JP2003501098A - Substrate-capturing protein tyrosine phosphatase - Google Patents

Abstract

  (57) [Summary] A novel substrate-capturing variant that has no catalytic function, but retains the ability to bind to a phosphotyrosine-containing protein substrate, and is further modified by replacing at least one tyrosine residue with a non-phosphorylatable amino acid Compositions and methods are provided for protein tyrosine phosphatase (PTP). The use of such PTPs for the identification of PTP substrates, the use of agents that alter PTP-substrate interactions, and methods of altering PTP activity are disclosed.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is related to US patent application Ser. No. 08/685, filed July 25, 1996.
U.S. Patent Application No. 09 filed September 1, 1998, which is a divisional application of 992.
/ 144,925 is a partial continuation application. This application is also incorporated by reference in US Provisional Application No. 60 / 137,319 filed June 3, 1999 (Attorney Docket Number CSHL99).
-06P) to claim the benefits. The teachings of these applications are incorporated herein by reference in their entirety.

US Government Statement of Rights The study described here is a research grant CA awarded by the National Institutes of Health.
Received government funding under 53840. The US Government may own some of the rights in this invention.

FIELD OF THE INVENTION The present invention generally relates to compositions useful for treating conditions associated with defects in the biochemical pathways of cells, such as those that control cell proliferation, cell differentiation and / or cell survival. And regarding the method. The invention more particularly relates to substrate-trapping variants of protein tyrosine phosphatase polypeptides, and variants thereof. The present invention also relates to the use of such polypeptides to identify antibodies and other agents, including small molecules, that alter biological signaling and cellular biochemical pathways.

BACKGROUND OF THE INVENTION Coordinated by the action of protein tyrosine kinase (PTK), which phosphorylates some tyrosine residues, and protein tyrosine phosphatase (PTP), which dephosphorylates some phosphotyrosine residues in polypeptides. Reversible protein tyrosine phosphorylation is a key mechanism in the regulation of many cellular activities. The diversity and complexity of PTP and PTK are comparable, and it is becoming apparent that PTP is equally important in delivering both positive and negative signals for proper functioning of cellular machinery. Regulation of tyrosine phosphorylation contributes to specific pathways of biological signaling, including those associated with cell division, proliferation and differentiation. Defects and / or dysfunctions in these pathways are responsible for some disease states in which effective means for interventional treatment remain ambiguous, including, for example, malignant diseases, autoimmune diseases, diabetes, obesity and infections. sell.

Enzymes of the protein tyrosine phosphatase (PTP) family have in common a highly conserved 250 amino acid PTP catalytic domain, but more than 500 structural members that show considerable variability in their non-catalytic segments. Consisting of different proteins (Charbonneau and Tanks, 1992 Annu. Rev.
． Cell Biol. 8: 463-493; Tonks, 1993 Semi.
n. Cell Biol. 4: 373-453). This structural diversity, in some cases, probably reflects the diversity of physiological roles of individual members of the PTP family, which have been shown to have specific functions in proliferation, development and differentiation. (Desai et al., 1996 Cell 84: 599-609; Kishih.
ara et al., 1993 Cell 74: 143-156; Perkins et al., 1
992 Cell 70: 225-236; Pingel and Thomas, 19
89 Cell 58: 1055-1065; Schultz et al., 1993 C.
ell 73: 1454-1454).

[0006] Recent studies have also provided considerable information on the structure, expression and regulation of PTP, but the nature of the tyrosine phosphorylated substrates for PTP to exert its action remains unclear. A limited number of studies on synthetic phosphopeptide substrates revealed some differences in the substrate selectivity of various PTPs (Cho et al., 1993 Protein Sci. 2: 977-984; Dechert et al., 1995 Eur. J. Biochem. 231: 673-681). PTP
Analysis of PTP Substrate Dephosphorylation Mediated by Peptide Suggests that the Presence of Certain Amino Acid Residues at Specific Positions of the Substrate Polypeptide to Phosphorylated Tyrosine Residues May Favor Catalytic Activity (Ruzzen et al., 1993 E
ur. J. Biochem. 211: 289-295; Zhang et al., 1994.
Biochemistry 33: 2285-2290). Therefore, although the physiological relevance of the substrates used in these studies is unknown, PTP is in
It exhibits some level of substrate selectivity in vitro.

The PTP family of enzymes comprises a common, evolutionarily conserved segment of approximately 250 amino acids known as the PTP catalytic domain. Within this conserved domain, a unique signature that is immutable in all PTPs.
) There is a sequence motif, [I / V] HCXAGXXR [S / T] G SEQ ID NO: 36. Cysteine residues within this motif are invariant in members of the family and are known to be essential for catalysis of phosphotyrosine dephosphorylation. Such a residue functions as a nucleophile that attacks the phosphate moiety present on the incoming substrate phosphotyrosine residue. Replacing a cysteine residue with a serine (eg, in a cysteine to serine or “CS” variant) or alanine (eg, a cysteine to alanine or “CA” variant) by site-directed mutagenesis The resulting PTP has a reduced catalytic activity, but retains the ability to complex or bind to its substrate, at least in vitro. Such variants are for example MKP-1 which is a member of the PTP family (Sun et al., 1993 Cell 75: 487-4).
93), as well as other PTPs. But these C
S mutants are generally capable of effectively binding in vitro to a phosphotyrosyl substrate to form a stable enzyme-substrate complex, although in many cases such a complex is found, for example, in mutant PTP and phosphotyrosyl protein substrates. When both are present together in one cell, they can be isolated in vivo. Therefore CS
Variants have limited utility and cannot be used to isolate all combinations of PTP and substrate.

[0008] Currently, desirable goals for elucidating the molecular mechanisms governing PTP-mediated cellular events are, inter alia, the determination of molecules with which PTP interacts, the determination of substrates and binding partners, and agents that modulate PTP activity. Including the identification of In certain situations, however, current approaches may lead to an understanding of some aspects of the regulation of tyrosine phosphorylation by PTP, but not specific intracellular tyrosine phosphorylation and / or dephosphorylation events. It would not be possible to provide a strategy to control.

Accordingly, there is a need in the art for improved ability to regulate phosphotyrosine signaling, including regulation of PTP. Increasing the understanding of PTP regulation is believed to facilitate the development of methods to regulate the activity of proteins involved in the phosphotyrosine signaling pathway and to treat conditions associated with such pathways. The present invention fulfills these needs and provides still other related benefits.

SUMMARY OF THE INVENTION The present invention is a substrate-capturing PTP (ST) that binds (captures) one or more substrates of PTP.
-PTP), which provides a novel substrate-capturing variant or modified form of mammalian PTP. The binding of ST-PTP to the PTP substrate forms a complex that can be easily observed and optionally isolated and identified. These P
Mutants of TP have attenuated catalytic activity (lacking or lacking catalytic activity) when compared to wild-type PTP, but retain the ability to bind tyrosine phosphorylated substrates of wild-type PTP is doing. ST-PTP is useful, for example, to measure the fine substrate specificity of one or more PTPs.

One aspect of the present invention is that the invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain is an amino acid that results in a decrease in Kcat of less than 1 per minute but not a significant change in the Km of the enzyme. Substituted; and a substrate-trapping mutant protein tyrosine phosphatase, wherein at least one wild-type tyrosine residue is replaced with an amino acid that is not phosphorylated.
In certain embodiments, at least one wild type tyrosine residue is alanine,
Cysteine, aspartic acid, glutamine, glutamic acid, phenylalanine,
Substituted with amino acids which are glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, arginine, valine or tryptophan. In certain other embodiments, at least one of the substituted tyrosine residues is located in the protein tyrosine phosphatase catalytic domain. In a particular embodiment, at least one of the substituted tyrosine residues is located in the protein tyrosine phosphatase active site, and in a further particular embodiment, at least one of the tyrosine residues is substituted with phenylalanine.
In certain other embodiments, at least one of the substituted tyrosine residues is a protein tyrosine phosphatase conserved residue, and in certain further embodiments, the conserved residue is amino acid position 676 of human PTPH1. Corresponds to tyrosine in position. In certain embodiments, at least one of the tyrosine residues.
Are substituted with amino acids that stabilize the complex containing protein tyrosine phosphatase and at least one substrate molecule. In a particular embodiment, the substrate-capturing variant has the variant PTPH1 and in a particular embodiment the substrate-capturing variant is PTP1B, PTP-PEST, PTPγ, MKP-1.
, DEP-1, PTPμ, PTPX1, PTPX10, SHP2, PTP-PE
Mutant protein tyrosine phosphatases which are Z, PTP-MEG1, LC-PTP, TC-PTP, CD45, LAR or PTPH1. In a particular embodiment, the substrate-capturing variant comprises a variant PTP-PEST phosphatase in which the amino acid at position 231 is replaced with a serine residue.

Another aspect of the invention is that (i) an invariant aspartic acid residue of the wild type protein tyrosine phosphatase catalytic domain reduces Kcat by less than 1 per minute, although the Km of the enzyme does not change significantly. Substituted with an amino acid that results in;
(Ii) at least one wild-type tyrosine residue under sufficient conditions and at such time to allow formation of a complex between the tyrosine phosphorylated protein and the substrate-trapping mutant protein tyrosine phosphatase. Combining at least one substrate-trapping mutant protein tyrosine phosphatase substituted with an amino acid that cannot be oxidized with a sample containing at least one tyrosine phosphorylated protein; and a tyrosine phosphorylated protein and a substrate-trapping mutation Determining the presence or absence of a complex having a somatic protein tyrosine phosphatase, wherein the presence of the complex indicates that the tyrosine phosphorylated protein is a substrate for its complexing partner, protein tyrosine phosphatase. Including,
Provided are methods for identifying tyrosine phosphorylated proteins that are substrates for protein tyrosine phosphatases. In certain embodiments, the substrate-capturing mutant is PTP1B, PTP-PEST, PTPγ, MKP-1, DEP-1, PTPμ.
, PTPX1, PTPX10, SHP2, PTP-PEZ, PTP-MEG1,
It includes a variant protein tyrosine phosphatase that is LC-PTP, TC-PTP, CD45, LAR or PTPH1. In a particular embodiment, the sample contains cells expressing tyrosine phosphorylated protein, and in another particular further embodiment, the cells are transfected with at least one nucleic acid molecule encoding said substrate. In another particular embodiment, at least one substrate-trapping mutant protein tyrosine phosphatase is expressed by a cell,
And in another more particular embodiment, the cells are transfected with at least one nucleic acid molecule encoding said substrate-capturing mutant protein tyrosine phosphatase. In certain other embodiments, the sample comprises (i) tyrosine phosphorylated protein that is a substrate for protein tyrosine phosphatase and (ii)
Contains cells expressing at least one substrate-trapping mutant protein tyrosine phosphatase. In certain other embodiments, the cells are transfected with (i) at least one nucleic acid encoding the substrate and (ii) at least one nucleic acid encoding a substrate-trapping mutant protein tyrosine phosphatase. In certain other embodiments, the sample contains a cell lysate containing at least one tyrosine phosphorylated protein, and in certain further embodiments,
Cell lysates are derived from cells transfected with at least one nucleic acid encoding a tyrosine phosphorylated protein. In certain further embodiments, cell lysates are derived from cells transfected with at least one nucleic acid encoding a protein tyrosine kinase. In yet another specific embodiment, at least one of the substrate-trapping mutant protein tyrosine phosphatases is present in a cell lysate, and in certain further embodiments, the cell lysate is a substrate-trapping mutant protein tyrosine. Derived from cells transfected with at least one nucleic acid encoding a phosphatase. In another embodiment, the tyrosine phosphorylated protein is VCP, ^p130cas , EGF receptor, p2
10 bcr: abl, MAP kinase, Shc (Tiganis et al., 1998.
Mol. Cell. Biol. 18: 1622-163) or the insulin receptor.

In another aspect, the present invention provides that in the absence and presence of a drug candidate, protein tyrosine phosphatase and tyrosine phosphorylated protein that is a substrate for protein tyrosine phosphatase undergoes detectable dephosphorylation of the substrate. For a sufficient period of time and for such a time (wherein the tyrosine phosphorylated protein, which is a substrate for protein tyrosine phosphatase, is (1) Group, the Km of the enzyme does not cause a significant change, but a Kcat of less than 1 per minute
At least one wild-type tyrosine residue is allowed to form a complex between the tyrosine phosphorylated protein and the substrate-trapping mutant protein tyrosine phosphatase. Combining at least one substrate-trapping mutant protein tyrosine phosphatase substituted with amino acids that cannot be phosphorylated under sufficient conditions and at such times with a sample containing at least one tyrosine phosphorylated protein; And (2) determining the presence or absence of a complex having a tyrosine phosphorylated protein and a substrate-trapping mutant protein tyrosine phosphatase, wherein the presence of the complex means that the tyrosine phosphorylated protein is its complexing partner. The group of a protein tyrosine phosphatase And the level of dephosphorylation of the substrate in the absence of the drug and the degree of dephosphorylation of the substrate in the presence of the drug (wherein substrate dephosphorylation is demonstrated). Differences in levels of oxidation indicate that the drug alters the interaction between protein tyrosine phosphatase and its substrate, including protein tyrosine phosphatase and tyrosine phosphorylated protein, the substrate for protein tyrosine phosphatase. Provided are methods of identifying agents that alter the action.

In another aspect, the present invention provides that (i) an invariant aspartic acid residue of a wild-type protein tyrosine phosphatase catalytic domain reduces Kcat by less than 1 per minute, although the Km of the enzyme does not change significantly. Substituted with an amino acid that results in
And (ii) a substrate-capturing mutant protein tyrosine phosphatase comprising a mutated protein tyrosine phosphatase in which at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. To allow the formation of a complex between protein tyrosine phosphatase and a substrate of protein tyrosine phosphatase in the presence and presence of tyrosine phosphorylated protein and a substrate-trapping mutant protein tyrosine phosphatase Contacting under sufficient conditions and for such a time; and comparing the level of complex formation in the absence of drug with the level of complex formation in the presence of drug (wherein the level of complex formation is The difference is that the drug is protein tyrosine phospha (Showing that it alters the interaction between protease and substrate), the present invention provides a method for identifying an agent that alters the interaction between protein tyrosine phosphatase and tyrosine phosphorylated protein that is a substrate of protein tyrosine phosphatase. To do.

In another aspect, the invention provides that (i) a constant aspartic acid residue of a wild-type protein tyrosine phosphatase catalytic domain reduces Kcat by less than 1 per minute, although the Km of the enzyme does not change significantly. Substituted with an amino acid that results in
And (ii) administering to the subject a substrate-capturing variant of a protein tyrosine phosphatase, wherein at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. Provided is a method for reducing the activity of tyrosine phosphorylated protein, wherein the interaction of somatic protein tyrosine phosphatase with tyrosine phosphorylated protein reduces the activity of tyrosine phosphorylated protein. In a particular embodiment, the tyrosine phosphorylated protein is V
CP, p130 ^cas , EGF receptor, p210 bcr: abl, MAP kinase, Shc (Tiganis et al. 1998 Mol. Cell. Bio.
l. 18: 1622-1634) or the insulin receptor. In certain other embodiments, the protein tyrosine phosphatase is PTP1B, PTP-P.
EST, PTPγ, MKP-1, DEP-1, PTPμ, PTPX1, PTPX
10, SHP2, PTP-PEZ, PTP-MEG1, LC-PTP, TC-P
TP, CD45, LAR or PTPH1.

In yet another aspect of the invention, the invention provides that (i) an invariant aspartic acid residue of a wild-type protein tyrosine phosphatase catalytic domain has a Km of the enzyme less than 1 per minute, although the Km of the enzyme does not change significantly. Substrate-capturing mutants of PTP-PEST substituted with amino acids that result in a decrease in Kcat of up to; and (ii) at least one wild-type tyrosine residue substituted with an amino acid that cannot be phosphorylated, p130 administered to a mammal capable of expressing a ^cas, includes thereby substrate trapping mutant reduces the transforming effects of at least one cancer gene involved in pl30 ^cas phosphorylation undergoes interaction with pl30 ^cas, Provided is a method for reducing the transforming effect of at least one oncogene involved in phosphorylation of ^p130cas . In a particular embodiment, the oncogene is v-crk, v-src or c.
-Ha-ras.

[0017]   In another aspect, the invention provides (i) a wild-type protein tyrosine phosphatase.
The invariant aspartic acid residue in the ze catalytic domain causes a marked change in the enzyme Km.
Not replaced by amino acids that result in a decrease in Kcat of less than 1 per minute
And (ii) at least one wild type tyrosine residue is phosphorylated.
A substrate-capturing mutant of PTP-PEST substituted with an amino acid that cannot
130^casWhen administered to an expressible mammal, whereby the substrate-capturing mutant is p130 ^cas Interaction with p130^casLowers the formation of signaling complexes involved in
P130, including reducing^casTo reduce the formation of signaling complexes involved in
Provide a way to

In another aspect of the invention, (i) the constant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain reduces Kcat by less than 1 per minute, although the Km of the enzyme does not change significantly. Substituted with an amino acid that results in
And (ii) administering a protein tyrosine phosphatase comprising administering to a mammal a substrate-trapping variant of a protein tyrosine phosphatase in which at least one wild-type tyrosine residue is replaced with an amino acid that is not phosphorylated. Provided are methods for reducing the cytotoxic effects associated with overexpression.

In another aspect of the invention, the invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain results in an amino acid that results in a decrease in Kcat of less than 1 per minute, while not significantly altering the Km of the enzyme. Replaced with; and
Provided is an isolated nucleic acid molecule encoding a substrate-trapping mutant protein tyrosine phosphatase in which at least one wild-type tyrosine residue is replaced with an amino acid that is not phosphorylated. In a particular embodiment, the invention provides an antisense oligonucleotide containing at least 15 contiguous nucleotides complementary to a nucleic acid molecule encoding a substrate-capturing mutant protein tyrosine phosphatase described above.

Another aspect of the invention is that the invariant aspartic acid residue of the wild type protein tyrosine phosphatase catalytic domain results in an amino acid that results in a decrease in Kcat of less than 1 per minute, while not causing a significant change in the Km of the enzyme. A fusion protein containing a polypeptide sequence fused to a substrate-trapping mutant protein tyrosine phosphatase in which at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated That is. In a particular embodiment, the polypeptide is an enzyme or variant or fragment thereof. In some embodiments, the polypeptide sequence fused to the substrate-trapping mutant protein tyrosine phosphatase is cleavable by a protease. In certain other embodiments, the polypeptide sequence is an affinity tag polypeptide that has an affinity for a ligand.

In yet another aspect, the present invention provides that the constant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain results in a decrease in Kcat by less than 1 per minute, although the Km of the enzyme does not change significantly. At least one promoter operably linked to a nucleic acid encoding a substrate-capture mutant protein tyrosine phosphatase, which is substituted with an amino acid; and at least one wild-type tyrosine residue is substituted with an amino acid that cannot be phosphorylated Recombinant expression constructs containing individual are provided. In certain embodiments, the promoter is a regulated promoter, and in certain further embodiments, the substrate-capture mutant protein tyrosine phosphatase is expressed as a fusion protein with the polypeptide product of the 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 recombinant viral expression construct. In certain other embodiments, the invention provides a host cell containing the recombinant expression construct described above. In a particular embodiment the host cell is a prokaryotic cell, and in a particular embodiment the host cell is a eukaryotic cell.

In another aspect of the invention, the invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain results in an amino acid that results in a decrease in Kcat of less than 1 per minute, although the Km of the enzyme does not change significantly. Replaced with; and
Recombinant containing at least one promoter operably linked to a nucleic acid sequence encoding a substrate-trapping mutant protein tyrosine phosphatase in which at least one wild-type tyrosine residue is replaced with an amino acid that is not phosphorylated Provided is a method of producing a recombinant substrate-trapping mutant protein tyrosine phosphatase comprising culturing a host cell containing an expression construct. In a particular embodiment, the promoter is a regulated promoter. In certain other embodiments, the invention provides a method of producing a recombinant substrate-capture mutant protein tyrosine phosphatase, the method comprising culturing a host cell infected with a recombinant viral expression construct as described above.

In another aspect of the invention, the constant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain, together with a pharmaceutically acceptable carrier or diluent, is used for 1 min, although the Km of the enzyme does not change significantly. Containing a substrate-trapping mutant protein tyrosine phosphatase substituted with amino acids that result in a decrease in Kcat of less than 1 per unit; and at least one wild-type tyrosine residue is substituted with an amino acid that cannot be phosphorylated A pharmaceutical composition is provided.

[0024] In yet another aspect, the present invention comprises a pharmaceutically acceptable carrier or diluent,
Invariant aspartic acid residues in the wild-type protein tyrosine phosphatase catalytic domain show Kca of less than 1 per minute, although the Km of the enzyme does not change significantly.
containing an agent that interacts with a substrate-trapping mutant protein tyrosine phosphatase substituted with an amino acid that results in a decrease in t; and at least one wild-type tyrosine residue is substituted with an amino acid that cannot be phosphorylated A pharmaceutical composition is provided. In certain other embodiments, the invention provides that (i) the invariant aspartate residue of the wild-type protein tyrosine phosphatase catalytic domain is the enzyme K
m is replaced with an amino acid that causes no significant change but a decrease in Kcat by less than 1 per minute; and (ii) at least one wild type tyrosine residue is replaced with an amino acid that is not phosphorylated. Protein tyrosine phosphatase comprising at least one substrate-trapping mutant protein tyrosine phosphatase; and an auxiliary reagent suitable for use in detecting the presence or absence of a complex of protein tyrosine phosphatase and tyrosine phosphorylated protein. A kit for identifying a tyrosine phosphorylated protein substrate of Escherichia coli is provided.

These and other aspects of the invention will be apparent upon reference to the following detailed description and attached drawings. All references (including websites) disclosed herein are incorporated by reference in their entirety as if each were individually incorporated.

DETAILED DESCRIPTION OF THE INVENTION The present invention shows that the invariant aspartic acid residue of the PTP catalytic domain does not result in a significant change in the Michaelis-Menten constant (Km) of the enzyme, but the catalytic rate constant (Km).
cat) a novel substrate capture derived from PTP mutated to be replaced with an amino acid that results in a decrease in the cat) and further mutated by replacing at least one tyrosine residue with an amino acid that cannot be phosphorylated. Targeted is a sex variant protein tyrosine phosphatase (PTP). The present invention is in part related to certain in
It is based on the unexpected finding that under vivo conditions the PTP enzyme itself may undergo tyrosine phosphorylation in such a way as to alter the interaction between PTP and other molecules, including PTP substrates. As defined herein, the phosphatase is
When it contains the signature motif of [I / V] HCXAGXXR [S / T] G (SEQ ID NO: 36), it is a member of the PTP family. Dual specific PTP,
Thus, PTPs that dephosphorylate both phosphorylated tyrosine and phosphorylated serine or threonine are also suitable for use in the present invention. Suitable PTPs are PTP1B, P
TP-PEST, PTPγ, MKP-1, DEP-1, PTPμ, PTPX1,
PTPX10, SHP2, PTP-PEZ, PTP-MEG1, LC-PTP,
Including but not limited to TC-PTP, CD45, LAR and PTPH1.

As mentioned above, the substrate-capturing mutant PTP is characterized in that the invariant aspartic acid residue of the catalytic domain of wild-type protein tyrosine phosphatase does not cause a significant change in the Km of the enzyme, but is less than 1 per minute. Is derived from a wild-type PTP that has been mutated so that at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated.
In this regard, amino acid sequence analysis of known PTPs revealed that there are 27 invariant residues within the PTP primary structure, including the aspartic acid residue of the catalytic domain, which is invariant in members of the PTP family. (Barford et al.,
1994 Science 263: 1397-1404; Jia et al., 1995.
Science 268: 1754-1758). When the amino acid sequences of multiple PTP family members are aligned (see, eg, FIGS. 1A-E; also see, eg, Bar.
ford et al., 1995 Nature Struct. Biol. 2: 1043
), This invariant aspartic acid residue is invariant among all PTPs, P
It can be easily identified in the catalytic domain region of each PTP sequence at a position corresponding to the TP signature sequence motif, [I / V] HCXAGXXR [S / T] G (SEQ ID NO: 36) (eg WO98 / 04712). Issue; Flint et al.
1997 Proc. Nat. Acad. Sci. 94: 1680 and references cited therein). However, the exact amino acid sequence position numbers of the invariant aspartic acid residues of the catalytic domain are believed to vary from PTP to PTP (variable by sequence shifts that would impose maximizing alignment of the various PTP sequences). For alignment of PTP sequences, see, eg, Barford et al., 1995 Nat.
ure Struct. Biol. 2: 1043).

In particular, the parts of the two PTP polypeptide sequences are numbered according to the amino acid position numbering of one PTP sequence and then the amino acids which are compatible or conserved residues with which the sequences to be compared are matched, eg Polarity at each position (eg D, E, K, R
, H, S, T, N, Q), hydrophobic (eg A, P, V, L, I, M, F, W, Y)
) Or neutral (eg C, G) residues are considered “corresponding” amino acid sequences, regions, fragments, etc. based on the convention of aligning to maximize the number of amino acids that remain. Similarly, candidate PTPs that mutate as provided herein.
Is a DNA sequence of known wild-type PTP.
According to the convention for numbering the position of a nucleic acid sequence in an NA sequence, it can correspond to a known wild-type PTP-encoding DNA sequence, whereby in a given sequence of at least 20 contiguous nucleotides of the sequence. At least 7
The DNA sequence of the candidate PTP is aligned with the known PTP DNA such that 0%, preferably at least 80%, more preferably at least 90% nucleotides are identical. In certain preferred embodiments, the candidate PTP DNA sequence is greater than 95% identical to the corresponding known PTP DNA sequence. In certain particularly preferred embodiments, the portion, region or fragment of the candidate PTP DNA sequence is identical to the corresponding known PTP DNA sequence. As is well known in the art, an individual whose DNA does not contain an irregularity (eg, common or predominant form) in the particular gene responsible for a given trait is considered to be wild-type genetically related to that gene. Although sometimes referred to as having a complement (genotype), the presence of an irregularity known as a mutation in the DNA of the gene, such as the substitution, insertion or deletion of one or more nucleotides, is abrupt The genotype of the mutated or mutant is shown.

As noted above, in some embodiments of the present invention, a substrate-trapping variant in which the constant aspartic acid and at least one tyrosine residue of the catalytic domain is replaced as provided herein. PTP is provided. Identification of catalytic domain invariant aspartic acid residues in PTP sequences other than those disclosed in Barford et al. gov / cgi-bi
n / BLAST), the BLAST algorithm (Altschul.
J. Mol. Biol. 219: 555-565, 1991: Henikoff
And Henikoff, Proc. Natl. Acad. Sci. USA 89:
This can be done by comparing the sequences using computer algorithms well known to those skilled in the art, such as 10915-10919, 1992).

Certain embodiments of the present invention, in part, where the invariant aspartic acid residue does not result in a significant change in the Km of the enzyme, but up to less than 1 per minute (less than 1 min ⁻¹ ) Kca.
It relates to novel PTPs substituted with amino acids that lead to a reduction in t. These P
TPs retain their ability to complex with or bind to their tyrosine phosphorylated substrates, but with diminished catalysis (ie,
The substrate-capturing mutant PTP retains a Km similar to the corresponding wild-type PTP, but 1
(Depending on the activity of the wild-type enzyme compared to a Kcat of less than 1 per minute, it has a Vmax reduced by at least 10 ² -10 ⁵ fold relative to the wild-type enzyme). This decay includes reduced or absent catalytic activity compared to wild-type PTP. For example, the invariant aspartic acid residue is replaced with alanine, valine, leucine, isoleucine,
It can be changed or mutated to proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine or histidine.

The invariant aspartic acid residue does not cause a significant change in the Km of the enzyme, but 1
Preferred as described herein, which is substituted with an amino acid that results in a decrease in Kcat of less than 1 per minute (less than 1 min ⁻¹ ) and at least one tyrosine residue is substituted with an amino acid that cannot be phosphorylated. The substrate-capturing mutant PTP can contain further mutations. In a particularly preferred embodiment, such additional mutations are for substitutions, insertions or deletions (most preferably substitutions) that help stabilize the PTP / substrate complex. For example, the mutation of a serine / threonine residue to an alanine residue in the signature motif (S / T → A mutant) is PT
The rate-limiting step of P-mediated substrate dephosphorylation can be altered. In the case of unmodified PTP, the formation of the transition state can be rate limiting, whereas in the case of the S / T → A mutant the decay of the transition state is rate limiting, thereby stabilizing the PTP / substrate complex. You can Such mutations allow for the substitution of invariant aspartic acid residues in the PTP catalytic domain and the substitution of PTP tyrosine, as provided herein, for example to stabilize the PTP-substrate complex and facilitate its isolation. Can be beneficially combined to As another example, any one or more other amino acids present in wild-type PTP that can be phosphorylated as provided herein (eg, serine, threonine, tyrosine) are phosphorylated. Substitution with amino acids that cannot be considered is considered desirable from the standpoint of stability of the PTP-substrate complex.

As mentioned above, the present invention provides a substrate in which the constant aspartic acid and at least one tyrosine residue of the catalytic domain are replaced, such tyrosine being replaced by an amino acid which is not phosphorylated. Provide a capture mutant PTP. Amino acids that cannot be phosphorylated are, in a preferred embodiment, alanine, cysteine,
It can be aspartic acid, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, arginine, valine or tryptophan. The desirability of tyrosine substitution is that under certain conditions in vivo, the PTP enzyme itself undergoes tyrosine phosphorylation so that it can alter the interaction between PTP and other molecules, including PTP substrates. It is derived from such findings. The PTP substrate may be any natural or unnatural tyrosine phosphorylated peptide, polypeptide capable of specifically binding to PTP as provided herein and / or dephosphorylated by PTP. It also includes proteins. Therefore, substitution of a tyrosine residue found in the wild-type amino acid sequence of a particular PTP with another amino acid as provided herein will result in the amount of complex present and / or the affinity of the mutant PTP for the substrate. The increased stability stabilizes the complex formed by the substrate-trapping mutant PTP of the present invention and the PTP substrate as compared to complex formation using PTP in which the tyrosine residue is not substituted.

As mentioned above, the present invention provides a method of identifying a tyrosine phosphorylated protein that is a substrate for wild-type PTP, in order to provide a substrate-trapping mutant PTP as described herein.
To use. In accordance with this aspect of the invention, a sample containing at least one tyrosine phosphorylated protein is combined with at least one substrate-trapping mutant PTP as provided herein to provide a substrate and mutant PTP. Examine for the presence of a complex containing. The binding interaction between PTP and PTP substrate results in the formation of a complex, which refers to the affinity interaction between PTP and PTP substrate. The complex may include a signaling complex, which refers to any complex that directly or indirectly provides a biological signal by its formation, stable association and / or dissociation. Such a signal is
For example, and not by way of limitation, intracellular and / or intercellular leading to molecular binding, covalent or non-covalent modification of molecular structure, gene expression, gene recombination, gene integration, nucleic acid synthesis or non-cellular particle assembly. Events, and may include endocytosis, phagocytosis, nucleolysis, proteolysis, lipolysis, hydrolysis, catalysis, or other regulatory events.

The measurement of the presence of a stable complex between PTP and PTP substrate can be performed according to the present disclosure using PT
Refers to the use of any methodology known in the art to reveal intermolecular interactions between P and PTP substrates. Such methodologies include, by way of example and not limitation, co-purification, co-precipitation, co-immunoprecipitation, radio or fluorescence assays, Western immunoblot analysis, solid-phase ligand-solvent.
counterligand sorbent) techniques, affinity chromatography and affinity capture including affinity techniques such as surface affinity plasmon resonance. With respect to these and other useful affinity procedures, details of procedures for isolating and characterizing the complex, including affinity procedures, can be found in, for example, Scopes, R .; K. , "Protein Purification: Principles and Practice"
"Pless and Practice)", 1987, Springer-Ve.
rlag, NY; Weir, D .; M. "Experimental Immunology Handbook (Handb
"ook of Experimental Immunology)", 198
6, Blackwell Scientific, Boston; and Herm
anson, G .; T. Et al., “Immobilized Affinity Ligand Technique (Immobi
"Lized Affinity Ligand Technologies)", 1
992, Academic Press, Inc. , California. PTP is equal to or greater than about 10 ⁴ M ^-1 , preferably equal to or greater than about 10 ⁵ M ^-1 , and more preferably equal to or greater than about 10 ⁶ M ^-1 Even more preferably, a K equal to or greater than about 10 ⁷ M ^-1 to 10 ⁹ M ^-1.
When bound to a substrate with a, PTP may interact with the PTP substrate via specific binding. The affinities of binding partners such as PTP and PTP substrates are determined by conventional techniques, eg Scatchard et al., Ann. N. Y. Acad. Sci. 51:
It can be easily measured by using the method described in 660 (1949).

Although not wishing to be bound by theory, a phosphorylated tyrosine residue that is part of the PTP molecule itself is a tyrosine phosphorylated protein that is capable of binding and / or dephosphorylating PTP molecules. It is believed that it may affect the interactions between the containing PTP substrate molecules. According to this non-limiting theory, a conserved tyrosine residue present in the primary structure of PTP is the result of an invariant cysteine residue found in the signature motif (as described above) located in the active site of the PTP catalytic domain. Radical and PT
It is believed to be a receptor for transferring phosphate groups from the highly reactive thiophosphate intermediate that can be formed between the phosphate groups present on the P substrate phosphorylated protein in the form of phosphotyrosine. Therefore, a conserved tyrosine residue in the PTP active site gives a hydrophobic interaction with the substrate phosphotyrosine, resulting in P for that substrate.
Although it is believed to promote intermolecular orientation of TP and also act as a phosphate acceptor, the invention is not so limited.

As mentioned above, the present invention provides a mutant PTP in which at least one tyrosine residue is replaced with an amino acid which cannot be phosphorylated. Preferably the tyrosine residues are located in the PTP catalytic domain, such domains being different P's as described above.
Refers to a region of approximately 250 amino acids that is highly conserved between TPs (eg Barfor
d, 1998 Ann. Rev. Biophys. Biomol. Struct
． 27: 133; Jia, 1997 Biochem. Cell Biol. 7
5:17; Van Vector et al., 1998 Curr. Opin Gene
t. Devel. See also 8: 112). More preferably, the tyrosine residue is located in the PTP active site and such site comprises a PTP signature motif,
And PTP binding site pockets or "cradles" for substrate binding and dephosphorylation
A PTP catalytic domain comprising an amino acid forming a "bend cage" and further comprising an invariant aspartate-containing loop (if present) and an adjacent peptide backbone sequence that contributes to substrate recognition and catalysis (see, eg, Jia, 1997). Refers to the area inside. In the most preferred embodiment, the tyrosine residue is replaced with phenylalanine, and in another most preferred embodiment, the tyrosine residue corresponds to the tyrosine located at position 676 of the amino acid sequence of human PTPH1 and at the same time in Figure 1. PTP to show
It is a conserved residue corresponding to the amino acid residue at position 46 of the -1B sequence. In another preferred embodiment, the tyrosine residues are PTP conserved residues, including 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 another preferred embodiment, the tyrosine residue is replaced with an amino acid that stabilizes the complex formed by PTP and at least one substrate molecule as provided herein.

As mentioned above, PTPs considered useful according to the invention include any PTP having an invariant aspartic acid residue and a tyrosine residue at the corresponding position in the catalytic domain. By way of illustration and not limitation, in some preferred embodiments of the invention, the substrate-trapping mutant PTP has at least one tyrosine residue found in the corresponding wild-type sequence substituted with phenylalanine. In some particularly preferred embodiments, the PTP is PTPH1, PTPH1 (Y) with invariant aspartic acid substituted with alanine and tyrosine at position 676 substituted with phenylalanine.
676F / D811A). In some other embodiments, the PTP is a mutation wherein the cysteine found in the corresponding wild-type sequence is replaced with serine and at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. Body PT
P-PEST phosphatase. However, variant PTPs other than those specifically mentioned herein also align the amino acid sequence of the PTP catalytic domain with the amino acid sequence of PTP described herein, including those provided in the cited references. By identifying an invariant aspartic acid residue and at least one tyrosine residue in the catalytic domain and altering these residues, for example by site-directed mutagenesis of the DNA encoding PTP. It must be recognized that it can be made in.

Modification of DNA uses primers that introduce and amplify modifications into the DNA template, such as site-specific or site-directed mutagenesis of DNA encoding PTP and PCR splicing (SOE) by overlap extension. Can be carried out by various methods including the DNA amplification method. Site-directed mutagenesis typically involves
It is carried out using phage vectors with single and double stranded forms, such as the well known and commercially available M13 phage vector. Other suitable vectors containing an origin of replication for single-stranded phage may also be used (eg, Veira et al., Me.
th. Enzymol. 15: 3, 1987). Site-directed mutagenesis is generally carried out by preparing a single-stranded vector encoding the protein of interest (eg, a member of the PTP family). An oligonucleotide primer containing the desired mutation in the region of homology to the DNA in the single-stranded vector is annealed to the vector and then a DNA polymerase such as E. coli DNA polymerase I (Klenow fragment). Add. Such a DNA polymerase uses the double-stranded region as a primer to create a heteroduplex in which one strand encodes a modified sequence and the other strand encodes the original sequence. Further disclosure regarding site-directed mutagenesis can be found, for example, in Kunkel et al. (Methods).
in Enzymol. 154: 367, 1987); and U.S. Pat.
18, 584 and 4,737,462. The heteroduplex is introduced into an appropriate bacterial cell and clones containing the desired mutation are selected. The resulting modified DNA molecule can be recombinantly expressed in a suitable host cell to produce the modified protein.

Specific substitutions of individual amino acids through the introduction of site-directed mutagenesis are well known and can be performed according to methodologies familiar to those of skill in the art. The effect of the resulting mutant PTPs on catalytic activity is as provided herein and as previously disclosed (eg, WO98 / 04712; Flint et al., 1997 Proc. Na).
t. Acad. Sci. 94: 1680), can be empirically determined by simply testing the resulting modified protein for conservation of Km and reduction of Kcat to less than 1 per minute. The effect on the ability of the resulting mutant PTP molecule to tyrosine phosphorylate is also as provided herein, for example, after exposure of the mutant to in vitro or in vivo conditions that can act as a PTK acceptor, simply Such variants can be empirically determined by testing for the presence of phosphotyrosine.

Specific examples of PTP variants described below are DA (aspartic acid to alanine) variants, YF (tyrosine to phenylalanine) variants, CS variants and combinations thereof, although the substrate of the invention It will be clear that the capture mutant PTPs are not limited to these amino acid substitutions. An invariant aspartic acid residue is any amino acid that does not result in a significant change in the Km of the enzyme, eg, by site-directed mutagenesis, but results in a decrease in Kcat of less than 1 per minute (less than 1 min ⁻¹ ). Can be changed to For example, an invariant aspartic acid residue is replaced with alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine or histidine, or a derivative or mutation. Other natural or unnatural amino acids known in the art, including the body and the like, can be changed or mutated. Similarly, substitution of at least one tyrosine residue cannot be phosphorylated (ie, a stable covalent modification of the hydroxyl of the amino acid side chain with a phosphate group) for any amino acid, such as alanine, cysteine, aspartic acid. , Glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, arginine, valine or tryptophan, or other natural or unnatural amino acids known in the art, including derivatives, variants, etc. Can be implemented by

The nucleic acid of the present invention may take the form of RNA or DNA, and such DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, but if single-stranded may be the coding strand or non-coding (antisense) strand. The invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain does not result in a significant change in the Km of the enzyme, but 1 per minute
A nucleic acid molecule encoding a substrate-trapping mutant PTP, which has been replaced with an amino acid that results in a decrease in Kcat by less than 1 and at least one wild-type tyrosine residue has been replaced with an amino acid that cannot be phosphorylated. Is identical to a coding sequence known in the art for any given PTP as described above, or
It may be the same PTP, but different coding sequences as a result of duplication or degeneracy of the genetic code.

The invention further provides variants of the nucleic acids described herein that encode fragments, analogs and derivatives of PTP polypeptides, including mutated PTPs such as the substrate-trapping variant PTP. Regarding A variant of a PTP-encoding nucleic acid can be a naturally occurring allelic variant or a non-naturally occurring variant of the nucleic acid. As is known in the art, allelic variants include P encoded by any of them.
It is an alternative form of nucleic acid sequence that may have at least one of one or more nucleotide substitutions, deletions or additions that does not significantly alter the function of the TP polypeptide.

Equivalent DNA constructs encoding various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences not required for biological activity are also included in the invention. For example, a sequence encoding a Cys residue that is not essential for biological activity can be deleted or replaced with another amino acid to prevent the formation of an incorrect intramolecular disulfide bridge during regeneration. It can be modified to prevent it. Other equivalents can be prepared by modifying flanking dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. EP
212,914 discloses the use of site-directed mutagenesis to inactivate the KEX2 protease processing site in proteins. KEX
The two protease processing sites are Arg-Arg, Arg-Lys, and L.
The ys-Arg pair is inactivated by deleting, adding, or substituting residues to modify the occurrence of these flanking basic residues. Lys-L
The ys pairing is much less susceptible to KEX2 cleavage, and Arg-Lys or Lys-
The conversion of Arg to Lys-Lys is the traditional and preferred approach to inactivate the KEX2 site.

The invention further provides a method for producing a recombinant PTP polypeptide by culturing a host cell containing a PTP polypeptide, including a substrate-trapping mutant PTP, in particular a PTP expression construct, and isolated. Recombinant PTP polypeptide. The polypeptides and nucleic acids of the invention are preferably provided in isolated form and, in some preferred embodiments, purified to homogeneity. When referring to PTP polypeptides or fusion proteins, including substrate-capturing mutant PTP, the terms "fragment,""derivative," and "analog" refer to essentially the same biological moieties as such polypeptides. Refers to any PTP polypeptide or fusion protein that retains function or activity. Thus, analogs include proproteins that can be activated by cleavage of the proprotein portion to produce an active PTP polypeptide. The polypeptide of the present invention may be a recombinant polypeptide or a synthetic polypeptide, but is preferably a recombinant polypeptide.

Fragments, derivatives or analogs of PTP polypeptides or fusion proteins, including substrate-capturing mutant PTPs, include (i) one or more amino acid residues with or without conserved amino acid residues. Substituted with groups (preferably conserved amino acid residues), such substituted amino acid residues being or not being encoded by the genetic code, or (ii) one or more Containing a substituent, or (iii) a PTP polypeptide fused to another compound, such as a compound (eg, polyethylene glycol) that extends the half-life of the polypeptide, or (Iv) additional amino acids are PT, including those used for purification of PTP polypeptide or proprotein sequences.
It may be fused to a P polypeptide. Such fragments, derivatives and analogs are considered to be within the skill of those in the art given the teachings herein.

Polypeptides of the present invention include PTP polypeptides and fusion proteins with amino acid sequences that are identical or similar to PTP sequences known in the art. For example, by way of illustration and not limitation, the human PT referred to below in the examples
P polypeptides (including substrate-capturing variant PTP) are polypeptides and portions of such polypeptides (such portions of PTP polypeptides described in the references and examples cited herein). Is generally at least 30 amino acids,
More preferably at least 50 amino acids) and at least 70% similar (preferably 70% identical), more preferably 90% similar (more preferably 90% identical), and even more It preferably has 95% similarity (even more preferably 95% identity) and is considered for use according to the invention.

“Similarity” between two polypeptides, as is known in the art.
By comparing the amino acid sequence of the polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide (eg, GENE as described above).
WORKS, Align or BLAST algorithms). Fragments or parts of the polypeptides of the invention may be used to produce the corresponding full-length polypeptide by peptide synthesis; thus, such fragments may be used as intermediates to produce the full-length polypeptide. Fragments or portions of the nucleic acids of the invention can be used to synthesize the full length nucleic acids of the invention.

The term “isolated” means that the material is removed from its original environment (eg, the natural environment if the material occurs naturally). For example, a naturally occurring nucleic acid or polypeptide present in a living animal has not been isolated, but the same nucleic acid or polypeptide separated from some or all of the coexisting substances in the natural system has been isolated. Such nucleic acid may be part of a vector and / or such nucleic acid or polypeptide may be part of a composition, but such vector or composition is in its natural environment for the nucleic acid or polypeptide. It is also isolated because it is not part. The term "gene" means the segment of DNA involved in the production of polypeptide chains; it is the "leader and trailer" region before and after the coding region and intervening sequences (introns) between individual coding segments (exons). )including.

As described herein, the present invention provides that the invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain does not result in a significant change in the Km of the enzyme, but up to less than 1 per minute. Poly fused to a substrate-capturing mutant PTP, which has been replaced with an amino acid that results in a decrease in Kcat, and in addition at least one wild-type protein tyrosine phosphatase tyrosine residue has been replaced with an amino acid that cannot be phosphorylated. A fusion protein comprising the peptide is provided. Such PTP fusion proteins are, for example, by way of example and not limitation, PTP polypeptides fused to additional functional or non-functional polypeptide sequences that allow the detection, separation and / or purification of PTP fusion proteins. Encoded by a nucleic acid having a substrate-trapping variant PTP coding sequence fused in frame to an additional coding sequence to provide for expression of the sequence. Such PTP fusion proteins have protein-protein affinity,
Peptide purification by polypeptide purification based on metal affinity or charge affinity
Specific protease cleavage of a fusion protein containing a fusion sequence that can be cleaved by a protease so that the polypeptide can be separated from the fusion protein may allow detection, isolation and / or purification of the PTP fusion protein.

Therefore, the PTP fusion protein can include an affinity-tagged polypeptide sequence,
This refers to a polypeptide or peptide that adds to PTP to facilitate detection and isolation of PTP through specific affinity interaction with a ligand. The ligand is
The affinity label can be any molecule, receptor, counter-receptor, antibody, etc. capable of interacting through a specific binding interaction as provided herein. Such peptides are described, for example, in US Pat. No. 5,011,912 and Hopp et al.
88 Bio / Technology 6: 1204).
His or antigen identification peptide, or XPRESS ^™ epitope tag (Inv
(itrogen, Carlsbad, CA). Affinity sequences can be used, for example, in the case of bacterial hosts, pB to facilitate purification of the mature polypeptide fused to the marker.
A hexahistidine tag as supplied by AD / His (Invitrogen) or pQE-9 vector, or, for example, the affinity sequence is a hemagglutinin (HA) when using a mammalian host, eg COS-7 cells. It can be a sign. The HA tag corresponds to an antibody defined epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984 Cell 3).
7: 767).

The PTP fusion protein may further comprise an immunoglobulin constant region polypeptide added to the PTP to facilitate detection, isolation and / or localization of the PTP.
The immunoglobulin constant region polypeptide is preferably fused to the C-terminus of the PTP polypeptide. Various parts of antibody-derived polypeptides (including Fc domain)
General preparation of fusion proteins comprising a heterologous polypeptide fused to
kenazi et al. (PNAS USA 88: 10535, 1991) and Byr.
n, et al. (Nature 344: 677, 1990). P
The gene fusion encoding the TP: Fc fusion protein is inserted into an appropriate expression vector. In a particular embodiment of the invention, PTP: Fc fusion proteins can assemble like antibody molecules, forming interchain disulfide bonds between Fc polypeptides, resulting in dimeric PTP fusion proteins.

A PTP fusion having a specific binding affinity for a preselected antigen by a fusion polypeptide comprising an immunoglobulin V region domain encoded by a DNA sequence linked in frame to a PTP encoding sequence. Proteins are also within the scope of the invention, including variants and fragments thereof as provided herein. General strategies for the construction of fusion proteins with immunoglobulin V region fusion polypeptides are described, for example, in EP 0318554; S. 5,132,405; U.S.P.
S. 5,091,513 and U.S.P. S. No. 5,476,786.

The nucleic acids of the invention may also encode a fusion protein comprising a PTP polypeptide fused to another polypeptide having the desired affinity properties, for example an enzyme such as glutathione-S-transferase. As another example, the PTP fusion protein can also be
It may include a PTP polypeptide fused to a Protein A polypeptide of Staphylococcus aureus; Uses are generally disclosed, for example, in US Pat. No. 5,100,788. Other useful affinity polypeptides for the construction of PTP fusion proteins are described, for example, in WO 89/03422; S. 5,489,528; U.S.P.
S. 5,672,691; WO 93/24631; U.S.P. S. 5,168,
049; U. S. Streptavidin fusion proteins and avidin fusion proteins as disclosed in US Pat. No. 5,272,254 and others (eg EP 511,
No. 747). As provided in the text and in the references cited, PTP polypeptide sequences, including substrate-capturing mutant PTPs, may be full length fusion polypeptides, or alternatively may be variants or fragments thereof. It can be fused to a polypeptide sequence.

The invention also provides that the fusion protein is secreted from the cell to the cell nucleus, in the lumen of the endoplasmic reticulum (ER), and from the cell through the classical ER-Golgi secretory pathway (eg von Heijne, J. Membrane Biol. 115: 195-2.
01, 1990), to associate with specific cytoplasmic components, including the cytoplasmic domain of the transmembrane cell surface receptor, for incorporation into the plasma membrane, or of various known intracellular protein sorting mechanisms familiar to those of skill in the art. To direct to specific non-cellular locations by either (eg, Rothman, Nature 372: 55-6.
3, 1994; Adrani et al., 1998 J .; Biol. Chem. 273:
10317, and references cited herein). Consider a PTP fusion protein comprising a directed polypeptide sequence. Thus, these and related embodiments are
Included in the compositions and methods of the invention are those directed to targeting the polypeptide of interest to a predetermined intracellular membrane or extracellular location.

The invention also relates to vectors and constructs comprising the nucleic acids of the invention, in particular “recombinant expression constructs”, comprising any nucleic acid encoding a PTP polypeptide according to the invention as provided above. The invention relates to host cells genetically engineered with the vectors and / or constructs of the invention and to the recombinant production of the PTP polypeptides and fusion proteins of the invention, or fragments or variants thereof. The PTP protein can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, in Sambrook et al., "Molecular Cloning: A Laboratory".
Oral Manual) ", 2nd Edition, Cold Spring Har
Bor, New York (1989).

Generally, recombinant expression vectors include origins of replication, selectable markers that allow transformation of host cells, such as the ampicillin resistance gene of E. coli and brewer's yeast (S.c.
erevisiae) TRP1 gene and a promoter derived from a highly expressed gene that directs transcription of downstream structural sequences. Such promoters can be derived from operons that encode glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat shock proteins, among others. Heterologous structural sequences are assembled at appropriate stages with translation initiation and termination sequences. Optionally, the heterologous sequence may encode a fusion protein containing an N-terminal identification peptide that provides desired properties, such as stabilization of the expressed recombinant product or easy purification.

Useful expression constructs for bacterial use express a structural DNA sequence encoding a desired protein with appropriate translational initiation and termination signals, in operable phase with a functional promoter. Constructed by inserting into the vector. The construct may contain one or more phenotypic selection markers and an origin of replication that ensures the maintenance of the vector construct and, if desired, confer amplification in the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis.
btilis), Salmonella typhimurii
um) and Pseudomonas, Streptomyces, and Staphylococcus.
Various species in s) are included, but others may be used as an option. Any other plasmid or vector can be used as long as they are replicable and viable in the host.

As a representative but non-limiting example, useful expression vectors for use in bacteria include selectable markers and the well known cloning vector pBR322 (ATCC 3
7017) and may contain a bacterial origin of replication derived from a commercially available plasmid containing the genetic element. Such commercially available vectors are, for example, pKK223-3 (Pha
rmcia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wisconsin, USA). The "backbone" portion of these pBR322 is combined with an appropriate promoter and expressing structural sequence.

After transformation of a suitable host strain and growth of the host strain to a suitable cell density, the selected promoter, if it is a regulated promoter as provided herein, is Induce by appropriate means (eg temperature shift or chemical induction) and culture the cells for a further period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells used in the expression of proteins can be disrupted by any conventional method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysing agents: such methods are known to those of skill in the art. Is well known to.

Thus, for example, a nucleic acid of the invention as provided herein is included in any one of a variety of expression vector constructs as a recombinant expression construct for expressing a PTP polypeptide. sell. Such vectors and constructs include chromosomal, non-chromosomal and synthetic DNA sequences such as derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmid and phage DNA, vaccinia, adenovirus. , Fowlpox virus and viral DNA such as pseudorabies. But any other vector
As long as it is replicable and viable in the host, it can be used for the preparation of recombinant expression constructs.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. Generally, the DNA sequence is inserted into the appropriate restriction endonuclease site by techniques known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases, and the like, and various separation techniques are well known to those of ordinary skill in the art and are commonly used. Is used for. Some standard techniques are described in, for example, Ausubel et al.
93, “Current Protocol in Molecular Biology (Current Protocol)
ol's in Molecular Biology) ", Greene Pu
bl. Assoc. Inc. & John Wiley & Sons, Inc
． Boston, MA); Sambrook et al. (1989, "Molecular Cloning", Second Edition, Cold Spring).
Harbor Laboratory, Plainview, NY); Man
iatis et al. (1982, “Molecular Cloning (Molecular Clon
ing) ", Cold Spring Harbor Laboratory,
Plainview, NY) and others.

At least one DNA sequence in the expression vector is used to direct mRNA synthesis.
Operatively linked to one suitable expression control sequence (eg a promoter or a regulated promoter). Representative examples of such expression control sequences, LTR or SV40 promoter, the E. coli lac or trp, is known to control phage .lambda.P _L promoter, and the expression of genes in prokaryotic or eukaryotic cells or their viruses Including other promoters. The promoter region can be selected from any desired gene using the CAT (chloramphenicol transferase) vector or other vector with a selectable marker. Two suitable vectors are pKK232-8 and pCM7. The specifically named bacterial promoters are:
lacI, lacZ, T3, T7, gpt, λP _R , P _L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, retrovirus-derived LTRs and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art, with some particularly preferred sets comprising at least one promoter or regulatable promoter operably linked to a nucleic acid encoding a PTP polypeptide. The preparation of recombinant expression constructs is described herein.

As mentioned above, in some embodiments the vector may be a viral vector, such as a retroviral vector. For example, retroviruses that can induce retroviral plasmid vectors are Moloney murine leukemia virus, splenic necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, gibbon ape leukemia virus, human immunodeficiency virus. Viruses, adenovirus, Myeloproliferative sarcoma virus, and mammary adenocarcinoma virus, but are not limited thereto.

Viral vectors contain one or more promoters. Suitable promoters that may be used include, but are not limited to, the retroviral LTR; SV40 promoter, and Miller et al., Biotechniques 7: 980-990.
(1989) human cytomegalovirus (CMV) promoter, or any other promoter (eg, but not limited to histone, po.
l III, cell promoters such as eukaryotic cell promoters, including β-actin promoter). Other viral promoters that may be used include, but are not limited to, adenovirus promoter, thymidine kinase (TK).
Examples of the promoter include B19 parvovirus promoter. Selection of the appropriate promoter will be apparent to those of skill in the art from the teachings contained herein and can be made from any of the aforementioned regulated promoters or the above promoters.

Retroviral plasmid vectors are used to transduce packaging cell lines to form production cell lines. Examples of packaging cells that may be transfected include, but are not limited to, Miller et al., Human Gene Therapy, 1:
PE501, PA317, Ψ-2, Ψ-AM, P, as described in 5-14.
A-12, T19-14X, VT-19-17-H2, ΨCRE, ΨCRIP,
GP + E-86, GP + envAm12, and DAN cell lines.
The vector may transduce the packaging cells by any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and calcium phosphate precipitation methods. As an option, the retroviral plasmid vector may be encapsulated in liposomes or attached to lipids and then administered to the host.

The producer cell line produces infectious retroviral vector particles that include a nucleic acid sequence encoding a PTP polypeptide or fusion protein. The retroviral vector particles may then be used to transduce eukaryotic cells in vitro or in vivo. Transduced eukaryotic cells express a nucleic acid encoding a PTP polypeptide or fusion protein. Eukaryotic cells that may be transduced include, but are not limited to, embryonic stem cells, embryonal tumor cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, keratinocytes, endothelial cells, bronchial epithelial cells and various other culture compatibles. Sex cell lines.

As another example of an embodiment of the invention for preparing a recombinant PTP expression construct using a viral vector, in one preferred embodiment, transduction with a recombinant viral construct that directs the expression of a PTP polypeptide or fusion protein. The engineered host cells will be able to produce viral particles that contain the expressed PTP polypeptide or fusion protein obtained from the portion of the host cell membrane that is taken up by the viral particles during viral budding. In another preferred embodiment, the nucleic acid sequence encoding PTP is cloned into a baculovirus shuttle vector, which is then recombined with baculovirus, eg, Baculovirus Expressi.
on Protocols, Methods in Molecular Bi
logic, Vol. 39, Christopher D.L. Richardso
n Press, Human Press, Totowa, NJ, 1995; Piwni
ca-Worms, “Expression of Proteins in Insect Cells Using Baculovirus Vectors (Expression of Proteins in I
nsect Cells Using Baculoviral Vector
s) ”, Chapter 16 §II,“ Short Protocols in Molecular Biology (Sho
rt Protocols in Molecular Biology) ", 2nd edition, edited by Ausubel et al., John Wiley & Sons, Ne.
w York, New York, 1992, p. Recombinant baculovirus expression constructs used to infect Sf9 host cells as described in 16-32 to 16-48 are generated.

In another aspect, the invention relates to a host cell containing the recombinant PTP expression construct. Host cells are genetically engineered (transduced, transformed or transfected) with the vectors and / or expression constructs of the invention, which can be, for example, cloning vectors, shuttle vectors or expression constructs. The vector or construct may be in the form of, for example, a plasmid, virus particle, phage or the like. The genetically engineered host cells are suitable for activating promoters, for selecting transformants or for amplifying specific genes such as those encoding PTP polypeptides or PTP fusion proteins. It can be cultured in a modified conventional nutrient medium. The culture conditions for a particular host cell selected for expression of temperature, pH, etc. will be readily apparent to those of ordinary skill in the art.

The host cell can be a higher eukaryotic cell such as a mammalian cell, or a lower eukaryotic cell such as a yeast cell, or the host cell can be a prokaryotic cell such as a bacterial cell. Representative examples of suitable host cells according to the present invention include, but are not limited to, bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; yeast and the like. Fungal cells of Drosophila S2 cells,
Insect cells such as Spodoptera Sf9; animal cells such as CHO cells, COS cells or 293 cells; adenoviruses, plant cells, or in vitro
Any suitable cell already adapted for growth in o or newly established as such may be mentioned. Selection of the appropriate host will be considered to be within the skill of those in the art given the teachings herein.

A variety of mammalian cell culture systems can also be used to express recombinant protein. Thus, the present invention provides that the constant aspartic acid residue of the wild type protein tyrosine phosphatase catalytic domain does not result in a significant change in the Km of the enzyme, but
Substrate-capturing mutation in which cat is replaced with an amino acid that results in a reduction of less than 1 per minute, and at least one tyrosine residue of the wild-type protein tyrosine phosphatase is replaced with an amino acid that cannot be phosphorylated. Of recombinant substrate-capturing mutant protein tyrosine phosphatase by culturing a host cell containing a recombinant expression construct containing at least one promoter operably linked to a nucleic acid sequence encoding a somatic protein tyrosine phosphatase. Part of the method. In some embodiments, the promoter may be a regulated promoter as provided herein, eg a tetracycline repressible promoter. In some embodiments, the recombinant expression construct is a recombinant viral expression construct as provided herein. Examples of mammalian expression systems include Gluz
man, Cell 23: 175 (1981), the COS-7 strain of monkey kidney fibroblasts, and other cell lines capable of expressing compatible vectors,
Examples include C127 cell line, 3T3 cell line, CHO cell line, HeLa cell line and BHK cell line. The mammalian expression vector contains an origin of replication, a suitable promoter and enhancer, and also contains any necessary ribosome binding sites, polyadenylation sites, as described herein for the preparation of PTP expression constructs, It also contains splicing donor, splicing acceptor sites, transcription termination sequences and 5'flanking nontranscribed sequences. DNA sequences derived from SV40 splicing and polyadenylation sites will be used to provide the necessary nontranscribed genetic elements. Introduction of the construct into the host cell can be performed by a variety of methods familiar to those of skill in the art including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation (Davis et al., 1986).
, Basic Methods in Molecular Biology)
.

Nucleic acid for use as antisense agents, comprising an antisense oligonucleotide and a ribozyme specific for a nucleic acid sequence encoding PTP (such as a substrate-trapping variant PTP) or variant or fragment thereof. Identification of molecules, as well as identification of DNA oligonucleotides encoding PTP genes (such as substrate-trapping mutant PTP) for targeted delivery in gene therapy includes methods well known in the art. For example, the desired properties, length and other characteristics of such oligonucleotides are well known. In certain preferred embodiments, such antisense oligonucleotides are substrate-capturing mutant P as provided herein.
It contains at least 15 contiguous nucleotides complementary to the isolated nucleic acid molecule encoding TP. Antisense oligonucleotides are typically phosphorothioates, methylphosphonates, sulfones, sulphates, ketyls, phosphorodithioates, phosphoramidates, phosphates, and other such linkages (eg, Agrwal et al., Tetrehedron Lett. 28:35
39-3542 (1987); Miller et al., J. Am. Am. Chem. Soc.
93: 6657-6665 (1971); Stec et al., Tetrehedron.
Lett. 26: 2191-2194 (1985); Moody et al., Nucl.
． Acids Res. 12: 4769-4782 (1989); Uznans.
ki et al., Nucl. Acids Res. (1989); Letsinger et al., Tetrahedron 40: 137-143 (1984); Eckste.
in, Annu. Rev. Biochem. 54: 367-402 (1985).
Eckstein, Trends Biol. Sci. 14: 97-100
(1989); Stein, "Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression (Oligodeoxynucleotides, Antis.
Sense Inhibitors of Gene Expression) "
Edited by Cohen, Macmillan Press, London, p.
97-117 (1989); Jager et al., Biochemistry 27:
7237-7246 (1988)) and is designed to withstand degradation by endogenous nucleolytic enzymes.

Antisense nucleotides are oligonucleotides that bind in a sequence-specific manner to nucleic acids such as mRNA or DNA. When bound to mRNA with complementary sequences, antisense interferes with translation of mRNA (eg, Altman).
Et al., U.S. Pat. No. 5,168,053; Inouye, U.S. Pat. No. 5,1.
90,931; Burch, US Pat. No. 5,135,917;
Smith, US Pat. No. 5,087,617, and Clusel et al., (1993) Nucl. A
cids Res. 21: 3405-3411). Triplex molecules refer to single DNA strands that bind to double-stranded DNA to form collinear triple-stranded molecules, thereby interfering with transcription (eg, on double-stranded DNA). Hogan et al., US Pat. No. 5,176,996, et al., Which describes methods for making synthetic oligonucleotides that bind to the target site of E. coli).

According to this aspect of the invention, particularly useful antisense nucleotides and triplex molecules include PTP polypeptides (substrate trapping mutants) such that inhibition of translation of the mRNA encoding the PTP polypeptide is effected. DNA encoding PTP, etc.
Or a molecule that is complementary to or binds to the sense strand of the mRNA.

Ribozymes are RNA molecules that specifically cleave RNA substrates such as mRNA, resulting in specific inhibition or interference with cellular gene expression. There are at least five known classes of ribozymes involved in RNA strand scission and / or RNA strand ligation. Ribozymes can target any RNA transcript and catalytically cleave such transcript (eg, US Pat. No. 5,272).
, 262; U.S. Pat. No. 5,144,019; and U.S. Pat. No. 5,168,053 to Cech et al., U.S. Pat. No. 5,180,818, U.S. Pat. 5,116,742 and US Pat. No. 5,093,
246, etc.). According to one aspect of the invention, any such PTP (such as substrate-capture mutant PTP) mRNA-specific ribozyme, or nucleic acid encoding such a ribozyme, is delivered to a host cell, resulting in inhibition of PTP gene expression. Could be Thus, ribozymes and the like can be delivered to host cells by DNA encoding the ribozyme linked to a eukaryotic promoter, such as a eukaryotic viral promoter, such that the ribozyme is directly transcribed when introduced into the nucleus.

The expressed recombinant PTP polypeptide or fusion protein (including substrate-trapping mutant PTP, etc.) is in intact host cells; intact cell organelles such as cell membranes, intracellular vesicles or other organelles. Or in disrupted cell preparations including but not limited to cell homogenates or lysates, microsomes, unilamellar and multilamellar vesicles or other preparations. On the other hand, the expressed recombinant PTP polypeptide or fusion protein may be subjected to ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxy. It can be recovered and purified from recombinant cell culture by methods such as apatite chromatography and lectin chromatography. Protein refolding steps can optionally be used in completing the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.

Focusing on another aspect of the present invention, tyrosine phosphorylated protein which is a substrate of PTP [tyrosine phosphorylated protei
n)] is provided. As used herein, "sample" refers to a biological sample containing at least one tyrosine phosphorylated protein, a blood sample, biopsy specimen, tissue explant derived from a subject or biological source. It can be provided by obtaining pieces, organ cultures or any other tissue or cell preparation. In addition, the sample may be morphologically complete or in a physical state such as dissociated, dissociated, solubilized, fractionated, homogenized, processed to treat the sample from a subject or biological source. It will refer to a tissue or cell preparation that has been disrupted by chemical or chemical extraction, milling, lyophilization, sonication or any other means. In some 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. is there. The subject or biological source can be a human or non-human animal, a primary cell culture or a genetically engineered cell that can include, but is not limited to, a chromosomally integrated nucleic acid sequence or an episomal recombinant nucleic acid sequence. Culture-compatible cell line (for which), immortalized cell line or immortalizable cell line, somatic cell hybrid cell line, differentiated cell line or differentiable cell line, transformed cell line, etc. May be Optionally, in certain circumstances, treating cells within the biological sample with pervanadate as described herein to enrich the sample for tyrosine phosphorylated proteins. Would be desirable. Other means can also be used to increase the percentage of tyrosine phosphorylated proteins present in the sample, the subject being a cell line transformed with at least one gene encoding a protein tyrosine kinase, or Includes the use of biological sources. Additionally or alternatively, tyrosine phosphorylation of a protein may be used in a subject using various methods well known in the art and any one or more of the compositions known in the art to stimulate protein tyrosine kinase activity. Alternatively, it may be stimulated in cells of a biological source. These stimuli would include, but are not limited to, exposure of cells to cytokines, growth factors, hormones, peptides, small molecule-mediated factors or other agents that induce PTK-mediated protein tyrosine phosphorylation. Such agents include, for example, interleukins, interferons, human growth hormone, insulin and fibroblast growth factor (FGF), as well as other agents familiar to those of skill in the art.

According to the invention, a sample containing at least one tyrosine phosphorylated protein has sufficient conditions and sufficient conditions to allow the formation of a complex between the tyrosine phosphorylated protein and the substrate-trapping mutant PTP. In time, it is mixed with at least one substrate-trapping mutant PTP provided herein. Suitable conditions for the formation of such complexes are readily determined based on the teachings provided herein known in the art, including solution conditions and methods for detecting the presence of the complex. be able to.
Next, the presence or absence of a complex containing the tyrosine phosphorylated protein and the substrate-trapping mutant PTP is determined, and the presence of the complex means that the tyrosine phosphorylated protein forms a substrate of PTP with which the complex is formed. Is shown.

Substrate-capturing mutant PTPs associated in complex with tyrosine phosphorylated protein substrates have been described in the art to elucidate intermolecular interactions between PTPs and PTP substrates, as described above. By any of a variety of known techniques, eg co-purification; co-precipitation; co-immunoprecipitation; radiometric or fluorometric assays; Western immunoblot analysis; solid phase ligand-compatible ligand adsorption techniques, affinity chromatography and It can be identified by affinity capture such as affinity techniques such as surface affinity plasmon resonance (see, eg, US Pat. No. 5,352,660). The determination of the presence of the PTP / substrate complex may use various antibodies such as monoclonal, polyclonal, chimeric and single chain antibodies that specifically bind to PTP or tyrosine phosphorylated protein substrates. Labeled PTP and / or labeled tyrosine phosphorylated substrate can also be used to detect the presence of the complex. The PTP or phosphorylated protein is labeled by covalently or non-covalently attaching a suitable reporter molecule or moiety such as any of various enzymes, fluorescent substances, luminescent substances, radioactive substances and the like. Can be done. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, biotin, alkaline phosphatase, β-galactosidase, acetylcholinesterase, and the like. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone,
Fluorescein, fluorescein isothiacinate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin and the like can be mentioned. Suitable emissive materials include luminol, and suitable emissive materials include radioactive phosphorus [ ³² P], iodine [ ¹²⁵ I or ¹³¹ I] or tritium [ ³ H].

Using such a method, a complex of PTP1B and p210bcr: abl, P
Complex with TP-PEST and pl30 ^cas, complex with TC-PTP and Shc (
For example, Tiganis et al., 1998, Mol. Cell. Biol. 18: 1
622 to 1634) and PTPH1 and pp97 / VCP complex, respectively, can be readily identified by Western immunoblot analysis as described below. These associations were observed, for example, in lysates and transfected cells from several cell lines, p210bcr: abl.
, P130 ^cas , Shc and VCP are PTP1B and PTP-PE, respectively.
It will be shown to represent the major physiologically relevant substrates for ST, TC-PTP and PTPH1. The compositions and methods of the invention that can be used as exemplified herein to identify specific tyrosine phosphorylation substrates for PTP1B, PTP-PEST and PTPH1 are not limited, Generally, TC-PTP, PTPγ, MKP-1, DEP-1, PTPμ, SHP2, P
TP-PEZ, PTP-MEG1, LC-PTP, CD45, LAR and PX
It can be applied to any member of the PTP family, including PX10.

In some embodiments of this aspect of the invention, the sample may contain cells that naturally express the tyrosine phosphorylated protein that is a substrate for PTP, while in some other embodiments, the sample is , May contain cells that have been transfected with one or more nucleic acid molecules that encode a substrate protein. For example, the sample may contain cells or populations of cells that have been transfected with a nucleic acid library, such as a cDNA library, containing 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 suitable substrates include, but are not limited to, p130 ^cas ,
pp97 / VCP, EGF receptor, p210bcr: abl, MAP kinase,
Examples include Shc and insulin receptor. Of particular interest are tyrosine phosphorylated proteins that are associated with mammalian diseases or disorders.

According to the present invention, substrates will include full-length tyrosine phosphorylated proteins as well as polypeptides and fragments (eg, portions), derivatives or analogs thereof that are capable of phosphorylating tyrosine residues. Such fragments, derivatives and analogs include, for example, any PTP substrate polypeptide that retains at least the biological function of interacting with PTP as provided herein by forming a complex with PTP. Is mentioned. A fragment, derivative or analog of a PTP substrate polypeptide containing a substrate which is a fusion protein has (i) at least one amino acid residue, which is a conserved amino acid residue or a non-conserved amino acid residue (preferably a conserved amino acid residue). A fragment, derivative or analog, which is substituted with and such substituted amino acid residue may or may not be an amino acid residue encoded by the genetic code, or (ii) Fragments, derivatives or analogs in which one or more amino acid residues contain a substituent, or (iii) a compound in which the substrate polypeptide increases the half-life of the polypeptide (eg, polyethylene glycol, etc.)
Or a fragment, derivative or analog, or (iv) an additional amino acid that is fused to another molecule, such as a detectable moiety, such as a reporter molecule, for purification of the substrate polypeptide or proprotein sequence. It may be a fragment, derivative or analog fused to a substrate polypeptide containing the amino acids and the like used for that purpose. Such fragments, derivatives and analogs are considered to be within the skill of those in the art.

The present invention also contemplates some embodiments in which the substrate-capturing mutant PTP (mixed with the sample) is a mutant PTP expressed by cells, wherein the cells are mutant PTPs.
Embodiments which are transfected with one or more nucleic acid molecules encoding TP are included. Thus, a method of identifying a tyrosine phosphorylated protein that is a substrate of PTP comprises, in some embodiments, mixing a sample containing a tyrosine phosphorylated protein with a mutant PTP, the sample comprising a tyrosine phosphorylated protein. And cells expressing either or both of the mutant PTPs. Optionally, the cells may be transfected with nucleic acids encoding either or both tyrosine phosphorylated protein and mutant PTP.

In another aspect, the invention provides agents that alter the interaction between PTP and a tyrosine phosphorylated protein that is a substrate of PTP, are candidates for altering (ie, increasing or decreasing) such interaction. Methods of identification are provided by the use of screening assays to detect the potency of a drug. The interaction between PTP and its substrate may be determined enzymatically, for example by detecting catalytic substrate dephosphorylation. On the other hand, the interaction between PTP (including substrate-trapping mutant PTP) and its substrate will be determined as a binding interaction, and in a preferred embodiment such interaction is described herein. The standards set out reveal the detection of complexes formed by PTP-substrate binding. Agents identified by these methods are agonists of PTP activity (eg, wild-type PTP
Agents or antagonists (eg wild type P) that enhance or increase the activity of
It may be a drug that inhibits or reduces the activity of TP). Agents will be identified among naturally occurring or unnatural compounds, including synthetic small molecules, as described below.

In some embodiments in which the screening assay is performed on the catalytic activity of PTP, the substrate for PTP, tyrosine phosphorylated protein, can be identified as described above, and such methods are disclosed herein. It is characterized by the use of a novel substrate-capturing mutant PTP as described above. Therefore, PTP and a tyrosine phosphorylated substrate are mixed in the absence and presence of a candidate drug, where the substrate is first identified using a substrate-trapping mutant PTP, as described above. There is. PTP and substrate are mixed under conditions that can allow detectable substrate dephosphorylation to occur.

Any suitable method can be used to detect dephosphorylation of phosphoproteins. Such methods include, but are not limited to, well known in the art and include, for example, radiometric, fluorometric, densitometric, spectrophotometric, chromatographic, electrophoretic, colorimetric, or biometric assays. As described above, the catalytic action of the substrate can be detected. The level of dephosphorylation of the substrate in the absence of the drug is compared to the level of dephosphorylation of the substrate in the presence of the drug, resulting in a difference in the level of dephosphorylation of the substrate (eg, statistically significant). An increase or decrease) indicates an agent that alters the interaction between protein tyrosine phosphatase and the substrate.

For example, an enzymatic activity assay in which wild type PTP is used can be performed in the absence and presence of a candidate agent. Enzyme activity assays known in the art include, for example, Flint et al. (1993, EMBO J. 12: 1937).
-1946), a PTP activity assay using a ³² P-labeled substrate in which tyrosine is phosphorylated, and the like. A decrease in PTP enzyme activity in the presence of a candidate drug indicates that the drug inhibits the interaction between PTP and its substrate. Conversely, increasing PTP enzyme activity in the presence of a candidate drug indicates that the drug enhances the interaction between PTP and its substrate.

In some other embodiments, where screening assays are performed to identify agents that can alter the substrate-trapping mutant PTP-substrate binding interaction (as described herein). Substrate-capturing mutant PTP and a tyrosine phosphorylated substrate allow the formation of a complex between the tyrosine phosphate protein and the substrate-capturing mutant PTP, thereby forming a conjugate The conditions are sufficient to generate a (combination), and they are mixed for a time. The formation of a complex containing tyrosine phosphorylated protein in the combination and the substrate-trapping variant protein tyrosine phosphatase is then determined (as also provided herein), where the drug That the agent alters (ie increases or decreases) the interaction of the protein tyrosine phosphatase with the substrate due to differences (eg, statistically significant differences) in the level of complex formation in the absence and presence of Indicates. Alternatively, competitive binding assays can be performed using the substrate-capturing mutant PTP in the absence and presence of the candidate agent. Competitive binding assays known in the art include, for example, US Pat. No. 5,352,660, which describes methods suitable for use with these aspects of the invention. A decrease in the degree of PTP-substrate binding in the presence of the drug under test indicates that the drug inhibits the interaction between PTP and its substrate. Conversely, an increase in the degree of PTP-substrate binding in the presence of the drug under test indicates that the drug enhances the interaction between PTP and its substrate.

Candidate agents for use in the methods of the invention to screen for agents that alter the interaction between PTP and its tyrosine phosphorylated protein substrate are "libraries" or collections of compounds, compositions or molecules. Will be offered as. Candidate agents capable of interacting with one or more PTPs, including agents that interact with substrate-trapping mutant PTPs as provided herein, include those that bind to PTP, Songyang et al. (1995, Nature, 375: 536-5
39; 1993, Cell, 72: 767-778), and the like, and members of the phosphotyrosyl peptide library. Peptides identified from such peptide libraries can then be evaluated to reveal whether tyrosine phosphorylated peptides containing these peptides are essentially present. On the other hand, a library of candidate molecules to be screened includes compounds known in the art as "small molecules" with a molecular weight of less than 10 ⁵ daltons (preferably less than 10 ⁴ daltons, even more preferably less than 10 ³ daltons). And the like will typically be mentioned. For example, a member of a library of test compounds may each comprise at least one substrate-trapping mutant PTP and at least one tyrosine phosphorylated protein that is a substrate for PTP as provided herein. Of the samples can then be assayed for their ability to enhance or inhibit the binding of mutant PTP to the substrate. A compound so identified as capable of altering a PTP-substrate interaction (eg, binding and / or substrate phosphotyrosine dephosphorylation) allows the compound to treat and / or detect a disease associated with PTP activity. Therefore, it is useful for therapeutic and / or diagnostic purposes. Such compounds are also useful in studies on molecular signaling mechanisms associated with PTP and on refinements in the discovery and development of future compounds with greater specificity.

In addition, candidate compounds will be provided as members of a combinatorial library which preferably comprises synthetic agents prepared according to a number of predetermined chemistries carried out in a number of reaction vessels. For example, solid phase syntheses, recorded random mix methodologies and recorded reaction resolution techniques that allow various starting compounds to undergo such that a given component can follow multiple permutations and / or combinations of reaction conditions. Will be prepared using one or more of The resulting product is a synthetic combinatorial library of peptides (eg, PCT / U
S91 / 08694, PCT / US91 / 04666, which are hereby incorporated by reference in their entirety), or other compositions that may include small molecules as provided herein (eg, , PCT / US94 / 0854
2, EP 0774464, US Pat. No. 5,798,035, US Pat. No. 5,789,172, US Pat. No. 5,751,629, which are hereby incorporated by reference in their entirety. Contained as a reference), after which the selection and synthesis procedures can be repeated. Those skilled in the art will appreciate that various assortments of such libraries can be prepared using established procedures and tested using the substrate-capturing mutant PTPs in accordance with the present disclosure.

[0090]   The present invention also provides (i) an unchanged aspartic acid residue of a wild-type PTP catalytic domain.
The group does not cause a significant change in the Km of the enzyme, but less than 1 per minute (1 min ^-1 Amino acids (eg, alanine residues) that result in a decrease in Kcat up to
Substituted and (ii) at least one wild type tyrosine residue may be phosphorylated
Administering a substrate-capturing mutant PTP that is substituted with a non-existing amino acid to a subject
A substrate-capturing mutant protein tyrosine phosphatase
The interaction between the protease and tyrosine phosphorylated protein
Method for reducing activity of tyrosine phosphorylated protein, which reduces activity of protein
Regarding In some preferred embodiments, the tyrosine phosphorylated protein is
VCP, p130^cas, EGF receptor, p210 bcr: abl, MAP kina
, Shc or insulin receptor. In some other preferred embodiments
The tyrosine phosphorylated protein is PTP1B, PTP-PEST, PTP.
γ, MKP-1, DEP-1, PTPμ, PTPX1, PTPX10, SHP2
, PTP-PEZ, PTP-MEG1, LC-PTP, TC-PTP, CD45
, LAR or PTPH1.

Without wishing to be bound by theory, such a mutant PTP forms a complex between the activity of the corresponding wild-type PTP and the wild-type PTP and a tyrosine phosphorylated protein substrate, thereby permitting the wild-type enzyme. It is reduced by providing a substrate that is not available for catalytic dephosphorylation by. Therefore, the substrate-capturing mutant PTP is
Without dephosphorylation (or catalyze dephosphorylation at a significantly reduced rate)
It binds to the phosphoprotein substrate, thereby interfering with the activity of the dephosphorylated protein substrate and reducing its downstream effects. As used herein,
"Reducing" includes both inhibiting or abolishing one or more activities or functions of phosphorylated protein substrates.

In one aspect of the method of reducing the activity of tyrosine phosphorylated protein, PTP
How to reduce the malignant transformation effect of the at least one cancer gene associated with phosphorylation of the substrate in which pl30 ^cas of -PEST is provided. Generally, the method replaces the constant aspartic acid residue of the catalytic domain of wild-type PTP with an alanine residue and at least one wild-type tyrosine residue with an amino acid that cannot be phosphorylated. And administering to the subject a substrate-capturing mutant PTP-PEST that is Wild type PTP-PEST binds to the substrate pl30 ^cas, dephosphorylated, thus, with respect to modulate the downstream biological effects of the substrate in the negative, substrate scavenging of the present invention PTP- PEST mutant, pl30 coupled to ^cas but (dephosphorylated at or very low speed) pl30 unable to dephosphorylate ^cas. By the non-limiting theory disclosed above, the substrate is then sequestered in complex with the substrate-capturing PTP-PEST and is unable to exert its downstream effects. In some aspects of the method, the oncogene is v-crk.
, V-src or c-Ha-ras.

Similarly, the present invention provides a substrate-capturing mutant PTP-PES as defined above.
The T comprising administering to a mammal, signaling complexes associated with ^{p130 cas (signaling complexes associated with p130} cas), particularly, a method for reducing the formation of these signaling complexes) which induce mitogenic pathways Regarding PTP
Is dephosphorylated binding and / or the pl30 ^cas to pl30 ^cas, thereby negatively regulates the downstream effects of pl30 ^cas, signal transduction complexes associated with ^{p130 cas (signaling complexes associated with p130} cas) Reduce the formation of. As another example, in some embodiments, the invention relates to regulation of the cell cycle by a substrate for PTPH1, pp97 / VCP, wherein the substrate-trapping mutant PTPH1 (as defined herein) ( That is, the double mutant in which the catalytic activity is weakened and the wild-type tyrosine is replaced is PTPH11 and VC.
The interaction with P may be altered.

As defined herein, the substrate-capturing mutant PTPs of the invention are PT
Biological regulation, including P-regulated signaling, is described in, for example, the corresponding wild-type P
It would be useful in virtually any situation involving alternatives to TP, or in addition to the corresponding wild-type PTP. Advantages of such utility of the present invention include, for example, wild-type PTP-mediated dephosphorylation without inducing the predominant adverse cytotoxic effects of wild-type PTP when administered and / or overexpressed. It is in the ability of the substrate-trapping mutant PTP to mimic the function of its corresponding wild-type enzyme in order to weaken the biological signaling activity of the tyrosine phosphorylated substrate that follows.
Therefore, the invention also relates to methods of reducing the cytotoxic effects associated with administration or overexpression of wild-type PTP. For example, CS variants of MKP-1 have been shown to have the same functional effects as wild-type MKP-1 without inducing potentially harmful side effects. Thus, the invariant aspartic acid residue of the catalytic domain of wild-type PTP does not cause a significant change in the Km of the enzyme, but an amino acid (changes in Kcat up to less than 1 per minute (less than 1 min ^-1 ) ( For example,
Alanine residue) and at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. The PTP described herein is the wild-type counterpart of the counterpart. Instead of enzymes, such counterparts (counterpart)
Can be used in many situations where the wild-type enzyme of E. coli can specifically interact with the same substrate as the mutant PTP.

Further, the substrate-capturing mutant PTP described herein is a nucleic acid encoding the substrate-capturing mutant PTP (or a functional portion thereof) which retains its ability to bind to a tyrosine phosphorylated substrate, For example, therapeutically in order to alter (ie, increase or decrease) the activity of a tyrosine phosphorylated protein, such as by introducing into a subject a recombinant expression construct as described above and expressing a gene therapy method or the like. May be used. The mutant PTP may partially or totally replace the corresponding host PTP enzyme normally produced in the subject, or may compete with the host PTP for binding to the substrate. For example, if a particular tyrosine phosphorylated protein substrate would be associated with a particular disease or condition, then at least one PTP could be identified that could dephosphorylate the suspect substrate. The corresponding substrate scavenging mutant PTP can be administered using the compositions and methods described herein, either directly or by gene therapy. Such mutant PTPs could mask tyrosine phosphorylated substrates, thereby inhibiting or reducing the role of the substrate in the disease process. In a preferred embodiment, the substrate-capture mutant PTPs of the present disclosure are administered in place of the corresponding wild-type enzyme in order to reduce the cytotoxic effects associated with overexpression of the wild-type enzyme. Gene therapy techniques are known in the art (see, eg, US Pat. No. 5,399,346) and are known for expressing substrate-capture mutant PTPs of the invention. Can be modified by known methods.

The method of the present invention comprises the phosphatases PTPH1, PTP1B and PTP-PE.
Specifically illustrated herein with respect to ST; however, the invention is
It is understood that it is applicable to all members of the PTP family, including but not limited to these particular PTPs. PTPH1, PTP1B and PTP-
Variant (ie, altered or substrate-trapping) forms of PTP that are less catalyzed but retain substrate-binding ability to identify potential substrates for PEST
H1, PTP1B and PTP-PEST are made as described herein.

In one aspect, the invention provides, in part, that the aspartic acid residue at position 181 of wild-type PTP1B is replaced with alanine, and further that the PTP tyrosine residue is not optionally phosphorylated. PTP1B (D181A) that may be replaced
Regarding In certain other aspects, the invention provides phosphatase PTP-PEST.
(D199A), and in certain other aspects, the invention relates to PTP-PEST (C231S), further comprising a PTP tyrosine residue optionally substituted by a residue that cannot be phosphorylated in either case. In a particularly preferred aspect, the invention relates to PTPH1 (Y676F / D811A).

As noted above, in one aspect, the invention features a substrate-trapping mutant PTP-PEST.
Regarding PTP-PEST is expressed everywhere in mammalian tissues (Yi et al., 1991, Blood 78: 2222-2228) and shows high specific activity when measured in vitro using an artificial tyrosine phosphorylated substrate. Show (Garton and Tonks, 1994,
EMBO J. 13: 3763-3771), 86 kDa cytosolic PTP (Charest et al.
1995, Biochem. J. 308: 425-432; den Hertog et al., 1992, Biochem. Biophys. Re.
s. Commun. 184: 1241-1249; Takekawa et al., 1992, Biochem. Biophys. Res. Com.
mun. 189: 1223-1230: Yang et al., 1993, J. Biol. Chem. 268: 6622-6628; Yang et al., 1992, J. Biol. Chem. 268: 17650). PTP-PEST is used in vitro and
It requires regulation by phosphorylation of Ser39 in vivo. This modification is catalyzed by both protein kinase C (PKC) and protein kinase A (PKA) and results in a decrease in PTP-PEST enzyme activity due to an increase in Km in the PTP-catalyzed dephosphorylation reaction ( Garton and Tonks, 1994, EMBO J
. 13: 3763-3771). An additional intracellular regulatory mechanism is tyrosine kinase 1
PTP-PEST mediated dephosphorylation of the above cytosolic substrates may be included.

PTP1B and PTPH as disclosed herein and described in the Examples.
The substrate specificity of 1 is a PTP catalyst and / or a binding interaction with a substrate, eg
It can be characterized by methods related to dephosphorylation and substrate capture in vitro and in vivo. PTP1B (eg Barford et al., 1994, Science 263:
1397; Jia et al., 1995, Science 268: 1754) and PTP1H (see, eg, US Pat. No. 5,595,911 and US Pat. No. 5,863,781) are well known to those of skill in the art. The substrate capture methods presented herein are generally applicable to any PTP due to the frequency of tyrosine in the invariant PTP catalytic domain aspartic acid residue and PTP amino acid sequence, and thus to other PTP family members. It should prove useful in illustrating the substrate priorities of In particular, the use of mutant PTPs with impaired catalysis that capture and thereby separate potentially potent substrates allows the identification of physiologically important substrates of individual PTPs, and cellular processes. It will lead to an improved understanding of the role of these enzymes in the regulation of. Furthermore, the replacement of PTP tyrosine residues by amino acids that cannot be phosphorylated provides substrate-trapping mutant PTPs that are not compromised in their ability to interact with tyrosine phosphorylated protein substrates.

The present invention also provides that (i) the invariant aspartic acid residue of the wild-type PTP catalytic domain does not cause a significant change in the Km of the enzyme, but less than 1 per minute (less than 1 min- ¹ ). An amino acid that is substituted with an amino acid that results in a decrease in Kcat (eg, an alanine residue) and (ii) at least one wild-type tyrosine residue cannot be phosphorylated (eg, serine or threonine or any other natural Or a non-natural amino acid that will be phosphorylated) which is substituted by the substrate-capturing mutant PTP. Therefore, the PT of the present invention
P can be formulated, for example, with a pharmaceutically acceptable carrier such as a pharmaceutically acceptable carrier or diluent for preparing pharmaceutical compositions.

For administration to a patient, one or more polypeptides (including substrate-trapping mutant PTPs), nucleic acid molecules (including recombinant expression constructs encoding substrate-trapping mutant PTPs) and / or regulatory agents (Including agents that interact with PTP and / or substrate-trapping variant PTP) are generally formulated as pharmaceutical compositions. The pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension or emulsifying agent which additionally contains a physiologically acceptable carrier (for example, non-toxic material that does not inhibit the activity of the active ingredient). Such compositions may be in solid, liquid or gas (aerosol) form. On the other hand, the composition of the invention may be formulated as a lyophilizate or the composition may be encapsulated within liposomes using well known techniques. The pharmaceutical composition within the scope of the present invention may also include other ingredients that may be biologically active or inactive. Such components include, but are not limited to, buffers (eg, neutral buffered saline or phosphate buffered saline), carbohydrates (eg, glucose, mannose, sucrose or dextran).
, Mannitol, proteins, polypeptides or amino acids such as glycine,
Antioxidants, chelating agents such as EDTA, or glutathione, stabilizers, dyes, flavors and suspending agents and / or preservatives.

Any suitable carrier known to those of ordinary skill in the art may be used in the pharmaceutical compositions of the present invention.
Carriers for therapeutic use are well known and also include, for example, Remingtons Pharmaceutica.
l Sciences. Mack Publishing Co. (edited by AR Gennaro, published in 1985). Generally, the type of carrier is selected based on the dosage form. The pharmaceutical composition is, for example, topical, oral, nasal, intraocular, intrathecal, rectal, vaginal, sublingual or subcutaneous, intravenous, intramuscular, intrasternal, intraurethral or intraurethral injection or infusion. May be formulated for any suitable method of administration including parenteral administration such as. For parenteral administration, the carrier is preferably water, saline, alcohol, fat,
Contains wax or buffer. For oral administration, any of the above carriers, or mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, kaolin, glycerin, starch dextrin, sodium alginate, carboxymethyl cellulose, ethyl cellulose, glucose, sucrose and / or carbonate. A solid support such as magnesium could be used.

The pharmaceutical composition (eg, oral administration or delivery by injection) may be in the form of a liquid (eg, elixir, syrup, solution, emulsifier or suspension). The liquid pharmaceutical composition is, for example, one or more of the following: water for injection, sterile excipients such as physiological saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, solvent or suspension. Fixed oils such as synthetic monoglycerides or diglycerides that act as liquids Polyethylene glycol, glycerin, propylene glycol or other solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, ethylenediaminetetraacetic acid (EDTA) Chelating agents such as, buffers such as acetates, citrates or phosphates, and agents for adjusting the tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The use of physiological saline is preferred, and the injectable pharmaceutical composition is
It is preferably sterile.

The compositions described herein may be formulated to be depot (ie, a capsule or sponge that provides a slow release of the compound after administration). Such compositions are generally prepared using well known techniques and may be administered, for example, by oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release preparations may include the drug dispersed in a carrier matrix and / or the drug contained within a reservoir surrounded by a rate controlling membrane. The carriers used in such formulations are biocompatible and may be biodegradable. Suitably the formulation provides a relatively constant level of active ingredient release. The amount of active ingredient contained within a sustained release formulation depends on the site of implantation, the rate and expected duration of release and the nature of the condition being treated or prevented.

For a pharmaceutical composition comprising a nucleic acid molecule that encodes a substrate-trapping mutant PTP polypeptide (such that the polypeptide is produced in vivo), the nucleic acid molecule is:
It may be present in any of a variety of delivery systems known to those of skill in the art, including nucleic acids, bacteria, viruses, and mammalian expression systems such as, for example, recombinant expression constructs presented in this application. Techniques for incorporating DNA into such expression systems are well known to those of skill in the art. The DNA is, for example, Ulme
R et al., Science 259: 1745-1749, 1993, and Cohen, summarized.
It is "naked" as described in e259: 1691-1692, 1993. Naked DNA
Uptake can be increased by coating its DNA on biodegradable beads that are efficiently transported into cells.

Within a pharmaceutical composition, a substrate-capturing mutant PTP polypeptide, a substrate-capturing mutant PTP encoding a nucleic acid molecule or modulator can be linked to any of a variety of compounds. For example, such a polypeptide, nucleic acid molecule or drug is a targeting moiety (eg, a monoclonal or polyclonal antibody, protein or liposome) that facilitates delivery of the drug to its target site.
Can be linked to. As used in this application, the term "targeting moiety", when linked to a drug, enhances the transport of that drug to target cells or tissues, thereby increasing the local concentration of that drug. It can be a substance (such as a compound or a cell). Target moieties include antibodies or fragments thereof, receptors, ligands and other molecules that bind to cells in or near the target tissue. The antibody targeting agent can be the entire molecule (whole), a fragment thereof, or a functional equivalent. Examples of antibody fragments are F (ab ') 2,-, which can be produced by ordinary methods or by genetic engineering or protein engineering.
Fab ', Fab and F [v] fragments. Coupling is generally covalent and can be accomplished, for example, by direct enrichment or other reaction, or a bifunctional or polyfunctional linker. Targeting moieties can be selected based on the cell (s) or tissue (s) for which the drug is expected to exert a therapeutic benefit.

The pharmaceutical composition can be administered by an appropriate method for the disease to be treated (or the subject to be prevented). The appropriate dose, appropriate duration, and frequency of administration will depend on the patient's condition,
It will be determined by such factors as the type and severity of the patient's disease, the particular form of active ingredient and the mode of administration. In general, the appropriate dosage and treatment regimen will be sufficient to provide the therapeutic and / or prophylactic benefit.
Supply (e.g., more frequent, complete or partial remissions or long term disease relief, and / or improved clinical outcomes such as overall survival). For prophylactic use, administration is sufficient to prevent, and defects in cell signaling, such as abnormal cell cycle control, proliferation, activation, differentiation, senescence, apoptosis, adhesion, metabolic activity, gene expression. It should be sufficient to delay the onset of, or reduce the severity of, the disease associated with defects that lead to

Optimal doses can generally be determined using experimental models and / or clinical trials. Generally, the amount of polypeptide present in a single dose, or
The amount of in vivo produced peripeptide by DNA present in a single dose is in the range of about 0.01 μg to about 100 μg / kg of host body weight, and typically,
It is about 0.1 μg to about 10 μg. The use of the lowest dose sufficient to provide effective therapy is usually preferred. Patients generally can be monitored for therapeutic or prophylactic effect using assays suitable for the condition being treated or prevented, which are familiar to those of skill in the art. Suitable doses will vary with the size of the patient, but are typically about 1 m for a 10-60 kg subject.
The range is from L to about 500 mL.

The following examples are provided for the purpose of illustrating the invention and should not be construed as limiting the scope of the invention. The teachings of all references cited in this application are incorporated herein in their entirety.

EXAMPLES Example 1 Production, Expression and Purification of Mutant PTP Proteins Plasmid Isolation; Competent Cell Production; Transformation and Related Manipulation for Cloning; Amplification; Recombinant Plasmids, Inserts, Vector Construction The nucleotide sequence determination and the like are published methods (Sambrook et al., Molecular Clonii).
ng, aLaboratoryManual, ColdSpringHarbo
rLaboratoryPress, ColdSpringHarbor, NY
, 1989; Ausubel et al., 1993 CurrentProtocolsinM.
olecular Biology, Greene Publ. Assoc. Inc
． & John Wiley & Sons. Inc. , Boston, MA). Human PTP-PEST (Garton et al., 1994 EMBOJ.13.
: 3763; Garton et al., 1996 Mol. Cell. Biol. 16: 6
408) and human PTP-1B (Brown-Shimer et al., 1990 Pr.
oc. Nat. Acad. Sci. USA 87: 5148), a recombinant nucleic acid expression construct was prepared as described.

Point mutations within the catalytic domain of PTP are standard techniques, eg PTP-PEST.
Of invariant aspartic acid (D) at amino acid position 199 of Alanine by substitution mutation
(A) was introduced by using (D199A) and the like. Therefore,
PTP-PEST (D199A), PTP-PEST (C231S), PTP1
Mutations producing B (D181A) and PTP1B (C215S) are Muta-G.
ene ™ in vitro mutagenesis kit (Bio-Ra
d, Richmond, CA) and by site-directed mutagenesis according to the manufacturer's instructions. The region containing this particular point mutation is then replaced with the corresponding wild-type base sequence in the appropriate expression vector, and the base sequence of the entire replaced mutant region is determined to confirm that there are no additional mutations. certified.

Full-length PTP-PEST protein (wild type and mutant proteins, Asp1
99-Ala mutation or Cys231-Ser mutation) and the wild-type PTP-PEST catalytic domain (amino acids 1-305) were added to recombinant baculovirus (BaculoGold ™, Pharmingen,
SanDiego, CA) and expressed in Sf9 cells and purified as described by Garton and Tonks (EMBO J. 13: 3763-3771, 1994). Truncated forms of wild-type and mutant PTP-PEST proteins containing amino acid residues 1 to 305 of PTP-PEST were derived from pGEX vector (Phar
maciaBiotech Inc. , Uppsala, Sweden) after subcloning of the GST PTP-PEST DNA in-frame downstream of GST.
E. coli as an ST fusion protein. expressed in E. coli. 25 ml of E. coli transformed with the appropriate vector. coli were grown to the logarithmic growth phase (OD _{60 0} about 0.5). The fusion protein expression was then induced by the addition of 0.2 mM isopropyl-1-thio-β-D-galactopyranoside and the cells were incubated at 30 ° C for 2
Grow for ~ 4 hours. Cells were collected by centrifugation and 50 mM Tris-HC
pH 7.4, 5 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 5 mg / ml leupeptin, 5 mg / ml aprotinin and 0.1% Tr.
50 μg in 3 ml buffer containing itonX-100 and 150 mM NaCl
/ Ml lysozyme and incubated and lysed by sonication (3 x 10 seconds). After removal of insoluble material by centrifugation (300,000 × g for 20 minutes), 100 ml of glutathione-Sepharose ™ beads (Ph.
armaciaBiotech Inc. , Uppsala, Sweden) and 4 ° C
The fusion proteins are separated by incubation for 30 minutes at 37 ° C., then the beads are collected by centrifugation and buffer A (20 mM Tris-HCl, p.
H7.4, 1 mM EDTA, 1 mM benzamidine, 1 mg / ml leupeptin, 1 mg / ml aprotinin, 10% glycerol, 1% Trito.
Wash 3 times with nX-100 and 100 mM NaCl). This procedure yielded an essentially homogeneous fusion protein at 1 mg protein / ml glutathione-Se.
Obtained at a concentration of pharose beads. PTP containing amino acids 1-321
1B protein (wild type and mutant) was transformed into E. E. coli and until uniform, Barford et al. (J. Mol. Biol. 239: 726-730 (19
94)).

Example 2 Regulation of PTP1B Expression Levels by p210BCR: ABL Chronic myelogenous leukemia (CML) has a Philadelphia chromosome in which the c-Abl proto-oncogene on chromosome 9 is linked to the bcr gene on chromosome 22. (P
encodes a protein tyrosine kinase (PTK), a clonal disorder of hematopoietic stem cells characterized by h). This is the bcr: abl fusion protein p
It results in the production of 210 bcr: abl and PTK activity is increased compared to c-Abl. This example shows that the phosphorylation-receptive p210bcr: abl protein specifically induces PTP1B expression.

BaF3 cells expressing a temperature-sensitive mutant of p210bcr: abl (Jai
N. et al., 1996 Blood 88: 1542) was shifted to a temperature at which p210 having PTK activity could be expressed, PTP1B mRNA and protein expression levels were observed to increase within 12 to 24 hours, which is the activity of PTK. Consistent with the development of the type (eg WO 98/04712; LaMon).
tagne et al., 1998 Mol. Cell. Biol. 18: 2965). An increase in PTP1B expression was also observed in Philadelphia chromosome-positive (Ph +) B lymphoid cells derived from CML patients, as compared to Ph- cells in the same patients. A change in PTP1B activity was also observed, which was the same criterion as a change in enzyme protein level. These changes are specific to PTP1B and are closely related homologs TC-PTP (having 65% amino acid sequence identity with PTP1B) or other PTs studied such as SHP-1, SHP-2, PTP-PEST.
Not found in Ps. p210bcr: PTP with ablPTK activity
The specificity of 1B induction was confirmed using Kinase deficient Rat1 cells (Pend.
ergast et al., 1993 Cell 75: 175). These cells express an inactive form of p210bcr: abl, which contains arginine enzyme at amino acid position 1172 instead of lysine residue and lacks PTK activity. Expression of this p210 mutant in Rat1 cells was unable to alter PTP1B expression levels.

Example 3 Interaction of p210BCR: ABL-Binding Substrate with Substrate-Capturing PTP Variants This example describes the search for substrate-interacting properties of substrate-capturing variant PTP to identify PTP substrates. It is a thing. Substrate-capturing PTP polypeptides and fusion proteins were prepared as described in Example 1.

Substrate-capture mutant PTP polypeptides or fusion proteins were contacted with lysates from various cell lines. Briefly, He was used as the starting material for cell lysates.
La and COS cells were treated with Dulbec containing 5% fetal bovine serum (FBS).
The cells were grown in modified Eagle's medium (DMEM). Rat1, Wi38,
C2C12, and MvLu cells were grown in DMEM containing 10% FBS. 293 cells were grown in DMEM containing 10% calf serum. MCF10A cells contain 50% DMEM 50% Ham containing 5% horse serum, 20 ng / ml epidermal growth factor, 10 mg / ml insulin, 0.5 mg / ml hydrocortisone, and 0.25 mg / ml fungizone. Were grown in F-12. BaF3 cells were maintained as previously described (Jain et al., 1996.
Blood 88: 1542). All media contained penicillin and streptomycin at concentrations of 100 U / ml and 100 mg / ml, respectively, and all cells were cultured at 37 ° C. CDNA encoding wild-type and mutant PTP-PEST proteins were cloned into C using calcium phosphate mediated transfection.
It was introduced into OS cells. These proteins are found in the plasmid pMT2 (Samb
look et al., Molecular Cloning, a Laboratory.
Manual. Cold Spring Harbor Laborator
y Press. Cold Spring Harbor, NY, 1989).
Subcloned into PTP-PEST cDNA (Garton et al., 19
96 Mol. Cell. Biol. 16: 6408), expression from this plasmid pMT2 is expressed in the adenovirus major late promoter (major).
late promoter). 20 μg of DNA was used for transfection of 10 cm of each cell plate. The level of expression of the PTP-PEST construct was similar in all cases.

Prior to cell lysis, 70-90% fusogenic cell culture was added to medium containing 50 mM of oxidized vanadate (pervanadate) (50 mM added to 10 ml of medium). Of sodium metavanadate (NaVO ₃ ) and 20 mM fresh solution containing 50 mM H ₂ O ₂ ) for 30 minutes. Treatment of cells with H ₂ O ₂ and vanadate increases phosphotyrosine concentrations synergistically, presumably because intracellular PTPs are inhibited by vanadate.
Heffezz et al., 1990 J. Biol. Chem. 265: 2896-29
02). At least 50 prominent phosphotyrosine protein bands appeared in all cell types upon pervanadate treatment, whereas untreated cells contained virtually undetectable phosphotyrosine concentrations.

Cells were lysed in buffer A containing 5 mM iodoacetic acid (see Example 1). After incubation at 4 ° C for 30 minutes, DTT was added to a final concentration of 10 mM. Next, insoluble substances were removed by centrifugation at 300,000 xg for 20 minutes. The resulting lysate was stored at -70 ° C for long (several months) storage and at 4 ° C.
Exogenously added PTP during long-term (at least 20 hours) incubation in
Was stable with respect to its phosphotyrosine content.

Pervanadate-treated HeLa cell lysates were loaded onto a MonoQ FPLC column (P
It was fractionated by anion exchange chromatography using H. Samples (50 mg total protein at a concentration of 3 mg / ml in buffer A before loading on the column)
3 mg of buffer B (20 mM tris-HCl, pH 7.4, 1 mM)
EDTA, 1 mM benzamidine, 1 mg / ml leupeptin, 1 mg / m
1 aprotinin, and 0.1% Triton X-100). Protein is a linear gradient of 0-0.5 M NaCl in buffer B, 1 m
Buffer B after elution over 20 fractions (fraction volume 1 ml) at a flow rate of 1 / min
Elute with a second gradient of 0.5-1.0 M NaCl in 5 fractions. Phosphotyrosine-containing protein was detected in fractions 7-21 by anti-phosphotyrosine immunoblotting. The same procedure was performed for PTP1B, except that the cells were not pervanadate treated.

For the dephosphorylation reaction, pervanadate-treated HeLa cell lysate (protein 1-2 mg / ml) containing tyrosine-phosphorylated protein was added to 2n on ice.
Incubated in the absence or presence of purified active PTP at a concentration of M. Dephosphorylation was terminated by removing aliquots (30 μg protein) in SDS-PAGE sample buffer, and the degree of dephosphorylation was determined using an immunoassay using phosphotyrosine-specific monoclonal antibody G104 produced as follows. It was measured by blotting. An assay for PTP activity using tyrosine phosphorylated ³² P-labeled reduced, carboxamidomethylated, maleylated lysozyme (RCM-lysozyme) as a substrate was performed using Flint et al. (1993, EMBOJ.1).
2: 1937-1946).

Antibodies and Immunoblotting : PTP-PEST specific monoclonal antibody AG25 was raised against purified full length PTP-PEST expressed by baculovirus. The anti-phosphotyrosine monoclonal antibody G104 uses phosphotyrosine, alanine, and glycine as antigens in a 1: 1: 1 ratio of 1-ethyl-3.
Produced using polymerized in the presence of keyhole limpet hemocyanin with-(3'-dimethylaminopropyl) carbodiimide, which is the method Kamps and Sefton first described (Oncogene 2: 305.
-315 (1988)). p130 ^cas monoclonal antibody is Transduc
obtained from Tion Laboratories (Lexington, Ky). The monoclonal antibody FG6 against PTP1B is Dr. David Hill (
Calbiochem Oncogene Research Product
S.Cambridge, MA). Visualization of proteins by immunoblotting was performed using HRP-conjugated secondary antibody (Amersham Life S).
science Inc. , Arlington Heights, Il) and SuperSignal® CL-HRP substrate system (Pierce, Ro.
ckford, Il) by enhanced chemiluminescence (ECL).

Immunoprecipitation and substrate capture : PTP from transfected COS cells
-PEST immunoprecipitation was performed using the chemical cross-linker dimethyl pimerimidate (Schn
eider et al. Biol. Chem. 257: 10766-10769 (1
982)) using Protein A-Sepharose beads (Pharmacia).
Biotech Inc. , Uppsala, Sweden) after covalently binding the monoclonal antibody AG25. Antibodies were first bound to Protein A-Sepharose at a concentration of 1 mg / ml bead volume, then unbound material was removed by washing 3 times with 0.2 M sodium borate, pH 9. Covalent binding was achieved by incubation for 30 minutes at room temperature in the presence of 20 mM dimethyl pimerimidate in 0.2 M sodium borate, pH 9. Then add these beads to an excess of 0.2
Unreacted cross-linking agent was blocked by incubation with M ethanolamine, pH 8 for 1 hour, washed 3 times with PBS, and stored at 4 ° C. 10 μl of A
G25 beads were used to precipitate transfected PTP-PEST from lysates containing approximately 0.375 mg protein.

Substrate capture was performed using various PTP affinity matrices. The full length PTP-PEST matrix is purified baculovirus-expressing PTP-PEST.
Covalently bound AG25-Protein A-Sepharose beads with bound protein were utilized. Add a fixed amount (10 μl) of AG25 beads to 5 μg of purified PTP.
-Incubated in 100 [mu] l buffer A in the presence of PEST (wild type or mutant) for 2 hours at 4 [deg.] C. Next, unbound PTP-PEST was removed by washing 3 times with 1 ml of buffer solution A. The resulting PTP-PEST-AG2
5 Protein A-Sepharose beads contain approximately 2 mg of PTP-for 10 ml.
Contains PEST. Substrate capture was also performed using glutathione-Sepharose beads coupled to a bacterially expressed GST fusion protein containing the catalytic domain of PTP-PEST.

PTP1B was also used in the substrate capture experiment. In this case, the monoclonal antibody FG6
Was pre-bound to Protein A-Sepharose without cross-linking agent (2 μg antibody / 10 μl beads), then purified PTP1B protein was added in excess and incubated at 4 ° C. for 2 hours. 10μ after removal of unbound PTP1B
One bead contained approximately 2 μg PTP1B.

Pervanadate-treated cell lysates, or column fractions, were used as the source of phosphotyrosine-containing proteins for substrate capture experiments. Generally 0.25-0.5 m in 0.5 ml Buffer A (containing 5 mM iodoacetic acid, 10 mM DTT).
Lysates containing g protein were incubated for 2 hours at 4 ° C. in 10 μl affinity matrix containing approximately 2 μg of the appropriate PTP protein.
Unbound protein was then removed from the sample by washing 3 times with 1 ml of buffer A and bound material was collected by the addition of 50 μL of SDS-PAGE sample buffer and then heated at 95 ° C. for 5 minutes. Next, the protein bound to the beads is
After S-PAGE, it was analyzed by immunoblotting.

In transient co-transfection experiments in COS cells, PTP1B dephosphorylates p210 bcr: abl but not v-abl. The PTP1B (D181A) mutant was expressed as a GST fusion protein, purified, and Mo7-p210 cells (overexpressing p210 bcr: abl).
Mutant PTP and p210 bcr: abl when incubated with lysates of
The complex was isolated. In contrast, tyrosine phosphorylated c-abl was also present in the lysate, which did not bind to mutant PTP. PTP1B (D18
The interaction of 1A) with p210 bcr: abl was blocked by vanadate, a finding suggesting that this interaction involves the active site of PTP.

After transient co-expression in COS cells, PTP1B (D181A) was added to p210.
A complex was formed with bcr: abl. The Y177F mutant of p210 bcr: abl did not interact with PTP1B (D181A), suggesting that this tyrosine residue is a component of the binding site in PTK. p21
This tyrosine residue in 0bcr: abl was phosphorylated in vivo and proved to serve as a docking site for GRB2 (Pendergast et al.,
1993 Cell 75: 175). p210 bcr: pTyr and G in abl
Direct interaction with the SH2 domain of RB2 is essential for the transforming activity of PTK. Interaction of PTP1B (D181A) with p210 bcr: abl interferes with PTK's association with GRB2. Taking all the above into consideration, these data suggest that p210 bcr: abl is a physiological substrate of PTP1B, and that PTP1B functions as an antagonist of the oncoprotein PTK in vivo. Vmax, K of a 37 kDa PTP1B mutant for RCML
m and Kcat are shown in FIG.

PTP1B and EGF Receptor : PTP1B (D181A in COS cells
Is expressed in the 180 kDa protein as well as 120 and 70 kDa.
Phosphorylation of tyrosyl residues in the protein of Escherichia coli is enhanced. GST-PTP1B (D
181A) When the fusion protein was expressed in COS cells and precipitated on glutathione-Sepharose®, this 180 kDa and lower p
120 and p70 co-precipitated. The p180 protein was identified as the epidermal growth factor (EGF) receptor by immunoblotting. But p120
And the identity of the p70 protein is unknown, the latter being neither src, p60 nor paxillin.

Expression of PTP1B (D181A) in COS cells induces tyrosine phosphorylation of the EGF receptor in the absence of its ligand, EGF, which
It shows that the mutant PTP has its effect in unchanged cells but not after lysis. An equivalent amount of PT in which the corresponding aspartic acid at position 199 was replaced with alanine
The P-PEST (D199A) mutant did not interact with the EGF receptor, a finding demonstrating the specificity of this substrate interaction.

Autophosphorylation of the EGF receptor is required for its interaction with PTP1B (D181A). Receptor mutants in which the kinase does not operate or the autophosphorylation site has been deleted do not interact with PTP1B (D181A). Excessive tyrosine phosphorylated protein was observed in v-src expressing cells, but phosphorylation of EGF receptor was not detected. Under such conditions, PTP
1B D181A predominantly binds to the 70 kDa tyrosine phosphorylated protein, and thus PTP1B appears to be able to regulate the EGF-induced signaling pathway.

Example 4 PTP-PEST Preferentially Dephosphorylates a 130 kDa Phosphotyrosine-Containing Protein To probe the in vitro substrate specificity of PTP-PEST, a constant amount of pervanadate-treated HeLa cell lysate was used. Were incubated on ice, producing 50-100 clearly distinguishable phosphotyrosine-containing proteins as determined by immunoblotting of cell lysates with the monoclonal anti-phosphotyrosine antibody G104. Next, either purified full-length PTP-PEST (expressed in Sf9 cells using recombinant baculovirus), PTP-PEST catalytic domain, or PTP1B catalytic domain (37 kDa type) was added to the lysate, An aliquot was taken out at various time points and analyzed by SDS-PAGE, followed by anti-phosphotyrosine immunoblotting.

Surprisingly, the prominent 130 kDa phosphotyrosine band (p130) was selectively dephosphorylated by PTP-PEST within 10 minutes, while the intensity of all other bands was 60 minutes incubation with PTP-PEST. It remained virtually unchanged thereafter. Prolonged incubation with higher concentrations of PTP-PEST (> 100-fold) resulted in complete removal of all phosphotyrosine bands from the lysate. However, under all conditions searched, p130 was found to be dephosphorylated more rapidly than all other present bands.

Selective dephosphorylation of p130 by PTP-PEST was also observed with a truncated phosphatase (amino acid residues 1-305) containing substantially only the catalytic domain of the enzyme. This result indicates that the remarkable substrate selectivity demonstrated by PTP-PEST in this analysis is an intrinsic property of the phosphatase catalytic domain, while the C-terminal 500 amino acid residues are the substrate specificity of the enzyme. Suggests that it has little detectable effect on.

The specificity of the interaction between PTP-PEST and p130 was probed using the catalytic domain of PTP1B in dephosphorylation (amino acid residues 1-321). P
When added at a molar concentration similar to that used in TP-PEST, PTP1B completely and rapidly (within 15 minutes) dephosphorylates most of the phosphotyrosine-containing proteins present in pervanadate-treated HeLa lysates. Was approved. Furthermore, the time course of p130 dephosphorylation was not significantly faster than the dephosphorylation of other phosphotyrosine bands dephosphorylated by PTP1B. Therefore, the availability of any substrate is different and isolated PTP
The range of PTP1B substrate specificities in vitro and in vivo may differ if the catalytic subunit is characterized.

Example 5 Identification of 130 kDa Substrate of PTP-PEST by Substrate Capture This example describes the use of substrate-capturing mutant PTP in an affinity matrix to identify PTP substrate in certain cell lysates. For the preparation of the substrate-capturing PTP affinity matrix, a mutant form of PTP-PEST (D199A) was generated by site-directed mutagenesis, and this mutant enzyme was purified after expression using recombinant baculovirus. . Tyrosine phosphorylated RCM-
When analyzed using lysozyme as a substrate, this purified mutant enzyme showed about 10,000-fold specific activity of the wild-type enzyme. The purified protein was bound to an affinity matrix consisting of anti-PTP-PEST monoclonal antibody (AG25) covalently bound to Protein A-Sepharose beads,
Next, MonoQ prepared from HeLa cell lysates as described in Example 3
Incubated with each of the fractions.

[0136] The pervanadate treated HeLa cell lysate fractionated by anion exchange chromatography (Example 3), after SDS-PAGE fractions by a constant amount an anti - immuno using phosphotyrosine or anti -P130 ^cas antibody It was analyzed by blotting. Next, for each fixed amount of all the analyzed samples, full-length PTP-PEST (D199A) in which Asp199 was changed to alanine was bound to covalently bound protein A-Sepharose / antibody (AG25) beads. Was incubated with an affinity matrix containing a substrate-capturing PTP-PEST variant consisting of After incubation for 45 minutes, mutant PTP
-PEST-associated proteins are harvested by centrifugation, the beads washed and SDS
-PAGE sample buffer was added. The associated proteins were then analyzed by immunoblotting with the monoclonal anti-phosphotyrosine antibody G104. By then SDS-PAGE the protein associated with PTP-PEST, then anti - were analyzed by immunoblotting with phosphotyrosine or anti -P130 ^cas antibodies.

By anti-phosphotyrosine immunoblotting of these column fractions, p
The 130 phosphotyrosine band was shown to elute as a single peak in fractions 11-14 (approximately 0.3 M NaCl). Given the abundance of tyrosine phosphorylated p130 in HeLa lysates, it is likely that p130 is a 130 kDa protein containing a previously identified phosphotyrosine. Several potential candidates have been found in the literature, and focal adhesion kinase p1
25 ^FAK , ras-GAP, gp130, p130 ^cas and the like. Of these candidate materials, pl30 ^cas has been identified as a particularly prominent phosphotyrosine band in a wide variety of systems, for example v-crk (Mayer and Hanaf
usa, Proc. Natl. Acad. Sci. USA 87: 2638-26.
42 (1990); Mayer et al., Nature 332: 272-275 (19.
88) and src (Kanner et al., Proc. Natl. Acad. Sci.
． USA 87: 3328-3332 (1990); Reynolds et al., Mol.
． Cell. Biol. 9: 3951-3958 (1989)) transformed fibroblasts, integrin-mediated cell adhesion (Nojima et al., J. Biol. Chem.
270: 15398-15402 (1995); Petch et al., J. Am. CellS
science 108: 1371-1379 (1995); Vuori and Ru.
Oslahti, J. et al. Biol. Chem. 270: 22259-22262 (
1995)) and PDGF stimulated 3T3 cells (Rankin and Rozeng.
urt, J .; Biol. Chem. 269: 704-710 (1994)) and the like.

[0138] Thus, pl30 phosphotyrosine band the possibility that true pl30 ^cas, was investigated by immunoblotting of MonoQ fractions using an antibody against pl30 ^cas. The 130 kDa band corresponding to p130 ^cas is p13.
It is considered that these proteins are the same because they eluted in the same fraction as the 0 tyrosine phosphorylated band and had apparent molecular weights that were almost the same. Furthermore, it was revealed that ^p130cas immunoprecipitated from these fractions was phosphorylated to a tyrosyl residue.

The mutant PTP-PEST protein has a molecular weight (130 kDa) and Mono.
It was found that the Q elution position (fractions 11-14) was associated with a single phosphotyrosine-containing protein that matched ^p130cas . This PT using ^p130cas antibody
Immunoblotting of P-PEST-associated proteins revealed mutant PTP-P
The 130 kDa tyrosine phosphorylated protein captured by EST proved to be in fact p130 ^cas . Therefore, p130 ^cas is PTP-PE
It appears to be a physiologically relevant substrate for ST.

[0140] The PTP-PEST in specific interactions with tyrosine phosphorylation pl30 ^cas structural features: the interaction of P130 ^cas and PTP-PEST, in order to precipitate the protein from pervanadate treated HeLa lysates Various purified mutant PTs
It was further searched in a substrate capture experiment using P-PEST. Several affinity matrices were incubated with the pervanadate treated HeLa cell lysate, the protein is associated with the beads, after SDS-PAGE, anti - were analyzed by immunoblotting with phosphotyrosine or anti -P130 ^cas antibodies.

[0141]   Wild-type full-length phosphatase is tyrosine phosphorylated p130^casAnd a stable meeting
It was found that PTP-PEST (D191A) mutant protein
, A mutant without an active site cysteine residue (C231S) was also extracted from the lysate with p130 ^cas Was specifically sedimented. Wild-type phosphatase causes tyrosine phosphorylation p13
0^casOf PTP-PEST and tyrosine phosphorylated p
130^casSeems to reflect that the normal interaction with
And this interaction is p130^casIs dephosphorylated by PTP-PEST
And it seems to end immediately.

The C-terminal 500 amino acids of PTP-PEST have src homology-3 (SH3
) Contains several proline-rich regions similar to the domain binding sequence,
The specificity of the interaction of PTP-PEST and pl30 ^cas is these segments were as some degree dependent on the association of the SH3 domain of pl30 ^cas.
Therefore, C-terminal, a segment of PTP-PEST is, the possibility to be involved in the specific interaction of PTP-PEST in the observed pl30 ^cas, wild-type and mutant (D199A) type both the PTP-PEST Substrate capture experiments with GST fusion proteins containing only the catalytic domain were also performed and searched. GS
The PTP-PEST mutant catalytic domain fused to T was found to specifically precipitate the ^p130cas phosphotyrosine band, but neither the wild-type fusion protein nor GST alone was able to precipitate ^p130cas . Thus, the specific interaction with PTP-PEST and pl30 ^cas observed in these experiments,
It appears to be an intrinsic property of the catalytic domain of PTP-PEST, comparable to the observed selectivity of the PTP-PEST catalytic domain active in in vitro dephosphorylation of ^p130cas .

[0143] Variants PTP-PEST and specificity of interaction with the tyrosine phosphorylation pl30 ^cas: Considering the fact that tyrosine phosphorylation pl30 ^cas is relatively abundant in pervanadate treated HeLa cell lysate, observed selective binding of pl30 ^cas of PTP-PEST inactive mutant proteins, rather than the enzyme directional (to reflect the true substrate selectivity of PTP-PEST), substrate directional (in the lysate (Reflecting the abundance of this potential substrate compared to other phosphotyrosine-containing proteins present). This possibility was examined in two ways. First, an inactive mutant form of the catalytic domain of PTP1B was used to capture a substance that was supposed to be a substrate of this enzyme from a pervanadate-treated HeLa lysate.
Again, wild-type phosphatase was found to be unable to interact stably with any phosphotyrosine-containing protein, while mutant variants of the PTP1B phosphatase domain (Cys or Asp mutations similar to those described above for PTP-PEST). Associated with many tyrosine phosphorylated proteins. This was especially clear for the aspartic acid variant of PTP1B (D181A), which appeared to precipitate virtually all phosphotyrosine-containing proteins from lysates with approximately the same degree of effectiveness. These data emphasize the specificity of the interaction of PTP-PEST and pl30 ^cas, than this rather feature that all PTP catalytic domains share, PTP-PE
It seems to be a property unique to the ST catalytic domain.

[0144] The specificity of the interaction between PTP-PEST and pl30 ^cas, several different cell lines with different sequences of tyrosine-phosphorylated proteins respectively (W1
38, 293, COS, MCF10A, C2C12, MvLu) were further investigated after pervanadate treatment. The lysates obtained were analyzed by anti-phosphotyrosine immunoblotting after SDS-PAGE. PTP-PES
Tyrosine phosphorylated protein associated with PTP-PEST was incubated with T (D199A) affinity matrix or a control matrix to give S
And DS-PAGE, anti - were analyzed as described above by immunoblotting with phosphotyrosine or anti -P130 ^cas antibodies.

In all cases, the D199A mutant PTP-PEST protein precipitated a single broad phosphotyrosine band with an apparent molecular weight of 120-150 kDa in various cell lines, whereas the affinity matrix alone showed a phosphotyrosine-containing protein. Could not be allowed to settle. Immunoblotting of this precipitate with ^p130cas antibody revealed that the proteins precipitated from all cell lysates corresponded to ^p130cas . Different molecular weights were observed in different cell lines, probably reflecting the different molecular weights of ^p130cas in different species or the expression of different alternatively spliced forms. Wax (Sakai et al., EMBO J. 13: 3748-
3756 (1994)).

[0146]   Tyrosine phosphorylated p130 in PTP-PEST sediment^casThe relative content of
P130 in the lysate^casIt seemed to show a substantial correlation with the protein content (
Data not posted). Surprisingly, tyrosine phosphorylated p130 in lysates^casIncluding
P130 regardless of quantity^casContains little protein, but is rich in other tyrosine
Even in 293 cell lysates showing a wide variety of highly phosphorylated proteins,
p130^casWas always the only phosphotyrosine-containing protein in the sediment.
Similarly, lysates of pervanadate-treated 293 cells (anti-phosphotyrosine of lysates
Tyrosine phosphorylated p130 in an amount undetectable by immunoblotting^casTo
Phosphotyrosylase was incubated with active PTP-PEST
However, dephosphorylation was not observed even in the band (Garton and
And Tonks, unpublished data). These results show that PTP-PEST p130 ^cas Has a significantly greater affinity for
And P130 of PTP-PEST^casSubstrate selectivity for
It is something that is emphasized.

[0147] inhibition by vanadate association with tyrosine phosphorylated pl30 ^cas and mutant PTP-PEST: in contrast to the inactive mutant PTP-PEST, the wild-type enzyme stability and tyrosine phosphorylation pl30 ^cas It was consistently observed that they could not associate as a complex, suggesting that the observed association was active site directed. To investigate this possibility, pervanadate treated HeL
Mutant PTP-PEST (D199A) was incubated with the PTP inhibitor vanadate at various concentrations prior to addition to a cell lysates (Denu et al. 1996.
Proc. , Natl. Acad. Sci. USA 93: 2493- 2498.
). It was then analyzed the degree of association of PTP-PEST for pl30 ^cas. A PTP-PEST affinity matrix composed of full-length PTP-PEST (D199A) bound to covalently bound Protein A-Sepharose / antibody (AG25) beads was used in the presence of various concentrations of sodium orthovanadate.
Incubated on ice for 10 minutes. The sample was then incubated with an aliquot of pervanadate-treated HeLa cell lysate. The associated protein was labeled with SDS-PA
GE and anti - were analyzed by immunoblotting with phosphotyrosine or anti -P130 ^cas antibodies. The activity of wild-type PTP-PEST was also measured under the same conditions using tyrosine phosphorylated ³² P-labeled RCM-lysozyme as a substrate.

The association was strongly disrupted by vanadate, a disruption of wild-type PTP-P.
It was found that EST showed a concentration dependence similar to vanadate inhibition, and complete destruction was observed at 10 mM vanadate.

Example 6 Mutant PTPs Transfected with Endogenous pl30 ^CAS in COS Cells
Meeting the above experiments -PEST suggest strongly that pl30 ^cas is representative of substrate under physiological conditions of PTP-PEST. To determine whether PTP-PEST interacts with ^{p130cas in} whole cells, wild-type or substrate-capturing mutants of PTP-PEST
COS cells were transformed with a plasmid encoding (D199A or C231S). The cells were treated with pervanadic acid for 30 minutes before lysing. The PTP-PEST protein was immunoprecipitated, and the obtained precipitate was subjected to anti-phosphotyrosine immunoblotting to analyze the associated tyrosine phosphorylated protein. Cell lysates were also incubated with covalently bound Protein A-Sepharose / anti-PTP-PEST (AG25) beads and associated proteins were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibody.

[0150]   Under these conditions, it was able to bind to the C231S PTP-PEST protein again
No, p130^casSince there was only a band containing phosphotyrosine corresponding to
When there are many other substances that can serve as substrates, intracellular PTP-PEST serves as the substrate.
P130^casIt has been shown that can be specifically selected. CO treated with pervanadic acid
In S cells, both wild-type PTP-PEST and D199A-type were tyrosine phosphorylated p130. ^cas And could not interact stably.

Under these conditions, pervanadate bound to the cysteine residue, which is the active site of PTP-PEST, is present, so the binding of the phosphotyrosine residue of p130 ^cas is efficiently eliminated, both wild-type and D199A form of PTP-PEST, most likely that the binding to the tyrosine phosphorylation pl30 ^cas is inhibited (Denu et al., Proc Natl Acad Sci USA 93: .... 2493-2498 (1996) ). C231S mutant PTP-PE
The ability of ST to bind to p130 ^{cas in the} presence of pervanadic acid to form a stable complex suggests that this mutant protein is hardly affected by pervanadic acid, and in the normal form. Inhibition of PTP by vanadates is critically dependent on the direct interaction between vanadates and the thiol anion of the cysteine residue, the active site of PTP. There is. Therefore, these observations further support the existence of an exclusive interaction between PTP-PEST and p130 ^cas , which seems to be exclusively related to the active site of PTP-PEST, and thereby PTP-PEST. The results reflect a physiologically very limited substrate selectivity for p130 ^cas of.

Example 7 Preparation of Substrate-Capturing PTP Mutant Generation of a mutant PTP capable of interacting with a substrate to form a stable complex was essentially as described by (Flint et al., 1997, Proc. Natl. Acad. Sci. USA 94: 1680; Garton et al., 1996.
, Mol. Cell. Biol. 16: 6408; Tiganis et al., 1997 J. Biol. Chem. 272: 21548;
See also PCT US97 / 13016). Plasmid isolation, production of competent cells, transformation, and manipulation of M13 followed published methods (Samb.
rook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Publishing, Cold Spring Harbor, NY.
Harbor Laboratory Press, Cold Spring Harbor, NY) 1989). Purification of DNA fragments was performed using the QIAEX® kit purchased from QIAGEN (Chatsworth, Calif.). Sequencing of the various constructs was performed according to the manufacturer's instructions using the Sequenase® kit (Amersham-Pharmacia, Piscataway, NJ).
am-Pharmacia)). Restriction and modification enzymes are available at Roche Molecular Biochemicals (Indianapolis, IN) and New England Biolabs (Beverly, MA).
) Purchased from.

Briefly, the pBlueScript plasmid (LaJoya, CA
Human PT ligated to Stratagene (Lla, CA)
PH1 cDNA (US Pat. No. 5,595,911) was prepared by site-directed mutagenesis according to the manufacturer's instructions according to Muta-Gene® (Hercules, CA) Bio-Rad. Rad Inc.)). The oligonucleotide used to mutate the cysteine at position 842 to serine in vitro is CCT AGT TCA CTC CAG TGC TGG AAT AG SEQ ID NO: 37, corresponding to PTPH1 2537-2562. The oligonucleotide for mutating the aspartic acid at position 811 to alanine is GCA TGG CCT GCC CAC GGT GTG C SEQ ID NO: 38, which corresponds to nucleotides 2445 to 2466 of PTPH1. Mutated replicative DNA
E. coli strain DH10B (LaJolla, CA)
, Stratagene, Inc., colonies were picked to confirm the mutations, and the Sequenase® kit (Piscataway, NJ, Amersham-Pharmacia (Piscataway, NJ) was used according to the manufacturer's instructions. Amersham-Pharmacia)) was used for sequencing by the dideoxy method. Amersham-Pharmacia (Amersham) of expression vector pGEX (Piscataway, NJ) in frame with wild-type and mutant PTPH1 gene sites encoding the PTP catalytic domain (amino acid residues 634 to 913).
-Pharmacia)) and three types of glutathione-S-transferase (
GST) fusion protein coding sequence: GST-PTPH1 (wild type), GST-PTPH1 (
D811A) and GST-PTPH1 (C842S) were prepared. The GST-PTPH1 fusion protein was expressed in E. coli and by affinity binding to glutathione immobilized on Sepharose® beads (Pharmacia, Piscataway, NJ) according to the manufacturer's protocol. Purification was performed.

Alternatively, wild-type and mutant PTPH1 constructs as described above for use in transfecting mammalian cells can be cloned with a nucleic acid sequence encoding a HA epitope into C
It was tagged with a sequence coding for the terminal side. This HA tag corresponds to the antibody-defined epitope: SYPYDVPDYAS SEQ ID NO: 39, derived from influenza hemagglutinin protein (Wilson et al., 1984, Cell 37: 767).

After confirmation by DNA sequencing, these constructs were constructed with the vector pCDNA3 (Invitrogen Corp., Carlsbad, Calif.).
gen)) and the retroviral vector pBSTR1 (S. of Massachusetts General Hospital, Boston, MA).
Reeves) during chrononing.

By the site-directed mutagenesis method as described above, nucleotides 2034 to 2034 of PTPH1
Oligonucleotide corresponding to 70: TTG GAC AAA AAC CGA TTT AAA GAT GTG CTG CCT TAT G SEQ ID NO: 40 was used to further modify the GST-PTPH1 (D811A) mutant construct and preserved PTP
A double mutant (Y676F / D811A) was prepared in which the tyrosine residue at position 676, which is the catalytic site, was replaced with phenylalanine.

Example 8 Effect of PTPH1 Expression on Cell Proliferation in Transfected Cells In this Example, overexpression of the transfected PTPH1 gene in cultured cells significantly inhibited cell proliferation, but it was transfected. It shows that inhibition does not occur when the mutant substrate-capturing PTPH1 gene is overexpressed.

Using the retroviral gene delivery system, the wild-type or substrate-trapping mutant PTPH1 GST fusion protein (Example 7) under the control of the tetracycline repressible promoter was used.
A stable NIH3T3 cell line was constructed (Paulus et al., 1996, J. Vir).
70:62; Wang et al., 1998, Genes Develop. 12: 1769). Briefly, viral packaging cell lines confluent in tissue culture dishes 10 cm in diameter.
LinX (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY in Cold Spring Harbor, NY)
G. Hannon) was transfected with 15 μg of wild-type or D811A mutant PTPH1 retroviral construct by calcium phosphate precipitation. In order to maintain the expression-suppressed state of the PTPH1 gene, in order to maintain a stable cell line in the presence of 2 μg / ml of tetracycline (Clontech of Palo Alto, CA), Was performed. Retroviruses were prepared by incubating transfected LinX cells at 30 ° C for 48 hours, and then adding virus-containing medium to a 0.45 μm filter (Millipore, Bedford, MA) to remove packaging cells. (Millipore)). To the virus supernatant was added 4 μg / ml of polybrene (Sigma, St. Louis, MO) and 10% fetal bovine serum (
Dulbecco's modified Eagle medium (DMEM, FBS, Gibco-BRL of Grand Island, NY) (DMEM,
NIH3T3 cells (GIBCO-BRL) maintained in NIH3T3 cells (GIBCO-BRL)
A stock strain of the Cold Spring Harbor Laboratory, originally used to infect the American Type Culture Collection (Rockville, MD). did. After overnight incubation at 30 ° C, the medium was replaced with fresh one and the cultures were incubated at 37 ° C. After 2 days, puromycin was added to a final concentration of 2 μg / ml and placed under selective conditions. Each colony was isolated and maintained in the presence of tetracycline and puromycin. PTPH
In order to induce 1 expression, cells were washed and replated in fresh dishes containing puromycin but no tetracycline.

Removal of tetracycline from the culture medium to induce expression of the catalytic domain of wild-type PTPH1 markedly inhibited cell proliferation (decreasing the number of accumulated cells by about 7-fold) (FIGS. 3 and 4). While inducing wild-type PTPH1 expression, about 10% of the cells gradually detached from the culture dish, so that these cells were non-viable as measured by the loss of their ability to eliminate trypan blue. In contrast, the PTPH1-D811A mutant (“DA”) with impaired catalytic function did not affect cell proliferation or viability. For each PTPH1 construct, similar results were obtained in three different cell lines produced from separately isolated colonies, so the difference in clonal populations resulted in differences between wild type and mutant (D811A). It has been shown that the phenotypic differences seen between cells transformed by PTPH1 cannot be explained. DNA fragmentation assay (Wyllie, 1980, Nature 284: 555; Arends et al., 1990, Am. J. Pathol. 136: 593.
) Was used to determine that cells induced to express PTPH1 did not undergo apoptosis.

Flow cytometric measurement of DNA using propidium iodide (Rabinovitc
h, 1994, Meths. Cell Biol. 41: 263-296) was performed on transfected cell populations in which expression of wild-type or mutant (D811A) PTPH1 was induced. Compared to control cells, the cell distribution at various cell cycle stages was unchanged, indicating that PTPH1-induced growth arrest did not act at specific cell cycle stages.

Cells in culture were also synchronized to measure the effect of PTPH1 expression on recovery from G1 / S arrest and cell cycle restart. PTPH1 (wild type or mutant D8
11A) 24 hours after induction of expression, 1 mM hydroxyurea (Calbiochem, San Diego, CA) was added.
The cells were synchronized by culturing for 18 hours. This drug is a G1 / S of the cell cycle
Arrest cells at the border of (Krek and DeCaprio, 1995, Meths. Enzymol. 254: 114.
). Inhibition by hydroxyurea is lost if the cells are washed 3 times with fresh medium. NP40 buffer (1% NP40, 10 mM sodium phosphate, pH 7.0, 150 mM NaCl, 2 mM EDTA) was used for immunoblot analysis using cyclin-specific antibodies at various time points after removal of cell cycle inhibition. , 50 mM NaF, 1 mM Na ₃ VO ₄ , 5
μg / ml leupeptin, 5 μg / ml aprotinin, 1 mM benzamidine, 1 mM PM
Cells were lysed in SF). Briefly, confluent cells in 10 cm diameter tissue culture dishes were lysed in 0.5 ml NP40 buffer for 10 minutes at 4 ° C,
The lysate was clarified by centrifugation at 10,000 xg for 10 minutes at 4 ° C. Aliquots of each lysate were standardized for protein concentration (BCA assay, Pierce Chemicals, Rockford, IL), sodium dodecyl sulfate (SDS) sample buffer (Laemmli, 1970, Nature 227).
: 680) and transferred to SDS polyacrylamide gel electrophoresis using 8% acrylamide gel and Immobilon-P PVDF membrane (Millipore, Bedford, MA). Analyzed by blot. Immunoblotting buffer (according to manufacturer's recommendations)
Contains 5% (w / v) skim milk, 150 mM NaCl and 0.05% Tween 20
Blots for 1 hour at room temperature using polyclonal rabbit anti-cyclin D1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in 20 mM Tris, pH 7.5). Probing. The blot was washed 3 times with the same buffer and the enhanced chemiluminescence (ECL) reagent and horseradish were added as previously described (Zhang et al., 1995, J. Biol. Chem. 270: 20067) according to the manufacturer's instructions. Peroxidase (HRP) conjugated secondary antibody (both Piscataway, NJ (Pisc
ataway, New Jersey) Amersham-Pharmacia Biotech (Amersh)
am-Pharmacia Biotech)).

As shown in FIG. 5, under conditions that do not allow expression of the wild-type PTPH1 transgene,
When the transfected cells were released from cell cycle inhibition by hydroxyurea, cyclin D expression gradually increased as the cells re-entered the cell cycle and progressed. However, when the cells were released from cell cycle inhibition under conditions permitting PTPH1 expression, all detectable cyclin D expression disappeared. this is,
It suggests that PTPH1 suppresses cell proliferation by disrupting cell cycle progression. Mutant PTPH in cells transfected with the mutant transgene
Expression of 1 (D811A) had no effect on the cell cycle.

Example 9 Using PTPH1 substrate-capturing mutants in vitro as a substrate for PTPH1
Identification of VCP In this example, using a substrate-capturing PTPH1 mutant in which the aspartic acid residue, which is the invariant PTP catalytic site, was replaced with alanine (D811A) was used to identify the substrate for PTPH1 in cell lysates. explain. Cell lysates were prepared as described in Example 3 above and contacted with the catalytic domain of wild-type or mutant PTPH1 to form PTP-
Substrate binding interactions were measured.

As described in Example 8 above, the substrate capture method using the mutant PTP in which the aspartic acid residue which is the invariant PTP catalytic site is replaced with alanine is as follows.
Except for PH1 (D811A), it is as described above (Flint et al., 1997, Proc
Natl. Acad. Sci. USA 94: 1680; Garton et al., 1996, Mol. Cell Biol. 16: 640.
8; Tiganis et al., 1997 J. Biol. Chem. 272: 21548; PCT US97 / 13016).

Cellular lysates treated with pervanadic acid were incubated with GST-PTPH1 catalytic domain fusion proteins immobilized on Sepharose® beads.
Briefly, subconfluent cultured mammalian cells were treated with 50 μM pervanadic acid (diluted from a 1: 1 mixture of 100 mM sodium vanadate and 100 mM H ₂ O ₂ in DMEM) for 30 minutes. , PBS, then substrate capture buffer (1% Triton X-100, 50 mM HEPES, pH 7.5, 5 mM as described in Example 8).
EDTA, 150 mM NaCl, 10 mM sodium phosphate, 50 mM NaF, 5 mM iodoacetic acid, 5 μg / ml leupeptin, 5 μg / ml aprotinin, 1 mM benzamidine and 1
The cells were lysed in mM PMSF). Add 10 mM DTT to the lysate, then 10,000 x
Clarified by centrifugation at g for 10 minutes. Glutathione under the conditions recommended by the supplier
-Sepharose beads (Piscataway, NJ)
Amersham-Pharmacia Biotech.
)) Purified GST-PTPH1 fusion protein or only GST, bound to 1% T
riton X-100 (Sigma, St. Louis, MO)
)), 2 mM dithiothreitol (DTT, Sigma), 5 μg / ml leupeptin, 5 μg / ml aprotinin, 1 mM benzamidine and 1 mM PMSF in phosphate-buffered saline (PBS). It was GST with cell lysate immobilized on beads
Alternatively, the beads were incubated with the GST-PTPH1 catalytic domain fusion protein for 2 hours at 4 ° C., and the beads were washed 4 times with the substrate capture buffer. The material bound to the beads is separated by SDS-PAGE and blotted onto Immobilon-P® (Millipore) membranes in Bedford, MA, prior to being recommended by the supplier. Concentration of phosphotyrosine-specific monoclonal antibody (G98, Tiga
nis et al., 1997 J. Biol. Chem. 272: 21548; 4G10, Upstate Bi Technology, Lake Placid, NY.
PY20, PY20, Transduction Laboratories, Lexington, KY, and probed with ECL reagent (Amersham-Pharmacia Biotech) as described in Example 7 above. Biotech. Piscataway, NJ))
Was developed using.

The prominent 97 kDa (pp97) tyrosine phosphorylated protein was specifically isolated from 293 cell lysates by the PTPH1 (D811A) mutant, but the wild-type PTPH1 or PTPH1 (C842S) mutant Was not isolated in (Fig. 6). Furthermore, pp97
PTPH1 was also a major tyrosine-phosphorylated protein from other mammalian cell lines investigated, including A431, COS-7, HepG2, MDCK, REF-52, Saos-2 and Vero cells.
(D811A) was consistently recovered by the mutant. The PTPH1 substrate-capturing mutant is one of the hundreds of tyrosine phosphorylated proteins present in cell lysates,
Both cell lysates used as starting material for substrate capture bound specifically and selectively to pp97, which is not the major protein component. Different amounts of other minor tyrosine-phosphorylated proteins were also detected in PTPH1-related materials from different cell lines.

The pp97 purification on cell lysate corresponding to 10 ⁸ 293 cells on immobilized PTPH1 (D811A) was scaled up and partial sequences were obtained by Edman degradation of the peptide degraded by K-endopeptidase. A sufficient amount of protein was obtained to determine (Russo et al., 1992, J. Biol. Chem. 267: 20317). All of them are ATPa
Each of the seven peptides known to have se activity and to match the amino acid sequence present in a membrane-bound protein known as p97 or VCP was sequenced (FIG. 7) (Egerton et al., 1992, EMBO J). . 11: 3533). The underlined sequence (Fig. 7) matches the sequence of mouse VCP searched from the NCBI database (http: //www/ncbi.nlm.nih.gov/, accession number Z14044) (SEQ ID NO: 42). did. CD
An ortholog of VCP in yeast, known as C48, has been well identified as a cell cycle regulatory protein (Patel et al., 1998, Trends Cell Bi).
ol. 8:65). A synthetic peptide corresponding to the C-terminal 15 residues of mouse VCP (Egerton et al., 1992) was prepared (Cold Spring Harbor Laboratories Core Peptide Facility, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY).
y Core Peptide Facility, Cold Spring Harbor, NY)), conventional method (Harlow and Lane
, Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory (Co
ld Spring Harbor Laboratory) 1988; Weir, DM, Handbook of Experimental
Polyclonal rabbit antisera according to Immunology, 1986, Blackwell Scientific, Boston, Boston.
SPDP (N-succinimidyl 3- [2-pyridyldithio] propionic acid, Pierce Chemicals of Rockford, IL) according to the manufacturer's recommendations for use as an immunogen to make CS531. (Pierce
Chemicals)) was used to bind keyhole limpet hemocyanin (KLH, Pierce Chemicals) .The VCP peptide immunogen had the sequence: GGSVYTEDNDDDLYG SEQ ID NO: 41.

Example 10 PTPH Using Substrate Capturing PTPH1 Double Mutants with Substituted Active Site Tyrosine
Identification of VCP as a Substrate of 1 This example describes the use of PTP double mutants to identify the interaction between PTP and its substrate and position in intact cells. More specifically, in this example, the aspartic acid residue that is the invariant PTP catalytic site is replaced with alanine (D8
11A) and a PTPH1 double mutant in which the conserved PTP catalytic site tyrosine at position 676 is replaced by phenylalanine.

Cultured 293 cells were transfected with the HA-tagged PTPH1 construct described in Example 8 and the expressed HA epitope-tagged proteins were previously described (Zhang et al., 1997.
, J. Biol. Chem. 272: 27281) (Stap.
hylococcal) protein A-bound HA-specific monoclonal antibody 12CA5 was immunoprecipitated and recovered. An immunoprecipitate was prepared from the cell lysate prepared as described in Example 3 above by a conventional method. Immunoprecipitates were analyzed for proteins containing phosphotyrosine by Western immunoblotting as described above. Surprisingly, the PTPH1 (D811A) mutant expressed in 293 cells contained a significant and easily detectable amount of phosphotyrosine (FIG. 8A), and the GST-PTPH1 (D811A) fusion protein expressed in E. coli had a , In contrast to the absence of detectable amounts of phosphotyrosine (Figure 6). From these results, it was not possible to determine the position of phosphorylated tyrosine in the primary structure of PTPH1. Furthermore, the PTPH1 (D811A) mutant expressed in 293 cells showed a detectable pp
97 / VCP was not coprecipitated (Fig. 8). Thus, PTPH1 (D811A) is
1 (D811A) was unable to capture sufficient pp97 / VCP detectable in vivo in a manner similar to the in vitro pp97 / VCP capture shown in vitro (Example 9).

As a result of analysis of the amino acid sequence of the catalytic domain of PTPH1, 67
A conserved tyrosine residue at position 6 was revealed (Barford et al., 1995.
, Nat. Struct. Biol. 2: 1043). The HA-tagged PTPH1 double mutant was constructed as described in Example 8, where the tyrosine at position 676 of PTPH1 (D811A) was replaced with phenylalanine, resulting in PTPH1 (Y676F / D811A). PTPH1 (Y6
76 / D811A) lysates of cell lysates from 293 cells transformed with the double mutant-encoding construct and immunoprecipitated with a monoclonal anti-HA antibody for the presence of phosphotyrosine as described above in the Western immunoblot. It was analyzed by the method. The immunoprecipitated substances were analyzed for the presence of pp97 / VCP using antiserum CS531 (Example 9) and for the presence of HA epitopes using monoclonal antibody 12CA5 (Zhang et al., 1997, J. Biol. Chem. 272: 27281).

Unlike 293 cells transformed with a single mutant PTPH1 (D811A), 293 cells transformed with a double mutant PTPH1 (Y676F / D811A) were immunoprecipitated with antiserum CS531. Specifically as revealed by immunoblot analysis of
It had acquired the ability to capture pp97 / VCP (Fig. 8). When analyzed for phosphotyrosine content, double mutant PTPH immunoprecipitated from transformed 293 cells
1 (Y676F / D811A) had a dramatic reduction in phosphotyrosine when compared to the single mutant PTPH1 (D811A) (FIG. 8B).

Example 11 Identification of Tyrosine Phosphorylation Sites on PTPH1 Substrate in Vivo Using Substrate Capturing PTPH1 Double Mutants In this example, substrate capturing PTPH1 double mutants were identified on pp97 / VCP. Used to functionally characterize the tyrosine phosphorylation site. Tyrosine at the C-terminal side of VCP (
Y796 and Y805) are major phosphorylation sites that may play a role in VCP in cell cycle regulation by previously uncharacterized molecular pathways (Egerton et al., 1994, J.
Biol. Chem. 269: 11435; Madeo et al., 1998, Mol. Biol. Cell 9: 131).

(I) Any of the HA-tagged PTPH1 constructs (wild type, D811A, or Y676F / D811A) described in Examples 8-10, and (ii) wild type VCP construct (VCPmyc) or two Cs. VCP construct (VCPmyc-FF), a double mutant (Y676F / D811A) in which the terminal tyrosine phosphorylation site is replaced with phenylalanine, National Institute of Health, Bethesda, Maryland.
L. Samelson), human 293 cells were co-transfected. VCP wild-type and mutant constructs were tagged with the Myc epitope recognized by monoclonal antibody 9E10 (American Type Culture Collection, Rockville, MD). Co-transfected cells were lysed as described in Example 3 and immunoprecipitated with antibody 12CA5 (anti-HA) as described (Zhang et al., 1997, J. Biol. Chem. 272: 27281).

Electrophoretically separated and blotted components were probed with anti-myc antibody 9E10 to identify VCP proteins that were co-precipitated (ie, “captured”) with PTPH1 protein, or anti-HA. Probing with PTPH in the immunoprecipitated material
1 It was confirmed that the protein was included. Wild-type and mutant PTPH1 proteins were expressed at levels comparable to the two forms of VCP. PTPH1 (Y676F / D811
A) The double mutant efficiently captured the wild-type VCP, but did not capture the double mutant VCP having no C-terminal tyrosine phosphorylation site (Fig. 9). Also, wild type PTPH1
Neither the single mutant PTPH1 (D811A) effectively captured VCP (Fig. 9).

Example 12 Selective Dephosphorylation of VCP by PTPH1 In this example, the effect of PTPH1 on the phosphorylation status of VCP was investigated.
A stable, transformed NIH3T3 that expresses full-length wild-type PTPH1 in the presence or absence of tetracycline under the control of the tetracycline repressible promoter.
Cell lines were pre-treated with 1 mM vanadate for 1 hour before cell lysis, then VCP was immunoprecipitated with rabbit CS531 antiserum. Cells instead of NP40 buffer
RIPA buffer (1% sodium deoxycholate and 0.1% SD in NP40 buffer)
Cell lysates and immunoprecipitates were prepared according to the standard procedure described in Example 3 except that S was added (with S added). Under conditions permitting PTPH1 expression (+), it was observed that the amount of phosphotyrosine in VCP decreased by 3 to 5 times as compared to when the expression of PTPH1 was suppressed (-) (Fig. 10A).

Lysates of NIH3T3 transformed cell lines were also immunoprecipitated with the anti-phosphotyrosine antibody PT66 (Sigma, St. Louis, Mo.),
Representative samples of tyrosine phosphorylated proteins were obtained from cells cultured in the presence (+) or absence (−) of PTPH1 expression (FIG. 10B). These immunoprecipitates
Immunoblot analysis with an antibody specific for VCP revealed that the amount of VCP was dramatically increased in tyrosine phosphorylated proteins immunoprecipitated from cells in which PTPH1 expression was induced, compared with the control (-) in which induction was not observed. It became clear that it decreased to (Fig. 1
0B). The apparent selective dephosphorylation of VCP by PTP H1 in these cells assesses the effect of PTPH1 induction on the extent of tyrosine phosphorylation of another tyrosine phosphorylated protein, the kinase FAK. Also indicated by. Induction of PTPH1 expression did not similarly reduce the amount of FAK immunoprecipitated by anti-phosphotyrosine antibody (FIG. 10B).

In addition, the effect of induced PTPH1 expression on the total tyrosine phosphorylated protein pool was 16% in DMEM containing 0.5% FBS under normal conditions (“untreated”).
Serum-depleted (“depleted”) by culturing for hours or depleted cells
10 μg / ml insulin (Roche Molecular Biochemicals
cular Biochemicals (Indianapolis, I.
N)) was stimulated with insulin for 10 minutes and then expanded, and then compared in NIH3T3 cells transformed with PTPH1. Aliquots of whole cell lysate were separated by electrophoresis and Immobilon-P® was transfected with the blot,
Two HRP-conjugated anti-phosphotyrosine antibodies, PY20 (Transduction Laboratories, Lexington, KY) and 4G10, (Lake Placid, NY), diluted according to the supplier's recommendations. ECL detection (Amersham, Cleveland, OH) was performed using a mixture of Upstate Biotechnology (Placid, NY) as a probe to induce overexpression of PTPH1. However, it is proliferating randomly ("untreated")
) Cells, depleted cells, or insulin-stimulated cells failed to alter the overall pattern of protein tyrosine phosphorylation (FIG. 11).
.

One of ordinary skill in the art will be able to recognize or identify numerous embodiments equivalent to the embodiments of the invention described herein without undue experimentation. Such equivalents would also fall within the scope of the following claims. Also, while specific embodiments of the invention are described herein for purposes of illustration, it will be appreciated that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the scope of the appended claims.

[Brief description of drawings]

1A-1E show an alignment of multiple amino acid sequences of the catalytic domain of various PTPs. The position of the amino acid residue of PTP1B that interacts with the substrate is indicated by a small arrow, and the number of the residue at the bottom of the alignment is PTP1B.
Equivalent to that of 1A-1E show multiple sequence alignments of the catalytic domain of PTP (SEQ ID NOs: 1-35). Domains 1 of cytosolic eukaryotic PTP and RPTP were combined into one group; domain 2 of RPTP was the second group and Yersinia PTP was the third. Invariant residues shared by all three groups are shown in lower case. Invariant residues and highly conserved residues within the groups are shown in italics and bold, respectively. Within the Yersinia PTP sequence, the constant or highly conserved residues between the cytosolic and RPTP domain sequences are shown in italics and bold, respectively.

FIG. 2 shows Vmax, Kcat and K of various PTP1B mutants against RCML (reduced, carboxamidomethylated, maleylated lysozyme).
indicates m.

FIG. 3 is a phase contrast micrograph showing the suppression of growth of a stable NIH3T3 cell line overexpressing PTPH1 (+, induced; −, non-induced).

FIG. 4 is a growth curve (mean value by triplicate plating) showing suppression of growth of a stable NIH3T3 cell line overexpressing PTPH1.

FIG. 5 shows suppression of cell cycle progression by overexpression of PTPH1 at a predetermined time after release from hydroxyurea block by immunoblot analysis using an antibody specific for HA epitope tag (PTPH1) or cyclin. Showing (+
, Induced;-, uninduced).

[FIG. 6] FIG. 6 shows in vitro analysis of 293 cell lysate protein captured by a substrate-trapping mutant PTPH1 (D811A) by anti-phosphotyrosine immunoblot analysis.
3 shows the identification of pp97 / VCP as a PTPH1 substrate in ro.

FIG. 7 shows the amino acid sequence of pp97 / VCP (ncbi database accession number Z14044) [SEQ ID NO: 42].

FIG. 8: Identification of pp97 / VCP as a PTPH1 substrate in vivo by immunoblot analysis of 293 cell protein that was captured by and co-immunoprecipitated with the substrate-capturing mutant PTPH1 (Y676F / D811A). Indicates.

FIG. 9 shows the localization of VCP tyrosine residues recognized by PTPH1 to the C-terminal region of VCP.

FIG. 10: VC in a stable NIH3T3 cell line expressing wild type PTPH1
Dephosphorylation of P is shown.

FIG. 11 shows the global profile of tyrosine phosphorylated proteins in a stable NIH3T3 cell line expressing wild-type PTPH1.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] July 27, 2001 (2001.27)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 36

[Correction method] Change

[Contents of correction]

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C12N 1/15 C12N 1/21 4C084 1/19 9/16 B 4H045 1/21 C12Q 1/42 5/10 G01N 33/15 Z 9/16 33/50 Z C12Q 1/42 33/566 G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A 33/566 A61K 37/52 (81) Designated country EP (AT , BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM) , GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA ( AM, AZ, B , KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ , DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Zhang, Xiao Hui California 92129 San Diego, Bavarian Drive 13415 F Term (reference) 2G045 AA40 BB20 DA2 0 DA36 DA78 FB01 FB03 4B024 AA01 AA11 AA12 BA11 CA06 CA07 DA02 DA05 DA12 EA02 EA04 FA02 GA11 HA11 4B050 CC04 CC05 DD07 LL01 LL03 4B063 QA19 QA20 QQ08 QQ13 QR13 QS02 QS33 QX02 4B057A90A90A72A02 BA22 BA44 DC25 NA14 ZB212 ZB262 4H045 AA10 AA30 BA41 CA11 CA40 DA89 EA22 EA50 FA74

Claims (54)

[Claims] 1. A) The invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain does not cause a significant change in the Km of the enzyme, but 1
Substrate-capture mutant protein tyrosine phosphatase, which is substituted with an amino acid that results in a decrease in Kcat of less than 1 per minute; and b) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. . 2. At least one wild-type tyrosine residue from alanine, cysteine, aspartic acid, glutamine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, arginine, valine and tryptophan. The substrate-capturing mutant according to claim 1, which is substituted with an amino acid selected from the group consisting of: 3. The substrate-capturing variant of claim 1, wherein at least one tyrosine residue that is replaced is located in the protein tyrosine phosphatase catalytic domain. 4. The substrate-capturing variant of claim 1, wherein the at least one tyrosine residue that is replaced is located in the protein tyrosine phosphatase active site. 5. The substrate-capture mutant protein tyrosine phosphatase of claim 1, wherein at least one tyrosine residue is replaced with phenylalanine. 6. The substrate-capturing mutant protein tyrosine phosphatase of claim 1, wherein the at least one tyrosine residue that is replaced is a protein tyrosine phosphatase conserved residue. 7. The substrate-capturing mutant according to claim 6, wherein the conserved residue corresponds to tyrosine which is the amino acid at position 676 of human PTPH1. 8. The substrate-capturing mutant according to claim 6, wherein at least one tyrosine residue is replaced with an amino acid that stabilizes a complex comprising a protein tyrosine phosphatase and at least one substrate molecule. . 9. The substrate-capturing mutant according to claim 1, which comprises a mutant PTPH1. 10. PTP1B, PTP-PEST, PTPγ, MKP-1,
DEP-1, PTPμ, PTPX1, PTPX10, SHP2, PTP-PEZ
, PTP-MEG1, LC-PTP, TC-PTP, CD45, LAR and P.
The substrate-capturing mutant according to claim 1, which comprises a mutant protein tyrosine phosphatase selected from the group consisting of TPH1. 11. The substrate-capturing mutant according to claim 1, which comprises a mutated PTP-PEST phosphatase in which the amino acid at position 231 is replaced with a serine residue. 12. A) containing at least one tyrosine phosphorylated protein under conditions and for a time sufficient to allow the formation of a complex between the tyrosine phosphorylated protein and the substrate-trapping mutant protein tyrosine phosphatase. And (i) an invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain is replaced with an amino acid that does not result in a significant change in the Km of the enzyme but results in a decrease in Kcat of less than 1 per minute. And (ii) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated,
Combining at least one substrate-capturing mutant protein tyrosine phosphatase, and b) measuring the presence or absence of a complex comprising tyrosine phosphorylated protein and substrate-capturing mutant protein tyrosine phosphatase, wherein: A method of identifying a tyrosine phosphorylated protein that is a substrate of protein tyrosine phosphatase, comprising: the presence of a complex indicates that tyrosine phosphorylated protein is a substrate of protein tyrosine phosphatase that forms a complex with it. 13. The substrate-capturing mutant is PTP1B, PTP-PEST or PTP.
TPγ, MKP-1, DEP-1, PTPμ, PTPX1, PTPX10, SH
P2, PTP-PEZ, PTP-MEG1, LC-PTP, TC-PTP, CD
The method according to claim 12, comprising a mutant protein tyrosine phosphatase selected from the group consisting of 45, LAR and PTPH1. 14. The method of claim 12, wherein the sample contains cells expressing tyrosine phosphorylated protein. 15. The method of claim 14, wherein the cell has been transfected with at least one nucleic acid molecule encoding a substrate. 16. The method of claim 12, wherein at least one substrate-trapping mutant protein tyrosine phosphatase is expressed by the cell. 17. The method of claim 16, wherein the cell has been transfected with at least one nucleic acid molecule encoding a substrate-trapping mutant protein tyrosine phosphatase. 18. The method according to claim 12, wherein the sample contains cells expressing (i) a tyrosine phosphorylated protein that is a substrate of protein tyrosine phosphatase and (ii) a substrate-trapping mutant protein tyrosine phosphatase. 19. The cell according to claim 18, wherein the cell is transfected with (i) at least one nucleic acid encoding a substrate and (ii) at least one nucleic acid encoding a substrate-trapping mutant protein tyrosine phosphatase. the method of. 20. The method of claim 12, wherein the sample contains cell lysate containing at least one tyrosine phosphorylated protein. 21. The cell lysate is from a cell transfected with at least one nucleic acid encoding a tyrosine phosphorylated protein.
The method described in 0. 22. The cell lysate is derived from cells transfected with at least one nucleic acid encoding a protein tyrosine kinase.
The method described in 0. 23. The method of claim 12, wherein at least one substrate-trapping mutant protein tyrosine phosphatase is present in a cell lysate. 24. The method of claim 23, wherein the cell lysate is from a cell transfected with at least one nucleic acid encoding a substrate-trapping mutant protein tyrosine phosphatase. 25. The tyrosine phosphorylated protein is VCP, p130 ^cas ,
13. The method according to claim 12, which is selected from the group consisting of EGP receptor, p210 bcr: abl, MAP kinase, Shc and insulin receptor. 26. (a) a protein tyrosine phosphatase and a substrate for the protein tyrosine phosphatase under conditions and for a time sufficient for the detectable dephosphorylation of the substrate to occur in the absence and presence of the candidate drug. A step of contacting with a tyrosine phosphorylated protein, wherein tyrosine phosphorylated protein which is a substrate of protein tyrosine phosphatase (1) forms a complex between tyrosine phosphorylated protein and a substrate-trapping mutant protein tyrosine phosphatase And (i) the invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain is associated with a significant Km of the enzyme under conditions and time sufficient to allow No change, but less than 1 per minute At least one substrate-capturing mutant protein tyrosine phosphatase, wherein (ii) at least one wild type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. And (2) the presence or absence of a complex containing a tyrosine phosphorylated protein and a substrate-capturing mutant protein tyrosine phosphatase is measured, wherein the presence of the complex indicates that the tyrosine phosphorylated protein is A step of demonstrating that it is a substrate of a protein tyrosine phosphatase forming a complex; and (b) the level of dephosphorylation of the substrate in the absence of the drug in the presence of the drug. Comparing the level of dephosphorylation of the substrate, where the level of dephosphorylation of the substrate The difference in that the drug alters the interaction between the protein tyrosine phosphatase and the substrate, including the interaction between a protein tyrosine phosphatase and a tyrosine phosphorylated protein that is a substrate for the protein tyrosine phosphatase. To identify drugs that alter the. 27. (a) Conditions sufficient to allow formation of a complex between a tyrosine phosphorylated protein and a substrate-trapping mutant protein tyrosine phosphatase in the absence and presence of a candidate agent and Contacting the protein tyrosine phosphatase with a tyrosine-phosphorylated protein that is a substrate of the protein tyrosine phosphatase at time, wherein the substrate-capturing mutant protein tyrosine phosphatase (i) Although the aspartic acid residue does not cause a significant change in the Km of the enzyme,
Containing a protein tyrosine phosphatase, which is substituted with an amino acid that results in a decrease in Kcat of less than 1 per minute and (ii) at least one wild type tyrosine residue is replaced with an amino acid that cannot be phosphorylated And (b) comparing the level of complex formation in the absence of the drug with the level of complex formation in the presence of the drug, wherein the difference in the level of complex formation is Identify agents that alter the interaction between a protein tyrosine phosphatase and a tyrosine phosphorylated protein that is a substrate for the protein tyrosine phosphatase, including, how to. 28. (i) an invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain which results in no significant change in the Km of the enzyme but an amino acid which results in a decrease in Kcat of less than 1 per minute. Substituting, and (ii) administering at least one wild-type tyrosine residue to a subject with a substrate-capturing variant of a protein tyrosine phosphatase, wherein the at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated, A method for reducing the activity of a tyrosine phosphorylated protein, whereby the interaction between the substrate-capturing mutant protein tyrosine phosphatase and the tyrosine phosphorylated protein thereby reduces the activity of the tyrosine phosphorylated protein. 29. The tyrosine phosphorylated protein is VCP, p130 ^cas ,
29. The method according to claim 28, which is selected from the group consisting of EGF receptor, p210 bcr: abl, MAP kinase, Shc and insulin receptor. 30. The protein tyrosine phosphatase is PTP1B or PT.
P-PEST, PTPγ, MKP-1, DEP-1, PTPμ, PTPX1, P
TPX10, SHP2, PTP-PEZ, PTP-MEG1, LC-PTP, T
29. The method according to claim 28, which is selected from the group consisting of C-PTP, CD45, LAR and PTPH1. 31. (i) an invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain which results in no significant change in the Km of the enzyme but an amino acid which results in a decrease in Kcat of less than 1 per minute. A variant of substrate-capturing PTP-PEST, which is substituted and (ii) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated, into a mammal capable of expressing ^p130cas. Associated with phosphorylation of p130 ^cas , comprising the step of administering, whereby the substrate-trapping variant interacts with p130 ^cas and reduces the malignant transformation effect of at least one oncogene associated with phosphorylation of p130 ^cas To reduce the malignant transformation effect of at least one oncogene. 32. The oncogenes are v-crk, v-src and c-Ha-.
32. The method of claim 31, which is selected from the group consisting of ras. 33. (i) an invariant aspartic acid residue of a wild-type protein tyrosine phosphatase catalytic domain that results in no significant change in the Km of the enzyme but an amino acid that results in a decrease in Kcat of less than 1 per minute. A variant of substrate-capturing PTP-PEST, which is substituted and (ii) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated, into a mammal capable of expressing ^p130cas. Reducing the formation of a signaling complex associated with p130 ^cas , wherein the substrate-trapping variant interacts with p130 ^cas and reduces the formation of a signaling complex associated with p130 ^cas. how to. 34. (i) an invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain which results in a significant change in the Km of the enzyme, but an amino acid which results in a decrease in Kcat of less than 1 per minute; A protein comprising the step of administering to a mammal a substrate-capturing variant of a protein tyrosine phosphatase, which is substituted and (ii) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. A method of reducing cytotoxic effects associated with administration or overexpression of tyrosine phosphatase. 35. a) The invariant aspartic acid residue of the wild type protein tyrosine phosphatase catalytic domain does not result in a significant change in the Km of the enzyme,
A substrate-trapping mutant protein tyrosine phosphatase, which is replaced with an amino acid that results in a decrease in Kcat of less than 1 per minute; and b) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. An isolated nucleic acid molecule that encodes. 36. An antisense oligonucleotide comprising at least 15 contiguous nucleotides complementary to the nucleic acid molecule of claim 35. 37. a) The invariant aspartic acid residue of the wild type protein tyrosine phosphatase catalytic domain does not result in a significant change in the Km of the enzyme,
Substrate-capture mutant protein tyrosine, which is substituted with an amino acid that results in a decrease in Kcat of less than 1 per minute and b) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. A fusion protein comprising a polypeptide sequence fused to a phosphatase. 38. The fusion protein of claim 37, wherein the polypeptide is an enzyme or variant or fragment thereof. 39. The fusion protein of claim 37, wherein the polypeptide sequence fused to the substrate-trapping mutant protein tyrosine phosphatase can be cleaved 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) The invariant aspartic acid residue of the wild type protein tyrosine phosphatase catalytic domain does not result in a significant change in the Km of the enzyme,
Substrate-capture mutant protein tyrosine, which is substituted with an amino acid that results in a decrease in Kcat of less than 1 per minute and b) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. A recombinant expression construct comprising at least one promoter operably linked to a nucleic acid encoding a phosphatase. 42. The expression construct of claim 41, wherein the promoter is a regulated promoter. 43. A substrate-capturing mutant protein tyrosine phosphatase
42. The expression construct of claim 41, which is expressed as a fusion protein with the polypeptide product of the second nucleic acid sequence. 44. The expression construct of claim 43, wherein the polypeptide product of the second nucleic acid sequence is an enzyme. 45. The recombinant expression construct of claim 41, wherein the expression construct is a recombinant viral expression construct. 46. A host cell comprising the recombinant expression construct of any one of claims 41-45. 47. The host cell of claim 46, wherein the host cell is a prokaryotic cell. 48. The host cell of claim 46, wherein the host cell is a eukaryotic cell. 49. a) The invariant aspartic acid residue of the wild type protein tyrosine phosphatase catalytic domain does not result in a significant change in the Km of the enzyme,
Substrate-capture mutant protein tyrosine, which is substituted with an amino acid that results in a decrease in Kcat of less than 1 per minute and b) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. Culturing a host cell comprising a recombinant expression construct comprising at least one promoter operably linked to a phosphatase-encoding nucleic acid sequence, the recombinant substrate-trapping mutant protein tyrosine A method of producing phosphatase. 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 the step of culturing a host cell infected with the recombinant viral expression construct of claim 45. 52. In combination with a pharmaceutically acceptable carrier or diluent: a) the invariant aspartic acid residue of the wild type protein tyrosine phosphatase catalytic domain does not result in a significant change in the Km of the enzyme, but 1 min. A substrate-trapping variant protein tyrosine phosphatase, which is substituted with an amino acid that results in a reduction of Kcat by less than 1 and b) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. A pharmaceutical composition comprising: 53. In combination with a pharmaceutically acceptable carrier or diluent: a) the invariant aspartic acid residue of the wild type protein tyrosine phosphatase catalytic domain does not result in a significant change in the Km of the enzyme, but 1 min. A substrate-capturing mutant protein tyrosine phosphatase, which is substituted with an amino acid that results in a reduction of Kcat by less than 1 and b) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated. A pharmaceutical composition comprising an interacting drug. 54. a) (i) The invariant aspartic acid residue of the wild-type protein tyrosine phosphatase catalytic domain does not cause a significant change in the Km of the enzyme, but results in a decrease in Kcat of less than 1 per minute. Replaced with an amino acid,
And (ii) at least one wild-type tyrosine residue is replaced with an amino acid that cannot be phosphorylated, and at least one substrate-capturing mutant protein tyrosine phosphatase; and b) protein tyrosine phosphatase and tyrosine phosphorylated protein. A kit for identifying a tyrosine phosphorylated protein substrate of protein tyrosine phosphatase, which comprises an auxiliary agent suitable for use in the detection of the presence or absence of a complex between and.