcutaneous. intravenous.
intramuscular. intrasternal. intracavernous. intrameatal or intraurethral injection or infiision. For parenteral administration. the carrier preferably comprises water, saline.
alcohol. a fat a wax or a buffer. For oral administration, any of the above carriers or a solid carrier. such as mannitol, lactose, starch, magnesium stearate, sodium saccharine.
talcum. cellulose, kaolin. glycerin. starch dextrins, sodium alginate, ' carboxymethylcellulose, ethyl cellulose. glucose, sucrose andlor magnesium carbonate.
may be employed.
~, pharmaceutical composition (e.o . for oral administration or delivery by injection) may be in the form of a liquid (e.o.. an eli.~cir, syrup.
solution. zmulsion or suspension). ..liquid pharmaceutical composition may include, for example. one or more of the following: sterile diluents such as water for injection- saline solution, 1 S preferably physiological saline, Ringer's solution. isotonic sodium chloride. tLYed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium. ' polyethylene glycols. glycerin. propylene glycol or other solvents;
antibacterial agents such as benzyi alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite: chelating agents such as ethylenediaminetetraacetic acid; bui~ers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral preparation can be enclosed in ampoules. disposable syringes or multiple dose vials made of Mass or plastic. The use of physiological saline is preferred- and an ~jec~le pharmaceutical composition is preferably sterile.
The compositions desi.ibed herein may be formulated for sustained release (l. e.. a formulation such as a capsule or sponge that erects a slow release of compound following administration). Such compositions may generally be prepared using well known technology and administered by. for example. oral- rectal or subcutaneous implantation. or by implantation at the desired target site.
Sustained-~0 release formulations may contain an agent dispersed in a carrier matrix and~'or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible. and may also be biode3adable: preferably the formulation provides a relatively constant level of active component release.
The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
For pharmaceutical compositions comprising a nucleic acid molecule encoding a substrate trapping mutant PTP polypeptide (such that the polypeptide is generated in situ), the nucleic acid molecule may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid, and bacterial. viral and mammalian expression systems such as, for example, recombinant expression constructs as provided herein. Techniques for incorporating DNA
into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described. for example. in Ulmer et al., Science Z59:174~-1749.
I~ 1993 and reviewed by Cohen, Science h9:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biode~adable beads, which are efficiently transported into the cells.
Within a pharmaceutical composition. a substrate trapping mutant PTP
polypeptide, a substrate trapping mutant PTP-encoding nucleic acid molecule or a modulating agent may be linked to any of a variety of compounds. For example.
such a polypeptide. nucleic acid molecule or agent may be linked to a targeting moiety (e.o , a monoclonal or polycional antibody, a protein or a Iiposome) that facilitates the delivery of the agent to the target site. As used herein. a "targeting moiety" may be any substance (such as a compound or cell) which, when linked to an agent enhances the ?5 transport of the agent to a target cell or tissue. thereby increasing the local concentration of the agent_ Targeting moieties include antibodies or fra~nents thereof.
receptors.
lisands and other molecules that bind to cells of. or in the vicinity of. the target tissue.
An antibody targeting agent may be an intact (whole) molecule. a fragment thereof. or a functional ecuivalent thereof. Examples of antibody fra~nents are F(ab')2. -Fab'. Fab and F[v] fragments. which may be produced by conventional methods or by genetic or WO 00/75339 ~ 1 PCT/US00/14211 protein engineering. Linkage is generally covalent and may be achieved by. for e:cample, direct condensation or other reactions. or by way of bi- or mufti-~nctional linkers. Targeting moieties may be selected based on the cells) or tissues) at which the agent is e:cpected to e:cezt a therapeutic benefit.
Pharmaceutical compositions may be administered in a manner appropriate to the disease to be treated (or prevented). An appropriate dosage and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient and the method of administration. In general. an appropriate dosage and treatment regimen provides the agents) in an amount sufficient to provide therapeutic andlor prophylactic benefit (e.,;., an improved clinical outcome.
such as more frequent complete or partial remissions. or loner disease-free and/or overall survival). For prophylactic use, a dose should be sufficient to prevent, delay the onset of or diminish the severity of a disease associated with a defect in cell si~aling, 1~ for e:cample a defect leading to abnormal cell cycle regulation.
proliferation. activation, differentiation, senescence. apoptosis, adhesion, metabolic activity. gene e:cpression or the like_ Optimal dosages may generally be determined using e:cperimental models and/or clinical trials. In general- the amount or polypeptide present in a dose. or produced in .nine by DNA present in a dose, ranges from about 0.01 p.g to about I00 ug per kg of host typically from about O.I ug to about 10 fig. The use of the minimum dosage that is sufficient to provide effective therapy is usually preferred.
Patients may ?enerally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those ~5 having ordinary skill in the art. Suitable dose sizes will vary with the size of the patient.
but will typically range from about 1 mL to about X00 mL for a 10-60 kg subject.
The following E.oamples are oiTered for the purpose of illustrating the present invention and are not to be construed to limit the scope of this invention. The teachinUs of all references cited herein are hereby incorporated by reference in their ;0 entirety.

WO 00/75339 ~ PCT/US00/14211 E'~A,~iPLES
E:WIPLE 1 GENER.~TION. E.'CPRESSION .aND PL:R1FIC.aTION OF ~IL;T.~NT PTP PROTEINS
Plasmid isolation. production of competent cells, transformation and related manipulations for the cloning, amplification- construction of recombinant plasmids, inserts and vectors. sequencing and the like. were carried out according to published procedures (Sambrook et al., :Llolecular Cloning, a Laboratory lLlanual, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, VY, 1989; Ausubel et al., Current Protocols in .Llolecular Biology. Greene Publ. Assoc. Inc. & John Wiley &
Sons. Inc., Boston. vIA). Recombinant nucleic acid expression constructs encoding human PTP-PEST (Garton et al., 1994 EyIBO J. 13:3763; Garton et al. 1996 :Llol. Cell.
Biol: 16:6408) and human PTP-1B (Brown-Shinier et al.. 1990 Proc. Nat. Acad.
Sci.
USA 87:~ 148) were prepared as described.
Point mutations within the catalytic domains of PTPs were introduced I ~ using standard procedures. for e~cample. the invariant aspattate (D) at amino acid position 199 in PTP-PEST being convened to alanine (A) by a substitution mutation (D I99A). Thus. mutations diving rise to PTP-PEST(D199A), PTP-PEST(C?3 I S), PTP I B(D 131 A) and PTP 1 B( C? 1 ~ S) were introduced by site-directed mutagenesis using the Vtuta GeneT''' in vitro mutagenesis kit (Bio-Rad, Richmond. CA) according to the manufacturer' s instructions. Regions containing the specified point mutation were they e:cchanged with the corresponding wild type sequences within appropriate e:cpression vectors. and the replaced mutant regions were sequenced in their entirety to verify the absence of additional mutations.
Full length PTP-PEST proteins (wild type and mutant proteins.
containinE either .asp 199 to .-Vila. or Cys231 to Ser mutations) and the wild type PTP-PEST catalytic domain (amino acids 1-30~) were e:cpressed in St~9 cells using recombinant baculovirus (BaculoGoldT-'". Phatmingen. San Die=o. C A). and purified a.s described in Garton and Tonks (E.LIBO J. 1~:3 X63-3771. 1994). Truncated iotms of >;
wild type and mutant PTP-PEST proteins. comprising amino acid residues 1-305 of PTP-PEST were also expressed in E. coli as GST fusion proteins following subcioning of PTP-PEST DNA, in-frame downstream of GST in pG~ vectors (Pharmacia Biotech Inc.. Uppsala. Sweden). Twenty-five ml of E: coli transformed with the appropriate vector were down to log Phase (ODboo aPpr°ely 0.~). Fusion protein expression was then induced by addition of 0.2 tnlVl isopropyl-1-thio-{3-D-galactopyranoside. and the cells were down for 2--~ hours at ~0°C. Cells were harvested by centrifugation, incubated with ~0 ~glml lysozyme in 3 ml buffer containing ~0 rt>1VI Tris-HC 1 > pH 7.-t, 5 rtuVt EDT, 1 mlVt P1~ISF, 1 m~l~t benzamid~e, ~ mg/ml leupeptin, ~ mglml aprotinin.
0.1% Triton X-100 and 1~0 mVI NaCl, then lysed by sonication (3 Y 10s).
Following removal of insoluble material by centrifugation (20 minutes at 300,000 Y ~), fusion proteins were isolated by incubation for 30 min at ~°C with 100 ml glutathione-SepharoseT'~ beads (Phatmacia Biotech Inc.. Uppsala, Sweden), and the beads were then collected by centrifugation and washed three times with Buffer A (20 mlVi Tris-HC1, 1 ~ pH 7.-t> 1 muI EDT:. 1 muI benzamidine. 1 mglml leupeptin, 1 mglml aprotinin. 10%
glycerol, 1% Triton X-100 and 100 mVt NaCI). This procedure yielded essentially homogeneous fusion protein at a concentration of 1 mg proteinlml ~utathione Sepharose beads. PTP 1 B proteins (wild type and mutant forms) comprising amino acids 1-X21 were expressed in E. coli and purified to homogeneity as described in Barford et al. (.l. :Llol. Biol. Z39:T_'6-'730 (1994)).
~1~IPLE 2 REGULATION OF PTP1B E.'CPRESSION LEVELS BY P210 BCR:ABL
Chronic myelogenous leukemia (CVIL) is a clonal disorder of the hematopoietic stem cell that is characterized by the Philadelphia chromosome (Ph), in ?5 which the c-:lbl proto-oncogene on chromosome 9. encoding a protein tyrosine kinase (PTK~. becomes linked to the bcr Qene on chromosome ''~. This results in the generation of a bcr:abl fusion protein. p?10 bcr:abl in which the PTK activity is enhanced relative to that of c-Abl. This e:cample demonstrates that phosphorylation competent p210 bcr:abl protein specifically induces PTP1B e:cpression.
When BaF; cells (lain et al.. 1996 Blood 88:1542) expressing a temperature-sensitive mutant form of p210 bcr:abl were shifred to the permissive temperature for e:cpression of p210 having PTK activity, PTP 1 B mRI~A and protein e:cpression levels were observed to increase within 12-24 hours. coincident with the appearance of the active form of the PTK (see, e.o . w098/0471?; LaiVlontagne et al., 1998 :Llol. Cell. Biol. 18:?965). The increase in e:~pression of PTP1B was also observed in Philadelphia chromosome-positive (Ph'-. ) B-lymphoid cells derived from a C:~. patient relative to Ph- cells from the same patient. Changes in PTP 1 B
activity.
which were commensurate with the charge in enzyme protein levels. were also observed. These changes were specific for PTP1B and were not seen in the closely related homologue TC-PTP (which shay 6~% ~o acid sequence identity with PTP1B) or in other tested PTPs. including SHP-1. SHP-2 and PTP-PEST. The specificity of PTP1B induction by p210 bcr:abl PTK activity was confirmed »sing kinase-detective Ratl cells (Pender?ast et al.. 1993 Cell 75:175). These cells e:cpress an inactive form of p210 -bcrabl_ which contains an ar°inine instead of a lysine residue at amino acid position 1172 and which lacks PTK activity. E.~tpression of this p210 mutant in Ratl cells failed to result in altered PTP1B expression levels.
E,yIPLE 3 P~ 10 BCR:.aBL BINDING SUBSTR.aTE 1NTER~.CTIONS WITH a SLBSTR.aTE TR~.PPtNG
PTP
VILT:~, iT
'This e:cample describes e:cploitation of substrate interacting properties of a substrate trapping mutant PTP to identify a PTP substrate. Substrate trappin_ PTP
?5 polypeptides and fusion proteins were prepared as desc:ibed in E:cample 1.
Substrate trapping mutant PTP polvpeptides or fusion proteins were contacted with lvsates derived from various cell lines. Briefly. as starring material for cell lysates. HeLa and COS cells were grown in Dulbecco's modified Ea=le's medium (DMEVI), containing ~% fetal bovine serum (FBS); Ratl. Wi38. C2C12 and VIvLu cells were down in DME1~L containing 10% FBS; 293 cells were grown in D1~IE1~I
containing 10% calf senun: MCF'.OA cells were grown in o0°,'°
DMEVI. ~0°,'° Ham's F-1'_' containing ~% hone serum.'_0 ng/ml epidermal gr°~h factor. 10 mglml insulin, 0-~
mgiml hydrocortisone and 0.?5 mgiml fun°-_izone; BaF~ cells were maintained as described (Jain et al., 1996 Blood 88:1 ~~2). All media also contained penicillin and streptomycin at 100 U/ml and 100 mg/ml_ respectively, and all cells were 'own at ;7°C. Calcium phosphate-mediated transfection was used to introduce cDNA encoding wild type and mutant PTP-PEST proteins into COS cells_ These were encoded by PTP-PEST cDNA (Garton et al., 1996 Viol. Cell. Biol. 16:6~08) subcloned into the plasmid pMT2 (Sambrook et al., ~Llolecular Cloning, a Laboratory :Llarruah Cold Spring Harbor Laboratory Press. Cold Spring Harbor. V~'I. 1989) from which expression was driven by an adenovirus .major late promoter, 20 ug DN ~ was used for transfection of each 10 cm plate of cells. The level of expression of PTP-PEST constructs was similar in all cases.
1~ Prior to cell lysis, 70-90% coniZuent cell cultures were treated for 30 minutes in medium containing 0.1 mVI o:cidized vanadate (pervanadate) (20 ~1 of a fresh solution containing ~0 ttllVl sodium metavanadate (NaVO;) and ~0 mVI
H,O=
added to 10 ml culture medium). Treatment of cells with H,O, and vanadate leads to a synergistic increase in phosphotyrosine levels. presumably due to inhibition of intracellular PTPs by vanadate (Heffetz et aL_ 1990 J. Biol. Chem. ?6~:?896-290?).
Pezvanadate treatment resulted in the appearance of at least ~0 prominent phosphotvrosine protein bands in all cell types. whereas untreated cells contained virtually undetectable levels of phosphotyrosine.
Cells were lysed in Buffer A (see Example 1) containing ~ mlVl iodoacetic acid. Following incubation at ~°C for 30 minutes. DTT was added to achieve a final concentration of 10 midi. Insoluble material was then removed by centrifusation for 20 minutes at 300.000 a e. The resultant lysates were stable with regard to their phosphotyrosine content during long term (several months) storage at -70°C and during prolonged (at least 20 hours) incubation at -~°C_ in the absence of exogenous added PTPs.

~6 Pervanadate-treated Hei.a cell lysate was fractionated by anion eYChange chromato~aphy using a Vlono Q FPLC column (Pharmacia). The sample (~0 mg total protein at 3 ma/ml in buffer A) was diluted in three volumes of buffer B (?0 rrWi tris-HC 1. pH 7.-~. 1 mVt EDT. 1 mVt benzamidine. 1 mJml leupeptin, 1 mglml aprotinin and 0.1% Triton ~-100) prior to loading. Proteins were eluted at a flow rate of 1 ml/min with a Linear gradient of 0-0.~ M NaCI in buffer B over 20 fractions ( 1 ml fraction volume), followed by a second gradient of 0.~-1.0 M NaCl in buffer B
over ~
fractions. Phosphotyrosine-containing proteins were detected within fractions according to anti-phosphotyrosine immunoblotting. The same procedures were followed for PTP1B, with the eYCeption that the cells were not treated with pervanadate.
For dephosphorylation reactions. Iysates of pervanadate-treated HeLa cells ( I -? mg protein/ml) containing tyrosine phosphorylated proteins were incubated on ice in the absence or presence of purified active PTPs at a concentration of ? nl~I.
Dephosphorylation was terminated by the removal of aliquots (30 ug protein) into SDS
1~ PAGE sample buffer. and the e:~tent of dephosphorylation was determined by itrununoblotting using the phosphotyrosine-specific monoclonal antibody G10~
generated as described below. Assays of PTP activity using tyrosine phosphorylated 'zP-labeled reduced and carborramidomethylated and maleylated lysozyme (RC~t lysozvme) as substrate were performed as described in Flint et al. ( 1993 E_LIBU J.
12:1937-1946).
Antibodies and ImmunoblottinQ: The PTP-PEST-specific monoclonal antibody AG25 was raised against baculovinLS-e:cpressed purified full-length PTP-PEST. The anti-phosphotyrosine monoclonal antibody 6104 was generated using as antigen phosphotyrosine. alanine and alycine. in a 1:1:1 ratio. polymerized in the ?5 presence of keyhole limpet hemocyanin with 1-zthyl-~-(~'-dimethvlaminopropyl)carbodiimide. a method originally described in Kamps and Setion (Oncogerre ?:30~-31 ~ ( 1983)). p 1 ~0~ monoclonal antibody was from Transduction Laboratories (Le:cington. Ky). Vlonocional antibody FG6 against PTP I B was provided by Dr. David Hill (Calbiochem Oncogene Research Products. Cambridge. Vt~).
Visualization of proteins by immunoblottina was achieved by enhanced chemiluminescence (ECL) using HRP-conjugated secondary antibodies (_~mersham Life Science Inc.. Arlington Heights. I1) and the SuperSignal"" CL-HRP
substrate system (Pierce. Rockford. I1).
Immunoyrecipitation and Substrate Trains: Immunoprecipitation of PTP-PEST from transfected COS cells was performed following covalent coupling of monoclonal antibody AG25 to protein A-Sepharose beads (Pharmacia Biotech Inc., Uppsala, Sweden) using the chemical cross-linking agent dimethyl pimelimidate (Schneider et al., J. Biol. Chem. ?~ % :10766-10769 (1982)). Antibody was first bound to protein A-Sepharose at a concentration of 1 mg/ml bead volume. and unbound material was then removed by three washes with 0.? M sodium borate, pH 9. Covalent coupling was achieved by incubation at room temperature for 30 minutes in the presence of ?0 mVI dimethyl pimeli.midate in 0.? VI sodium borate. pH 9. The beads were then incubated for 1 hour with an excess of 0.? YI ethanolamine, pH 3, to block any unreacted cross-linker. and washed three times with PBS prior to storage at ~°C. Ten q1 of AG25 beads were used to precipitate transfected PTP-PEST from lysates containing approximately 0.375 mg protein.
Substrate trapping was performed using various PTP affinity matrices.
The full-length PTP-PEST matrix utilized covalent coupled AG25-protein A-Sepharose beads to which purified baculovims-expressed PTf-PEST protein was bound.
Aliquots (10 p.1) of AG25 beads were incubated for ? hours at -~°C in 100 q1 buffer A in the presence of 5 p.g of purified PTP-PEST (wild type or mutant forms); unbound PTP-PEST was then removed by washing three times 'nnth 1 ml buffer A_ The resultant PTP-PES'L AG25-protein A-Sepharose beads contained approximately 2 mg of PTP-PEST
per 10 ml aliquot_ Substrate trapping was also carried out with glutathione-Sepharose 'S beads bound to bacterially-e:cpressed GST fusion proteins containing the catalytic domain of PTP-PEST.
PTP 1 B was also used in substrate trapping e:cperiments. In this case. the monoclonal antibody FG6 was precoupled to protein A-Sepharose in the absence of c: oss-linker (~' ug antibodvi 10 u1 beads). then purified PTP t B proteins were added in ~8 excess and incubated at ~°C for ? hours. Following removal of unbound PTP 1 B, 10 u1 beads contained approximately ? ug PTP1B.
Pervanadate-treated cell lysates. or column fractions. were used as a source of phosphotyrosine-containing proteins for substrate trapping e:cpenments. In general. lysates containing 0.25-0. mg protein in 0.~ ml buffer ~ (including ~
mlV1 iodoaceric acid, 10 mIVI DTT) were incubated at ~°C for 2 hours in the presence of 10 ~1 of a~nitY matrix containing approxunazeiy 2 ug of the appropriate PTP
protein.
Unbound proteins were then removed from the samples by washing three rimes with 1 ml buffer :~, and bound material was collected by addition of ~0 ~1 SDS-PAGE
sample buffer followed by hearing at 95°C for ~ minutes: proteins bound to the beads were then analyzed by SDS-PAGE followed by immunoblotting.
In transient cotrsnsfection experiments in C OS cells. PTP 1 B
dephosphorylates p210 bcr:abl but not v-abl. When the PTP1B(D181~) mutant was expressed as a GST fusion protein, purified and incubated with lysates of VIo7-p210 I~ cells (which overexpress p210 bcr:abl), a complex of the mutant PTP and p210 bcr:abl was isolated. In conk ~S~e phosphorylated c-abl. which was also present in the lysates. did not bind to the mutant PTP. The interaction between PTP1B(D131A) and p210 bcr:abl was blocked by vanadate. suggesting that the interaction involved the active site of the PTP.
Following transient coe:cpression in COS cells. PTP1B(D131A) formed a comple:c with p210 bcr:abl. The YI77F mutant form of p210 bcr:abl did not interact with PTP1B(D181~), suggesting that this tvrosme residue is a component of the b~~g ~m ~ the PTK. 'Ibis tyrosine residue in p210 bcr:abl is phosphorylated in vivo and has been demonstrated to serve as a docking site for GR.B2 (Pende~ast et al.. 1993 ~5 Cell 75:175). Direct interaction of the pTyr in p210 bcr:abl and the SH2 domain of GR.B2 is essential for the transforming actl~ry of the PTI~ Interaction of PTP 1 B(D 131:0 with p=10 bcr:abl interferes with the association of the PT's ~"i~
GRB=. Taken together. these data suggest that p? 10 bcr:abl is a physiological substrate of PTP 1 B and that PTP 1 B may function as an antagonist of the oncoprotein PTK in ~9 vivo. The Vma.Y, Km and Kcat of 37 kDa PTP1B mutants toward RCV1L are shown in Figure ?.
PTP 1 B and the EGF Receptor. Expression of PTP 1 B(D 181 A) in COS
cells leads to enhanced phosphorylation of tyrosyl residues in a 180 kDa protein and in proteins of 1?0 and 70 kDa. When a GST-PTP1B(D181A) fusion protein is e:cpressed in COS cells and precipitated on ~utathione-SepharoseT'~', the 180 kDa, and smaller quantities of pl?0 and p70, were coprecipitated. The p180 protein was identified as the epidermal ~owth factor (EGF) receptor by immunoblotting. The identity of the p gad p70 proteins is unclear, however, the latter is not src, p62 or paxillin.
E.Ypression of PTP1B(D181A) in COS cells induces tyrosine phosphorylation of the EGF receptor in the absence of its ligand. EGF, indicating that the mutant PTP is eYertins its efFects in the intact cell and not post-lysis.
The equivalent PTP-PEST(D 199A) mutant_ which has the corresponding aspartate at position 199 replaced with alanine, does not interact with the EGF receptor, indicating the specificity of this substrate interaction.
Autophosphorylation of the EGF receptor is required for the interaction with PTP 1 B(D 181 A). Mutants of the receptor that are either kinase-dead or in which the autophosphorylation sites have been deleted do not interact with PTP 1 B(D
181 A).
In v-src-eapressin~ cells. a plethora of tyrosine phosphorylated proteins were observed.
but phosphoryiation of the EGF receptor was not detected. Under these conditions, PTP 1 B D 181 A bound predominantly to a 70 kDa tyrosine phosphorylated protein.
PTP 1 B thus appears capable of modulating EGF-induced signaling pathways.
E~.-WtPLE ~
PTP-PEST PREFER~NThLLY DEPHOSPHORYL.aTES ~1 1~0 KD.4 PHOSPHOTYROSINE
7j CO'iT.~lN1'iG PROTEIN
In order to investisate the substrate specificity of PTP-PEST in vitro.
aliquots of pervanadate-treated HeLa cell lvsates were incubated on ice.
yielding ~0-100 distinct phosphotyrosine-~:ontaining proteins as judged by immunoblottina of the cell WO 00/75339 6o PCT/US00/14211 lysate using the monoclonal anti-phosphotyrosine antibody G10~. Purified full-len'th PTP-PEST (expressed in SP9 cells using recombinant baculovirus), PTP-PEST
catalytic domaitL or PTP1B catalytic domain (~~ kDa form) was then added to the lysate.
and aliquots were removed at various time points for analysis by SDS-PAGE followed by anti-phosphotyrosine immunoblotting.
Surprisingly, a prominent 130 kDa phosphotyrosine band (p130) was selectively dephosphorylated by PTP-PEST within 10 minutes, whereas the intensity of all the other bands was essentially unchanged even after 60 minutes of incubation with PTP-PEST. Long incubations with higher concentrations of PTP-PEST (eater than I00-fold) resulted in the complete removal of alI phosphotyrosine bands from the lysate. However, under all conditions tested, pl.i0 was found to be dephosphorylated more rapidly than all other bands present.
The selective dephosphorylation of p130 by PTP-PEST was also observed using a truncated form of the phosphatase (amino acid residues 1-30~) which I S essentially contains only the catalytic domain of the enzyme. This result suggests that the striking substrate preference displayed by PTP-PEST in this analysis is an inherent propemr of the phosphatase catalytic domain, whereas the C-terminal X00 amino acid residues have little discernible effect on the substrate specificity of the enzyme.
The specificity of the interaction between PTP-PEST and p 130 was examined using the catalytic domain of PTP 1 B (amino acid residues 1-X21 ) in dephosphorylation reactions. When added at a similar molar concentration to that used for PTP-PEST. PTP1B was found to dephosphorylate fully and rapidly (within 1~
minutes) most of the phosphot~rrosine-containing proteins present in the pervanadate treated HeLa lysate. In addition- the time course of dephosphorylation of p 1 ~0 was not 2~ significantiv_ more rapid that. that of the other phosphotyrosine bands dephosphorylated by PTP 1 B . The range o f PTP 1 B substrate specificity in vitro and in vivo thus can differ where availability of a Given substrate may vary. and where an isolated PTP
catalytic subunit is characterized_ EY~vIPLE ~
IDEVTIFIC.aTION OF A I~O KDA SL.BSTR.1TE OF PTP-PEST BY SUBSTR.~TE TRAPPING
This e:cample describes the use of a substrate trapping mutant PTP in an affinity matti.Y. to identify a PTP substrate in a cell lysate. For preparation of the substrate trapping PTP at~nity mama, a mutant form of PTP-PEST (D 199A) was generated by site-directed mutagenesis. and the mutant enzyme was purified following e:~pression using recombinant baculovirus. When assayed using tyrosine phosphorylated RC1~I-Lysozyme as substrate. the purified mutant enzyme exhibited a specific activity which was approxunately 10.000 fold lower than that of the wild type enzyme. This purified protein was bound to an afEnity matri:c comprised of an anti-PTP-PEST monoclonal antibody (.-~G25) covalently coupled to Protein :~-Sepharose beads. then incubated with each of the Vlono Q fractions prepared from HeLa cell lvsates as described in E~ataple 3.
Pervanadate-treated Hei.a cell lysate was fractionated by anion e:cchange chromatography (E.~cample 3) and aliquots of the fractions were analyzed by SDS
PAGE followed by immunoblotting with anti-phosphotvrosine or anti-p130'~
antibodies. Aliquou of all samples analyzed were then incubated with an affinity matrix containing a subswate trapping PTP-P~ST mutant. comprising full length PTP
PEST in which Asp199 is changed to alanine (D199A), bound to covalently coupled protein A-Sepharoserantibody (AG2~) beads. After ~5 minutes of incubation.
proteins associating with the mutant PTP-PEST were collected by centrifugation. the beads were washed, and SDS-PAGE sample buffer was added. Associated proteins were then analyzed by itnmunoblotting using the monoclonal anti-phosphotyrosine antibody G10~. Proteins associated with PTP-PEST were then analyzed by SDS-P.aGE
followed ~5 by immunoblotting with anti-phosphotyrosine or anti-pl.i0'~ antibodies.
anti-phosphotvrosine immunoblotting of the column fractions showed that the p 1 ~ 0 phosphoy rosine band eluted as a single peak in fractions 11-1 ~ (approx.
0.~ VI V aC 1 ). In view of the abundance of tyrosine phosphorylated p 1 ~ 0 in HeLa lysates. it appeared likely that p 1 ~ 0 represents a previously identified phosphotyrosine-6' containing 130 kDa protein. Several potential candidates were identified in the literature. including the focal adhesion kinase p 1?5F''~". ras-GtIP. op 130 and p 130'. Of these candidates. p 130' has been identified as a particularly prominent phosphotyrosine band in a wide variety of systems, including v-crk (~Iayer and Hanafusa. Proc. ~Vatl. .cad Sci. LSd 8::?638-2642 (1990); Vlayer et al., :Vature 332:272-275 (1988) and src (Kanner et al., Proc. Natl. cad Sci. G'S~ 87:3328-(1990); Reynolds et al., nlol. Cell. Biol. 9-.3951-3958 (1989)) transformed fibroblasts, integrin-mediated cell adhesion (Nojitna et al.. J. Biol. Chem. 270:15398-1502 (I995);
Perch et al.; .I. Cell Science 108:1371-1379 ( 1995); Vuori and Ruoslahti, J.
BioL Chem.
Z~O:~~~ 9-?262 (1995)) and PDGF stimulated 3'I ~ cells (Rankin and Rozengurt, J.
Biol. Chem. 269:704-7I0 (1994)).
Therefore. the possibility that the p 130 phosphotyrosine band corresponds to p130'y was tested by immunoblotting the Vlono Q fractions using an antibody to p130'~. The 130 kDa band corresponding to p130'u eluted in the same fractions as the p 130 tyrosine phosphorylated band, and displayed a similar apparent molecular weight, suggesting that they might represent the same protein.
Furthermore, p130'~ immunoprecipitated from these fractions was found to be phosphorylated on tyrosyl residues.
The mutant PTP-PEST protein was found to associate with a single phosphotyrosine-containing protein. the molecular weight (I30 kDa) and Mono Q
elution position (fractions 11-1=~) of which coincided with those of p 130.
Immunoblotting of the PTP-PEST-associated proteins using the p130~ antibody demonstrated that the 130 kDa tyrosine phosphorylated protein trapped. by the mutant PTP-PEST is indeed p130'u. Therefore it appears that p130"~ is a physiologically relevant substrate for PTP-PEST.
Structural Features of PTP-PEST in S ecinc Interaction with Tyrosine Phosphorv lated o 130'w The interaction between P 130'' and PTP-PEST was investisated further in substrate trapping experiments using various purified mutant forms of PTP-PEST to precipitate proteins from pervanadate-treated HeLa Lvsates.
Several ai~nity matrices were incubated with pervanadate-treated HeLa cell tysaie. and 6~
proteins associated with the beads were analyzed by SDS-PAGE followed by itnmunoblotting with anti-phosphotyrosine or anti-p130"' antibodies.
The wild type full-length phosphatase was found to be incapable of stable association with tyrosine phosphorylated p130"~, whereas both the PTP-PEST
(D 199A) mutant protein and a mutant lacking the active site cysteine residue (C231 S) specifically precipitated p130'~ from the lysate. The inability of the wild type phosphatase to precipitate tyrosine phosphorylated p130"~ presumably reflects the transient nature of the normal interaction between PTP-PEST and tyrosine phosphorylated p1~0~, which is likely to be concluded as soon as p130"~ is dephosphorylated by PTP-PEST.
Since the C-terminal X00 amino acids of PTP-PEST contain several proline-rich regions which resemble src homology-~ (SH3) domain binding sequences.
it appeared plausible that the specificity of the interaction between PTP-PEST
and p130'~ might depend to some extent on association of these segments with the SHS
1 ~ domain of p 130". The possible contribution of the C-terminal se~nent of PTP-PEST
in the observed specific interaction of PTP-PEST with p130~ was therefore addressed in further substrate trapping experiments using GST fusion proteins containing the catalytic domain of PTP-PEST alone, in both wild type and mutant (D199A) forms.
The mutant catalytic domain of PTP-PEST fused to GST was found to precipitate the p130"~ phosphotyrosine band specifically, whereas both the wild type fusion protein and GST alone failed to precipitate p130'~. The specific interaction between PTP-PEST and p130"~ observed in these experiments therefore appears to be an intrinsic propem of the catalytic domain of PTP-PEST, emulating the observed preference of the active PTP-PEST catalytic domain for dephosphorylation of p130°s in vitro.
S ecificitv of Interaction Between Mutant PTP-PEST and Tyrosine Phosnhorvlated t?l30'w In view of the relative abundance of tyrosine phosphorylated p l ~ 0'~ in the pervanadate-treated HeLa cell Ivsate. the possibility that the observed selective binding of PTP-PEST inactive mutant proteins to pI.O''~ was substrate-directed (;retlecrin~ the abundance of this potential substrate relative to the other phosphotyrosine-containing proteins present in the lvsate) rather than enzyme-directed 6~
(reflecting a genuine substrate preference of PTP-PEST) was considered; this possibility was addressed in two ways. First inactive mutant fotTns of the catalytic domain of PTP I B were used to trap potential substrates for this enzyme from the pervanadate-treated HeLa lysates. Again it was found that the wild type phosphatase was incapable of stable interaction with any phosphotyrosine-containing protein. whereas mutant variants of the PTP1B phosphatase domain (comprising Cys or Asp mutations analogous to those described above for PTP-PEST) associated with many tyrosine phosphorylated proteins. This was especially apparent for the aspartic acid mutant of PTP 1 B (D 181 A), which appeared to precipitate essentially all phosphotyrosine-IO containing proteins from the lysate with similar efficacy. These data emphasize the specific nature of the interaction between PTP-PEST and p 1 ~ 0~. which appears to be a propem peculiar to the PTP-PEST catalytic domain. rather than a feature shared by all PTP catalytic domains.
The specificity of the interaction between PTP-PEST and p 130'u was 1~ addressed further following pervanadate-zr~azment of several different cell lines (Wi38, 293, COs. MCF10A, C2C12. MvLu), yielding a different array of tyrosine phosphorylated proteins in each case: the resultant lysates were analyzed by SDS
PAGE followed by anti-phosphotyrosine itnmunoblotting. Aliquots were incubated with PTP-PEST (D 199A) a~niry matri:c or control matri.~c. and tyrosine phosphorylated 20 proteins associating with PTP-PEST were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine or anti-p130''~ antibodies as described above.
In each case. the D 199A mutant PTP-PEST protein precipitated a single broad phosphotyrosine band with an apparent molecular weight between 1?0 and kDa in different cell lines. whereas the affinity matri.Y alone failed to precipitate any ~5 phosphotyrosine-containing protein. Immunoblottins of the precipitates with a p130"5 antibody revealed that the protein precipitated from all cell lysates corresponded to p 130"S; the observed molecular weight variation between different cell Lines presumably retlec~~s either species differences in the molecular weight of p130"s or e:cpression of different alternatively spliced forms (Sakai et al.. E LIBO J.
I ~ :37-~8-:'~ d 30 ( 1990).

The relative abundance of tyrosine phosphorylated p 1 ~ 0'~ in the PTP-PEST precipitates appeared to correlate approximately with the abundance of p130'~
protein in the lvsates (data not shown). Surprisingly. re5~~ess of the abundance of tyrosine phosphorylated p130"~ in the lysates. p130''~ was invariably the only phosphoryrosine-containing protein in the precipitates. even in ?93 cell lysates which contained very little p I ~ 0'u protein but which displayed a wide variety of other abundantly tyrosine phosphoryiated proteins. Similarly, when lysates of pervanadate-treated 293 cells (containing tyrosine phosphoryiated p130'~ in amounts which are undetectable by anti-phosphoryrosine immunobiotting of the lysate) were incubated with active PTP-PEST, no visible dephosphorylation of any phosphotyrosine band occurred (Garton and Tonks, unpublished data). These results indicate that the affinity of PTP-PEST for p130"~ is substantially 'eater than for any other substrate present and further emphasizes the remarkable substrate selectivity of PTP-PEST for p 130'.
Vanadate Inhibition of Tyrosine Pho horvlated 130' :association with 1 ~ Mutant PTP-PEST: A consistent observation was that in contrast to the inactive mutant PTP-PEST, the wild type enzyme failed to associate in a stable comple:c with tyrosine phosphorylated p 130', suggesting that the observed association is active site directed. In order to investigate this possibility. mutant PTP-PEST (D 199A) was incubated with the PTP inhibitor vanadate (Denu et al., 1996 Proc.. Natl. .cad Sci GSA
93:?493-?498), at various concentrations prior to addition of pervanadate-treated HeLa cell lysate. The e:ctent of association of p130~ with PTP-PEST was then analyzed.
PTP-PEST affinity matri.~c. comprising full length PTP-PEST (D 199A) bound to covaleatly coupled protein A-Sepharose~antibody (AG25) beads, was incubated for 10 minutes on ice in the presence of varying concentrations of sodium orthovanadate. The ''_5 samples were then incubated with aliquots of pervanadate-treated HeLa cell lysate:
associated proteins were analyzed by SDS-P AGE and immunoblotting with ann-phosphoryrosine or anti-p 130' antibodies. The activity of wild type PTP-PEST
was also determined ;order the same conditions. using tyrosine phosphoryiated '-P-labelled RCS(-lvsozyme as substrate.

The association was found to be potently disrupted by vanadate. with a concentration-dependence similar to that of vanadate inhibition of wild type PTP-PEST.
and complete disruption being observed at 10 tniVt vanadate.
E~,;~tPLE 6 ASSOCIATION OF EVDOGF'10US P1~0''~ 'KITH TR~.NSFECTED MLT.~NT PTP-PEST IN
COS CELLS
The work described above strongly suggests that p 130"s represents a physiological substrate of PTP-PEST. In order to assess whether PTP-PEST
interacts with p130'~ in intact cells. COS cells were transfected with plasmids encoding wild type or substrate trapping mutant forms (D 199A or C?31 S) of PTP-PEST. The cells were treated with pervanadate 30 minutes prior to lysis. PTP-PEST proteins were immunoprecipitated. and associated tyrosine phosphorylated proteins were analyzed by anti-phosphotyrosine immunoblotting of the resultant precipitates. Lysates were also incubated with covalently coupled protein A-Sepharoselanti-PTP-PEST (AG25) beads and associated proteins were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibody.
Under these conditions. the phosphotyrosine-containing band corresponding to p 1.i0'~ was again unique in its ability to associate with the CZ~ 1 S
PTP-PEST protein. indicating that p 130"s can be specifically selected by PTP-PEST as a substrate in an intracellular conte.~ct in the presence of a lame number of alternative possible subsnates. Neither the wild type nor the D199A form of PTP-PEST was capable of a stable interaction with tyrosine phosphorylated p 130" in pervanadate-treated COS cells.
The binding of both wild type and D 199 PTP-PEST to tyrosine ?5 phosphorylated p1~0'~ under these conditions is most likely prohibited by the presence of pervanadate bound to the active site cysteine residue of PTP-PEST (Denu et :11..
Proc. Varl. .-lcad. Se;:. LSD 93:'~9~-?~93 ( 1996)). which effectively e:ccludes the binding of phosphotyrosine residues of p 130'. The ability of the C'_'~ 1 S
mutant PTP

PEST to associate in a stable complex with p130"~ in the presence of pervanadate suggests that this mutant protein is largely unaffected by pervanadate, indicating that the normal mode of inhibition of PTPs by vanadate ions depends critically on direct interactions between vanadate and the thiolate anion of the PTP active-site cysteine residue. These observations therefore lend further support to the existence of an exclusive interaction between PTP-PEST and p130~. which appears exclusively to involve the PTP-PEST active site, and therefore reflects the physiological.
highly restricted substrate preference of PTP-PEST for p130°'.
E~~1~LE 7 ~~ PREPAR.aTION OF SLBSTR:.TE TR:IPQiVG PTP MZ;TaNTS
Generation of mutant PTPs capable of interacting with substrates in a stable complex was essentially as described (Flint et al.. 1997 Proc. Vat. .-lcad Sci.
USA 9:1680; Garton et al., 1996 :Llol. Cell Biol. 16:608: Tiganis et al., 199?
J. Biol.
Chem. Zi2:215~8; see also PCT US97/13016). Plasmid isolation. production of 1~ competent cells, transformation and M13 manipulations were carried out according to published procedures (Sambrook et al., .ylolecular Cloning, a Laboratorv :Llam,~al, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY, 1989). Purification of DN
fragments was achieved using a QIAEX~' kit purchased from QI~GEN. Inc.
(Chatsworth. Cue). Sequencing of the different constructs was performed using a ?0 SequenaseT'~' kit (~mersham-Pharmacia. Piscaraway, N~ according to the manufacturer's instructions. Restriction and modification enzymes were purchased from Roche Molecular Biochemicals (Indianapolis. IN) and New England Biolabs (Beverly. l~fr1).
Brietlv. human PTPH1 cDNA (U.S. Patent No. ~.~95.911) liaated into ?s plasmid pBlueScript (Stratagene. LaJolla. Ca) was mutated by site-directed mutagenesis using the Viuta-GeneT'~' kit (Bio-Rad. Inc.. Hercules. C.~) according to the supplier's instructions. T'ne oli~onucleotide used for in vitro mutaUenesis of cysteine 8-~~ to serine was:

CCT SGT TC ~ CTC CMG TGC TGG :~1T ~.G SEQ ID N0:37 which spans nucleotides ?~~7-?562 of PTPH1. The oligonucleotide for mutagenesis of asparcate 311 to alanine was:
GCA TGG CCT GCC CAC GGT GTG C SEQ ID N0:~8 which spans nucleotides 2~~-?~6 of PTPH1. The mutated replicative form DNr1 was transformed into E coli strain DH10B (Stratagene, La Jolla. CA) and colonies were picked and dideoxy sequenced using a SequenaseT'~' kit (Amersham-Pharmacia. Piscataway> NJ) according to the manufactures s instructions for verification of the mutation. The portions of the wildtype and mutated PTPH1 ?enes encoding the PTP catalytic domain (amino acid residues 63~ to 913) were ligated in-frame into the 1 ~ e:cpression vector pGE.~ (~mersham-Pharmac~a, Piscataway, Nn to generate three glutathione-S-transferase (GST) fusion protein encoding sequences: GST-PTPH1(w-ildtype), GST-PTPHl(D811:~) and GST-PTPH1(C842S). GST-PTPH1 fusion proteins were e:cpressed in E. coli and purified by affinity binding to ?lutathione immobilized on Sepharose'''~' beads (Pharmacia_ Piscatawa; NJ) according to the manufacturer s protocol.
:alternatively. wildtvpe and mutant PTPH1 constructs as described above to be used for transfection of mammalian cells were tagged at the C-terminal encoding ends with nucleic acid sequences encoding the HA epitope_ The HA tag corresponds to an antibody -de:ined epitope derived from the influenza hemaaglutinin protein (~Nilson .5 et a1._ 198- Call 3; :767):
SYPYDVPDY:~S SEQ ID N0:39 after confirmation by DNA sequencins, these constructs we:e cloned into vector pCDN.~~ (InvitroQen. Carlsbad_ C.~) and retroviral vector pBSTRI
~S.
Reeves. Massachusetts General Hospital. Boston. VLF).

PTPH 1 (D811 ~) mutant constructs were further modified by size directed mutagenesis as described above but using the oligonucleotide:
TTG GAC ~ ~1C CGA TTT :~ GAT GTG CTG CCT TAT G SEQ ID NO::~O
which spans nucleotides 2034-2070 of PTPH1 to ?enerate a double mutant (Y676F1D811A) in which the conserved PTP catalytic site tyrosine residue at position 676 is replaced with phenvlalanine.
P~WIPLE 8 INFLUENCE OF PTPHI EXPRESSION ON CELL GROWTH IN TR~NSFECTED CELLS
IO This example shows that overeapression of a transfected PTPH1 gene in cultured cells markedly impairs cell ~owth. while overe:cpression of a transfected mutant substrate-trapping PTPHI gene does not.
Stable ~2T3 cell lines e;cpressing wildtype or substrate trapping mutant PTPH1 GST fusion proteins (see E:cample 7) under the control of a tetracycline 1 ~ repressible promoter were constructed using a retroviral gene delivery system (Paulus et al.. 1996 ,I. virol. l 0:62; Wang et al.. 1998 Genes Develop. I ?: I769).
Briefly.
confluent 10 cm diameter tissue culture plates of the viral packaging cell line Lin:~ (G.
Hannon_ Cold Spring Harbor Laboratory, Cold Spring Harbor. NY) were transfected by calcium-phosphate precipitation with 1~ ~ of either the wildtype or mutant 20 PTPHI retroviral constructs. To maintain repression of PTPHI gene e:cpression. the following steps of establishing and maintaining the stable cell Iines were performed in the presence of 2 uQlml tetracvciine (Clontech. Palo :alto. C:~). Retroviruses were produced by culturing the transfected Lin.~ cells at 30°C for ~3 hours afrer which culture fluids containing virus were filtered using a 0.~.~ um filter (vlillipore. Bedford.
''_S VIA) to remove packaging cells. The viral supernatants were supplemented with -~
uQiml polvbrene (Sigma. St_ Louis. VIO) and were used to infect ~iIH3T: cells (Cold Spring Harbor Laboratory stock. orisinally obtained from :American Type Culture Collection. Rockville, vfD) maintained in Dulbecco's modified Eagle's Vfedium (DIvIEVt, GIBCO-BRL, Grand Island. N~ supplemented with 10% fetal bovine setwn (FBS, GIBCO-BRL). Infection took place overnight at 30°C, after which the medium was replaced with fresh medium and cultures were incubated at 37°C. Two days later selective conditions were imposed by supplementing the medium with puromycin to a final concentration of 2 ugiml. Individual colonies were isolated and maintained in the presence of both tetracycline and puromycin. To induce PTPHI expression, cells were washed and re-seeded in new dishes in the presence of puromycin. but in the absence of tetracycline.
10 Cell ~owth was markedly ~bited (approximately seven-fold decrease in accumulated cell number) when wildtype PTPH1 catalytic domain expression was induced by removal of tetracycline from the culture media (Figures ~ and -f).
Approximately 10% of the cells ~adually detached from the culture dish during induction of wildtype PTPH1 e:cpression_ and these cells were non-viable as determined 1 ~ by their inability to exclude trypan blue. In contrast expression of the catalytically impaired PTPHI-D81 IA mutant ("DA~7 had no effect on cell ~owth or viability.
For each PTPH1 construct, similar results have been obtained in three separate cell lines generated from distinct isolated colonies. indicating that differences among clonal populations do not account for the phenotypic differences observed between cells 20 transfected with wildtvpe and mutant (D811?.) PTPH1. Using a DNA
fra~entanon assay (Wyllie. 1980 :Varz~re ?8-I:5>j: Arends et al.. 1990 Vim. ,I. Parhol.
136:~9~), it was determined that cells in which PTPH1 expression was induced did not undergo apoptosis.
Cell cycle analysis by flow cvtotluorimetric measurement of DNA
~5 content using propidium iodide (Rabinovitch. 199 _~leths. Cell Biol. ~1:?63-?96) was performed on populations of transfected cells in which wiidtype or mutant (D811~) PTPH1 e:cpression was induced. The distribution of cells amonQSt various phases of the cell cycle was not altered relative to control cells. indicating that PTPH1-induced growth arrest did not operate in a particular cell cycle phase.

Cells in culture were also synchronized to determine the effects of PTPH1 expression on re-entry into the cell cycle during recovery from Gl/S
arrest.
Following a ?~-hour period of induced PTPHl (wildtype or mutant D811A) expression, cells were 5-vnchronized by cultivation for 18 h in the presence of 1 tnVl hydroxvurea (Calbiochem, San Diego. C A) : this agent arrests cells at the G1/S boundary in the cell cycle (Kreck and DeCaprio, 1995 .Lleths. En.:-ymol. ?5~:11~) . The hydroxyurea block was released by washing the cells with flesh medium three yes. At various tune points following removal of the cell cycle block. cells were lysed in NP40 buffer (1%
NP40. 10 nuVl sodium phosphate-pH 7.0, 1~0 tnVt NaCl. ? tnul EDTA, ~0 tnul NaF, 1 mVI Na~VO~, ~ ~g/ml leupeptin, ~ ~glm1 aprotuiin. 1 mui benzamidine, 1 muI
P'~ISF) for immunoblot analysis using a cyciin-specific antibody. Briefly, confluent cells in a 10 cm diameter tissue culture plate were lysed at -~°C for I O min in 0.~ ml ~iP~O buffer.
and the lysates were clarified by centrifugation at 10.000 Y g for 10 min at =~°C.
Aliquots of each lysate were normalized for protein concentration (BCA assay.
Pierce 1~ Chemicals, Rockford_ IL), diluted in sodium dodecylsulfate (SDS) sample buffer (Laemmli. 1970 Nature 227:680), resolved by SDS poiyacrylamide gel electrophoresis using 8% acrylamide gels and blot transferred onto Immobilon-P PVDF membranes (Llillipore, Bedford. VIA). Polvcional rabbit anti-cvclin D 1 antibodies (Santa Cruz Biotechnology. Santa Cruz. CA) diluted according to the supplier's recommendations in immunoblot buffer ('_'0 mVI Tris-pH 7.~ containing ~% (wiv) nonfat dry milk.
1~0 muI
NaCI and 0.0~% Tween ?0) were used to probe the blot for 1 hour at room tempezature.
The blot was washed three times in the same buffer and developed using enhanced chemiluminescence (ECL) reagents and horseradish peroxidase (HRP)-coupled secondary antibodies (both from Amersham-Pharmacia Biotech. Piscataway, New ''S Jersey) according to the supplier s instructions. as previously described (Zhang et al..
199 J. Biol. Chem. ?-0:'_'006.
As shown in Figure ~. when transfected cells were released from the hydroxvurea cell cycle block under conditions non-permissive for expression or the w-ildtvpe PTPH1 transVene. cvclin D expression gradually increased as cells reentered ~0 and progressed through the cell cycle. When. however. cells were released from the cell 7?
cycle block under conditions permissive for PTPHl eYpression_ all detectable cyciin D
expression was abolished. suggesting that PTPH1 retards cell ~owth by disrupting cell cycle progression. E.~cpression of a mutant PTPH1(D811~) in cells transfected with the mutant transgene had no effect on the cell cycle.
E~C.:~vIPLE 9 IDE~ITIFIC.3TION OF VCP ~s ..~ PTPH1 SussTx~,TE UsmrG .~ PTPH1 SussT~TE
'fR;,ppn~tG ViUT~NT IN VITRO
'This e:cample describes identification of a PTPH1 substrate in cell lysates, using a substrate trapping PTPH1 mutant having the invariant PTP
catalytic site aspartate residue replaced with alanine (D811 ~). Cell lysates were prepared as described above in E.~campie 3, and then contacted with wildtype or mutant catalytic domains to determine PTP-substrate binding interactions.
Substrate trapping methodologies using mutant PTPs in which the invariant catialytic domain asparate residue is replaced with alanine were as described 1~ (Flint et al.. 1997 Proc. Vat. .-lcad Sci. USA 9:x:1680; Garton et al..
1996 :Llol. Cell Biol: 16:608: Tiganis et al., 1997 J. Biol. Chem. ?; ?:21 ~~8; see also PCT
US97/13016) except that the mutant PTP was PTPH1 (D811A) as described above in Example 8.
Pervanadate-treated cell lysates were incubated with GST-PTPHI
catalytic domain fusion proteins immobilized on SepharoseT'2 beads. Briefly, subconfluent mammalian cell cultures were treated with ~0 E.Wf pervanadate (diluted from a 1:1 mi.~cture of 100 rm~i sodium vanadate and 100 rW( H,0= in DhfEW) for ~0 min_ washed with PBS and lysed_ as described in E.Yample 8. in substrate-trapping buffer (1°,'o Triton Y-100. ~0 mVI HEPES-pH 7.~. ~ mV( EDT:. 1~0 rruW
NaCI. 10 mNl via phosphate_ ~0 mW VaF. ~ mu( iodoacetic acid_ ~ uJml leupeptin. ~ uglml aprotinin. 1 mW benzamidine and 1 WI PNISF. Lysates were made l0 cnVl DTT and ciari~ied by centrifu=anon for 10 min at 10.000 ~c g. Purified GST-PTPH1 fusion proteins. or GST alone. bound to ~lutathione-Sepharose beads ( ~mersham-Pharmacia 7~
Biotech, Piscataway. NJ) under conditions recommended by the supplier were e:ctensivelv washed with phosphate buffered saline (PBS) containing 1°%
Triton X-100 (Si~na- St. Louis. V10), ? ttWl diththiothreitol (DTT. Sigma), ~ uglml leupeptin, ~
~g/ml aprotinin, 1 cniVl benzamidine and 1 tnlVl PI~ISF. Lysates were incubated with the bead-immobilized GST or GST-PTPH1 catalytic domain fusion proteins for 2 h at -~°C, and the beads were washed four times with substrate-trapping buffer. Material bound to the beads was resolved by SDS-PAGE and blotted onto Immobilon-PT'~' (l~fillipore, Bedford, MA) membranes, then probed with phosphotyrosine-specific monoclonal antibodies at concentrations recommended by the suppplier (G98. Tiganis et al.. 1997 J.
Biol. Chem. l l 2:21 X48; =1G10, Upstate Biotechnology. Lake Placid, NY;
P'j?0.
Transduction Laboratories. Lexington, K'~ and developed using ECL reagents (A.mersham-Pharmacia Biotech, Piscataway, NJ) as described above in E.Yample 7.
~, prominent, tyrosine-phosphorylated protein of 97 kDa (pp97) was specifically isolated by the PTPH1(D811~-1) mutant from 293 cell lysates. but not by 1~ either the wildtype PTPH1 or the PTPH1(C8~2S) mutant (Figure 6).
Furthermore, pp97 was also consistently recovered by PTPH1(D811A) as the major tyrosine-phosphorylated protein from other mammalian cell lines tested. including A431, COS-7, HepG2, MDCK. REF-~2. Saps-2 and Vero cells. The PTPH1 substrate trapping mutant specifically and preferentially bound to pp97, which was one of several hundred tyrosine-phosphorylated proteins present in the cell lysates; pp97 was not a major protein component in any of the cell lysates used as a starting material for substrate trapping. Variable amounts of other, minor tyrosine-phosphorylazed proteins were also detected in the PTPH1-associated materials from the various cell lines.
Purification of pp97 on immobilized PTPH1(D811r~) from lysates representing 10g 29~ cells was scaled up to obtain sufficient protein for partial sequencing by Edman de~adation of K-endopeptidase-digested peptides (Russo et 31..
199'_' J. Biol Chem. .6 7 :20317). Sequences of seven individual peptides were determined '(Figure ~l. all of which were Found to match amino acid sequences present in a membrane-associated protein having .~TPase activity and known as p97 or V
CP
~0 (E~erton zt al.. 1992 FLIBU f 11:333). Underlined sequences (Fig. 7) matched the mouse VCP sequence retrieved from the ~1CBI database (httPV~~~ncbi.nlm.nih.jov~, accession number Z1~044)(SEQ ID VO:=~2). The Yeast ortholog of VCP, known as CDC~B, is a well established cell cycle revelatory protein (Patel et al., 1998 Trends Cell Biol. 8:6~). A synthetic peptide corresponding to the C-terminal 1 ~ residues of marine VCP (Egerton et a1._ 199?) was prepared (Cold Spring Harbor Laboratory Core Peptide Facility, Cold Spring Harbor, N'~ and conjugated using SPDP (N-succinimidyl 3-{2-PYTidyldithio]proprionate, Pierce Chemicals, Rockford. IL) according to the m~tt~acturer's recommendations to keyhole limpet hemocyanin (KLH, Pierce Chemicals) for use as an immunogen according to standard procedures (Harlow and 10. Lane, Antibodies: ~ Laboratory ~Llam~al, Cold Spring Harbor Laboratory, 1988; Weir.
D.M., Handbook of Experimental Immunology. 1986, Blacltweil Scientific.
Boston) to generate polycional rabbit antiserum CS~31. The VCP peptide immunogen had the sequence:
GGSVYTEDNDDDLYG SEQ ID NO:~I
1~ E~WiPLE 10 IDENTIFICATION OF VCP ~S A PTPH1 SUBSTRATE USING ~ SUBSTRATE TRAPPING
PTPHl DOUBLE MUTA'tT HAVING SUBSTITUTED ACTIVE SITE TYROSINE
'Ibis e:cample describes identification of an interaction between a PTP
and its substrate in intact cells, using a substrate trapping PTP double mutant. More 20 specifically, this e:cample employs the PTPH1 double mutant having the invariant PTP
catalytic site aspartate residue replaced with alanine (D811A) and also having the conserved PTP catalytic site tyrosine residue at position 676 is replaced with phenylalanine.
Cultured 293 cells were transfected using the HA-tagged PTPH1 ?5 constructs described in E.Yample 8. and the e:cpressed HA epitope tagged proteins were recovered by immunopmcipitation with HA-specific monoclonal antibody 1=C A
bound to immobilized staphylococcal protein A as described (Zhang et al.. 1997 J. Biol.

Chem. ~i?:?7281). Immtmoprecipitates were prepared according to stand~d procedures from lysates produced as described above in Example Immunoprecipitates were analyzed for phosphotyrosine-containing proteins by western immunoblot methods as described above. Surprisingly, the PTPH1 (D811 A) mutant e:cpressed in 393 cells contained significant and readily detectable levels of phosphotyrosine (Figure 8A), which contrasted with the absence of detectable phosphotyrosine in the GST-PTPH1(D811A) fusion protein expressed in E toll (Figure 6). From these results. the location of phosphorylated tyrosine in the PTPH1 primary structure could not be determined. Additionally, the PTPH1(D811A) mutant expressed 10 in 293 cells did not co-precipitate detectable pp97/VCP (Figure 8).
PTPHl(D811A) thus failed to e~ciently trap detectable pp97/VCP in vivo in a manner commensurate with the in vitro pp97/VCP trapping exhibited by PTPH1(D81 1A) in vitro (Example 9).
Analysis of the PTPH1 catalytic domain amino acid sequence revealed the presence of a conserved tyrosine residue at position 676 in the PTP active site 15 (Barford et al., 1995 Vat. Struct. Biol. ?:1043). An HA-tagged PTPHl double mutant was constructed as described in E.~camPle 8, in which the tyrosine at position 676 of PTPH1(D811A) was replaced with phenylalanine to provide PTPHl(Y676F/D811A).
Cell lysates from ?93 cells transfected with a construct encoding the PTPH1 (Y6 7 61D811A) double mutant were lysed. immunoprecipitated with monoclonal anti-~p HA antibody and analyzed by western immunoblot methodologies as described above for the presence of phosphotyrosine. Immunoprecipitated materials were also analyzed for the presence of pp97/VCP using antisezum CS531 (E.oample 9), and for the presence of the HA epitope using monclonal antibody 12CA~ (Zhang et al., 1997 J. Biol.
Chem.
l i?:?7?81).
L mike the X93 cells transfected with the single mutant PTPH 1 (D811 A).
293 cells transfected with the double mutant PTPH1(Y676F,~D811A) had gained the ability to specifically trap pp97~CP- as demonstrated by immunoblot analysis of the itnmunoprecipate using antiserum CS>>1 (Figure 81. When analyzed for phosphotyrosine content. the double mutant PTPHl(Y676F-D811 A) immunoprecipitated from transfected 293 cells e:chibited dramatically reduced phosphoryrosine, relative to the single mutant PTPH1(D811A) (Figure 8B).
E~,~IPLE 11 IDE~(TIFIC~.TION OF TYROSINE PHOSPHORYL.aTION SITES ON ~ PTPH1 SUBSTR.aTE IN
VIVO USING .a SUBSTRATE TRAPPING PTPH1 DOUBLE MUT.aNT
In this e:cample, a substrate trapping PTPH1 double mutant is used to functionally characterize tyrosine phosphoryiation sites on pp97/VCP. The ryrosines (Y796 and Y805) at the C-terminus of VCP are major phosphorylation sites that may be responsible for VCP roles in cell cycle regulation via heretofore uncharacterized molecular pathways (Eaerton et al.. 199 J. Biol. Chem. 169:11>>; Madeo et al., ~Llol. Biol. Cell 9:131 ).
Human 293 cells were co-transfected with (l) one of the HA-tagged PTPH1 constructs (wildtype, D81 l~ or Y676FID811~) as described in E:camples 8-10, and (ii) either a wildtype VCP construct (VCPmyc) or a double mutant (Y796F/Y805F) VCP construct (VCPmyc-FF, L. Samelson, National Institutes of Health.
Bethesda.
Maryland) in which the two C-terminal tyrosine phosphorylation sites are replaced with phenylalanines. The VCP wildtype and mutant constructs were tagged with the Vlyc epitope recognized by monoclonal antibody 9E10 (American Type Culture Collection.
Roclcville. Maryland). Co-transfected cells were lysed as described in E.Yample 3 and itamunoprecipitated with antibody 12C.~ (anti-HA) as described (Zhang et al., 1997 J.
Biol. Chem: ?: ?:?7281). .
Electrophoretically resolved and blotted components were then probed with anti-mvc antibody 9E10 to identify VCP proteins that co-precipitated with (I.e..
were "upped" by) the PTPH1 protein. or with anti-H~ to confirm the presence of ~ PTPH1 proteins in immunoprecipitated material. Wildtype and mutant PTPH1 proteins were e:cpressed at comparable levels. as were the two forms of VCP. The PTPH11Y6 7 6F,'D811 _~) double mutant trapped wildrype VCP efficiently. but did not trap the double mutant VCP. which lacks two C-terminal tyrosine phosphorylation sites (Figure 9). tllso. neither wildtype PTPH1, nor the single mutant PTPH1(D811?.), effectively trapped VCP (Figure 9).
E~.:WLPLE 1?
SELECTIVE DEPHOSPHOItYL.~TION OF VCP BY PTPH1 In this e:cample, the effect of PTPH1 on the phosphorylation state of VCP was e:camined. Stable ~T~ cells, transfected with and expressing full length wildtype PTPH1 under control of a tetracycline-repressible promoter in the presence or absence of tetracycline. were pretreated with 1 rWI vanadate for 1 hour prior to lysis.
and VCP was immunoprecipitated using rabbit CS>; l antiserum. Lysates and immunoprecipitates were prepared according to st~~d Precedttres as described above in Example 3. except cells were Iysed in RIPA buffer (i~1P40 buffer supplemented with 1% sodium deo:rycholate and 0.1% SDS) instead of ~1P40 buffer. Under conditions permissive for PTPH1 e:cpression ('), a three- to five-fold decrease in the 1~ phosphotvrosine level of VCP was observed. relative to that seen when PTPHl expression was repressed (-) (Figure 10A).
Lysates from the ~T~ t~fec'ants were also immunoprecipitated with anti-phosphotyrosine antibody PT66 (Sigma, St_ Louis. MO) to obtain a representative sample of tyrosine-phosphorylated proteins from cells cultured in the presence (~-) or absence (-) of PTPH1 e.~cpression (Figure 10B). Immunoblot analysis of these immunoprecipitates with antibodies specific for VCP revealed dramatically reduced levels of VCP among tyrosine-phosphoryiated proteins immunoprecipitated from cells in which PTPH1 expression was induced (~-) relative to uninduced controls (-(Figure 10B). The apparently selective dephosphorylation of VCP by PTPH1 in these ?5 cells was also shown by assessing the effect of PTPH1 induction on the decree of tyrosine phosphorylation of a distinct tyrosine-phosphoryiated protein. the kinase F ~K.
Induction of PTPH1 expression did not cause a corresponding decrease in the level of F ~K that was immunoprecipitated by anti-phosphotyrosine antibodies (Figure I
OB).

The effects of induced PTPH1 expression on total tyrosme-phosphorylated protein pools were also compared in PTPH1-transfected VIH~T3 cells down under normal conditions ("untreated', under serum starvation by cultivation in DhfEVI containing 0.~% FBS for 16 hours (''starved'' or following insulin stimulation of starved cells by 10 u~/ml insulin (Roche Molecular Biochemicals.
Indianapolis. N) for 10 minutes. Aliquots of total cell lysates were electrophoretically resolved, blot transferred to Immobilon-PT''' and probed with a mixture of two HRP-conjugated anti-phosphotyrosine antibodies. PY'_'0 (Transduction Laboratories, Le:cington, KY) and 4610 (Upstate Biotechnology Inc., Lake Placid, ~ diluted according to the suppliezs' recommendations. followed by ECL detection (Amersham, Cleveland. OH). The induction of PTPH1 overexpression failed to alter the global pattern of protein tyrosine phosphorylation in randomly bowing ("unu'eated'~, starved or insulin-stimulated cells (Figure 11).
1~ Those skilled in the art will recognize. or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Also. it will be appreciated that. although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly. the present invention is not limited except as by the appended claims.

Claims (54)

What is claimed is:

1. A substrate trapping mutant protein tyrosine phosphatase in which a) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute; and b) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated.

2. The substrate trapping mutant of claim 1 in which at least one wildtype tyrosine residue is replaced with an amino acid selected from the group consisting of alanine.
cysteine, aspartic acid, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine; methionine, asparagine, proline, arginine valine and tryptophan.

3. The substrate trapping mutant of claim 1 wherein at least one tyrosine residue that is replaced is located in a protein tyrosine phosphatase catalytic domain.

4. The substrate trapping mutant of claim 1 wherein at least one tyrosine residue that is replaced is located in a protein tyrosine phosphatase active site.

5. The substrate trapping mutant protein tyrosine phosphatase of claim 1 wherein at least one tyrosine residue is replaced with phenylalanine.

6. The substrate trapping mutant protein tyrosine phosphatase of claim 1 wherein at least one tyrosine residue that is replaced is a protein tyrosine phosphatase conserved residue.

7. The substrate trapping mutant of claim 6 wherein the conserved residue corresponds to tyrosine at amino acid position 676 in human PTPH1.

8. The substrate trapping mutant of claim 1 wherein at least one tyrosine residue is replaced with an amino acid that stabilizes a complex comprising the protein tyrosine phosphatase and at least one substrate molecule.

9. The substrate trapping mutant of claim 1 comprising a mutated PTPH1.

10. The substrate trapping mutant of claim 1 comprising a mutated protein tyrosine phosphatase selected from the group consisting of PTP1B, PTP-PEST, PTP.gamma., MKP-1, DEP-1, PTPµ, PTPX1, PTPX10, SHP2, PTP-PEZ, PTP-MEG1, LC-PTP, TC-PTP, CD45, LAR and PTPH1.

11. The substrate trapping mutant of claim 1 comprising a mutated PTP-PEST phosphatase in which the amino acid at position 231 is replaced with a serine residue.

12. A method of identifying a tyrosine phosphorylated protein which is a substrate of a protein tyrosine phosphatase, comprising the steps of:
a) combining a sample comprising at least one tyrosine phosphorylated protein with at least one substrate trapping mutant protein tyrosine phosphatase, in which (i) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute. and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated, under conditions and for a time sufficient to permit formation of a complex between the tyrosine phosphorylated protein and the substrate trapping mutant protein tyrosine phosphatase: and b) determining the presence or absence of a complex comprising the tyrosine phosphorylated protein and the substrate trapping mutant protein tyrosine phosphatase, wherein the presence of the complex indicates that the tyrosine phosphorylated protein is a substrate of the protein tyrosine phosphatase with which it forms a complex.

13. A method according to claim 12, wherein the substrate trapping mutant comprises a mutated protein tyrosine phosphatase that is selected from the group consisting of PTP1B, PTP-PEST, PTP.gamma., MKP-1, DEP-1, PTPµ, PTPX-1, PTPX10, SHP2, PTP-PEZ, PTP-MEG1, LC-PTP, TC-PTP, CD45, LAR and PTPH1.

14. The method of claim 12 wherein the sample comprises a cell that expresses the tyrosine phosphorylated protein.

15. The method of claim 14 wherein the cell has been transfected with at least one nucleic acid molecule encoding the substrate.

16. The method of claim 12 wherein at least one substrate trapping mutant protein tyrosine phosphatase is expressed by a cell.

17. The method of claim 16 wherein the cell has been transfected with at least one nucleic acid molecule encoding the substrate trapping mutant protein tyrosine phosphatase.

18. The method of claim 12 wherein the sample comprises a cell that expresses (l) the tyrosine phosphorylated protein which is a substrate of the protein tyrosine phosphatase, and (ii) the substrate trapping mutant protein tyrosine phosphatase.

19. The method of claim 18 wherein the cell has been transfected with (i) at least one nucleic acid encoding the substrate, and (ii) at least one nucleic acid encoding the substrate trapping mutant protein tyrosine phosphatase.

20. The method of claim 12 wherein the sample comprises a cell lysate containing at least one tyrosine phosphorylated protein.

21. The method of claim 20 wherein the cell lysate is derived from a cell transfected with at least one nucleic acid encoding the tyrosine phosphorylated protein.

22. The method of claim 20 wherein the cell lysate is derived from a cell tansfected with at least one nucleic acid encoding a protein tyrosine kinase.

23. The method of claim 12 wherein at least one substrate trapping mutant protein tyrosine phosphatase is present within a cell lysate.

24. The method of claim 23 wherein the cell lysate is derived from a cell transfected with at least one nucleic acid encoding the substrate trapping mutant protein tyrosine phosphatase.

25. A method according to claim 12 wherein the tyrosine phosphorylated protein is selected from the group consisting of VCP,p130cas, the EGF
receptor, p210 beriabl, MAP kinase, Shc and the insulin receptor.

26. A method of identifying an agent which alters the interaction between a protein tyrosine phosphatase and a tyrosine phosphorylated protein which is a substrate of the protein tyrosine phosphatase, comprising:
(a) contacting in the absence and in the presence of a candidate agent a protein tyrosine phosphatase and a tyrosine phosphorylated protein which is a substrate of the protein tyrosine phosphatase under conditions and for a time sufficient for detectable dephosphorylation of the substrate to occur, wherein the tyrosine phosphorylated protein which is a substrate of the protein tyrosine phosphatase is identified by (1) combining a sample comprising at least one tyrosine phosphorylated protein with at least one substrate trapping mutant protein tyrosine phosphatase, in which (i) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute, and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated, under conditions and for a time sufficient to permit formation of a complex between the tyrosine phosphorylated protein and the substrate trapping mutant protein tyrosine phosphatase; and (2) determining the presence or absence of a complex comprising the tyrosine phosphorylated protein and the substrate trapping mutant protein tyrosine phosphatase, wherein the presence of the complex indicates that the tyrosine phosphorylated protein is a substrate of the protein tyrosine phosphatase with which it forms a complex; and (b) comparing the level of dephosphorylation of the substrate in the absence of the agent to the level of dephosphorylation of the substrate in the presence of the agent, wherein a difference in the level of substrate dephosphorylation indicates the agent alters the interaction between the protein tyrosine phosphatase and the substrate.

27. A method of identifying an agent which alters the interaction between a protein tyrosine phosphatase and a tyrosine phosphorylated protein which is a substrate of the protein tyrosine phosphatase, comprising:
(a) contacting in the absence and in the presence of a candidate agent, a protein tyrosine phosphatase and a tyrosine phosphorylated protein which is a substrate of the protein tyrosine phosphatase under conditions and for a time sufficient to permit formation of a complex between the tyrosine phosphorylated protein and the substrate trapping mutant protein tyrosine phosphatase, wherein the substrate trapping mutant protein tyrosine phosphatase comprises a mutated protein tyrosine phosphatase in which (i) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute. and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated: and (b) comparing the level of complex formation in the absence of the agent to the level of complex formation in the presence of the agent wherein a difference in the level of complex formation indicates the agent alters the interaction between the protein tyrosine phosphatase and the substrate.

28. A method of reducing the activity of a tyrosine phosphorylated protein, comprising administering to a subject a substrate trapping mutant of a protein tyrosine phosphatase in which (i) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute, and (ii) at least one wildtype tyrosine residue is replaced with an ammo acid that is not capable of being phosphorylated, whereby interaction of the substrate trapping mutant protein tyrosine phosphatase with the tyrosine phosphorylated protein reduces the activity of the tyrosine phosphorylated protein.

29. A method according to claim 28, wherein the tyrosine phosphorylated protein is selected from the group consisting of VCP, p130cas, the EGF
receptor, p210 bcr~abl, MAP kinase, Shc and the insulin receptor.

30. A method according to claim 28, wherein the protein tyrosine phosphatase is selected from the group consisting of PTP1B, PTP-PEST, PTP.gamma., MKP-1, DEP-1, PTPµ, PTPX1, PTPX10, SHP2, PTP-PEZ. PTP-MEG1, LC-PTP, TC-PTP, CD45, LAR and PTPH1.

31. A method of reducing a transforming effect of at least one oncogene associated with p130cas phosphorylation comprising:
administering to a mammal capable of expressing p130cas a substrate trapping mutant of PTP-PEST in which (i) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute. and (ii) at least one wildtype tyrosine residue is replaced with an ammo acid that is not capable of being phosphorylated:

whereby the substrate trapping mutant interacts with p130cas to reduce the transforming effect of at least one oncogene associated with p130cas phosphorylation.

32. A method according to claim 31 wherein the oncogene is selected from the group consisting of v-crk, v-src and c-Ha-ras.

33. A method of reducing formation of signaling complexes associated with p130cas, comprising administering to a mammal capable of expressing p130cas a substrate trapping mutant of PTP-PEST in which (i) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute, and (ii) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated;
whereby the substrate trapping mutant interacts with p130cas to reduce the formation of signaling complexes associated with p130cas.

34. A method of reducing cytotoxic effects associated with protein tyrosine phosphatase administration or overexpression, comprising administering to a mammal a substrate trapping mutant of a protein tyrosine phosphatase in which (i) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute, and (ii) at least one wildtype tyrosine residue is replaced with amino acid that is not capable of being phosphorylated.

35. An isolated nucleic acid molecule encoding a substrate trapping mutant protein tyrosine phosphatase in which a) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute; and b) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated.

36. An antisense oligonucleotide comprising at least 15 consecutive nucleotides complementary to the nucleic acid molecule of claim 35, wherein at least one of said nucleotides is complementary to a nucleotide in the nucleic acid molecule of claim 35 that encodes the amino acid that is not capable of being phosphorylated.

37. A fusion protein comprising a polypeptide sequence fused to a substrate trapping mutant protein tyrosine phosphatase in which a) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute; and b) at least one wildtype protein tyrosine phosphatase tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated.

38. The fusion protein of claim 37 wherein the polypeptide is an enzyme or a variant or fragment thereof.

39. The fusion protein of claim 37 wherein the polypeptide sequence fused to a substrate trapping mutant protein tyrosine phosphatase is cleavable by a protease.

40. The fusion protein of claim 37 wherein the polypeptide sequence is an affinity tag polypeptide having affinity for a ligand.

41. A recombinant expression construct comprising at least one promoter operably linked to a nucleic acid encoding a substrate trapping mutant protein tyrosine phosphatase in which a) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute; and b) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated.

42. The expression construct of claim 41 wherein the promoter is a regulated promoter.

43. An expression construct according to claim 41 wherein the substrate trapping mutant protein tyrosine phosphatase is expressed as a fusion protein with a polypeptide product of a second nucleic acid sequence.

44. The expression construct of claim 43 wherein the polypeptide product of said second nucleic acid sequence is an enzyme.

45. A recombinant expression construct according to claim 41 wherein the expression construct is a recombinant viral expression construct.

46. A host cell comprising a recombinant expression construct according to any one of claims 41-45.

47. A host cell according to claim 46 wherein the host cell is a prokaryotic cell.

48. A host cell according to claim 46 wherein the host cell is a eukaryotic cell.

49. A method of producing a recombinant substrate trapping mutant protein tyrosine phosphatase. comprising:
culturing a host cell comprising a recombinant expression construct comprising at least one promoter operably linked to a nucleic acid sequence encoding a substrate trapping mutant protein tyrosine phosphatase in which a) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute; and b) at least one wildtype protein tyrosine phosphatase tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated.

50. The method of claim 48 wherein the promoter is a regulated promoter.

51. A method of producing a recombinant substrate trapping mutant protein tyrosine phosphatase, comprising:
culturing a host cell infected with the recombinant viral expression construct of claim 45.

52. A pharmaceutical composition comprising:
a substrate trapping mutan protein tyrosine phosphatase in which a) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute; and b) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated.
in combination with a pharmaceutically acceptable carrier or diluent.

53. A pharmaceutical composition comprising an agent that interacts with a substrate trapping mutant protein tyrosine phosphatase in which a) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than I
per minute: and b) at least one wildtype tyrosine residue is replaced with an amino acid that is not capable of being phosphorylated, in combination with a pharmaceutically acceptable carrier or diluent.

54. A kit for identifying a tyrosine phosphorylated protein substrate of a protein tyrosine phosphatase comprising:
a) at least one substrate trapping mutant protein tyrosine phosphatase in which (i) the wildtype protein tyrosine phosphatase catalytic domain invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute. and (ii) at least one wildtype tyrosine residue is replaced with an ammo acid that is not capable of being phosphorylated; and b) ancillary reagents suitable for use in detecting the presence or absence of a complex between the protein tyrosine phosphatase and a tyrosine phosphorylated protein.