Reversible oxidation of protein tyrosine phosphatases

A method for identifying a protein tyrosine phosphatase that is reversibly modified by a PTP active site-binding agent in a cell, comprising:

• contacting a PTP active site-binding agent that is capable of reversibly modifying a sulfhydryl group of a protein tyrosine phosphatase active site invariant cysteine with a biological sample comprising a cell that comprises at least one protein tyrosine phosphatase;
• isolating the protein tyrosine phosphatase in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a protein tyrosine phosphatase active site invariant cysteine;
• and
• determining, under conditions that are capable of reversing a reversible modification of a sulfhydryl group of a protein tyrosine phosphatase active site invariant cysteine, a level of dephosphorylation of a detectably labeled protein tyrosine phosphatase substrate by the protein tyrosine phosphatase, wherein detectable substrate dephosphorylation indicates that an active protein tyrosine phosphatase is present, and therefrom identifying a protein tyrosine phosphatase that is reversibly modified by a PTP active site-binding agent in a cell.

A method for identifying a protein tyrosine phosphatase that is a reversibly modified component of an inducible biological signaling pathway in a cell, comprising:

• contacting a biological sample comprising a cell that comprises at least one protein tyrosine phosphatase with a stimulus that induces a biological signaling pathway under conditions and for a time sufficient to induce the biological signaling pathway and thereby reversibly protect a protein tyrosine phosphatase active site invariant cysteine from modification;
• isolating the protein tyrosine phosphatase in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a protein tyrosine phosphatase active site invariant cysteine;
• and
• determining, under conditions that reverse the reversible protection of the protein tyrosine phosphatase active site invariant cysteine from modification, a level of dephosphorylation of a detectably labeled protein tyrosine phosphatase substrate by the protein tyrosine phosphatase, wherein detectable substrate dephosphorylation indicates that an active protein tyrosine phosphatase is present, and therefrom identifying a protein tyrosine phosphatase that is a reversibly modified component of an inducible biological signaling pathway in a cell.

A method for identifying an agent that alters an inducible biological signaling pathway, comprising:

• (a) identifying a protein tyrosine phosphatase that is reversibly oxidized in a cell according to a method comprising:
• (i) contacting a first biological sample comprising a cell that comprises at least one protein tyrosine phosphatase with a stimulus under conditions and for a time sufficient to induce reversible oxidation of at least one protein tyrosine phosphatase in the cell;
• (ii) isolating the protein tyrosine phosphatase in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a protein tyrosine phosphatase active site invariant cysteine;
• (iii) determining under reducing conditions a level of dephosphorylation of a detectably labeled protein tyrosine phosphatase substrate by the protein tyrosine phosphatase, wherein detectable substrate dephosphorylation indicates that an active protein tyrosine phosphatase is present, and therefrom identifying a protein tyrosine phosphatase that is reversibly oxidized in a cell;
• (b) contacting, in the presence and absence of a candidate agent, a second biological sample comprising a cell that comprises the PTP that is reversibly oxidized as identified according to the method of (a) with the stimulus under conditions and for a time sufficient to induce reversible oxidation of the PTP;
• (c) isolating the protein tyrosine phosphatase in the presence of a sulfhydryl-reactive agent that is capable of covalently modifying a sulfhydryl group of a protein tyrosine phosphatase active site invariant cysteine;
• and
• (d) determining under reducing conditions a level of dephosphorylation of a detectably labeled protein tyrosine phosphatase substrate by the protein tyrosine phosphatase,
• wherein a level of substrate dephosphorylation that is decreased when the second sample is contacted with the stimulus in the presence of the candidate agent relative to the level of substrate dephosphorylation when the sample is contacted with the stimulus in the absence of the agent indicates that the agent is an inhibitor of an inducible biological signaling pathway,
• and wherein a level of substrate dephosphorylation that is increased when the sample is contacted with the stimulus in the presence of the candidate agent relative to the level of substrate dephosphorylation when the sample is contacted with the stimulus in the absence of the agent indicates that the agent is a potentiator of an inducible biological signaling pathway.

A method for identifying a SHP-2 protein tyrosine phosphatase (SHP-2) that is reversibly oxidized in a cell, comprising:

• contacting a biological sample comprising a cell that comprises SHP-2 with a stimulus under conditions and for a time sufficient to induce reversible oxidation of SHP-2 in the cell;
• isolating anaerobically SHP-2 in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a SHP-2 active site invariant cysteine;
• and
• determining under reducing conditions a level of dephosphorylation of a detectably labeled SHP-2 substrate by SHP-2, wherein SHP-2 comprises a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS:
• 14, 16, 26, 28, 30, and 32, wherein detectable substrate dephosphorylation indicates that an active SHP-2 is present, and therefrom identifying a SHP-2 that is reversibly oxidized in a cell.

A method for identifying a PTP1B protein tyrosine phosphatase (PTP1B) that is reversibly oxidized in a cell, comprising:

• contacting a biological sample comprising a cell that comprises PTP1B with a stimulus under conditions and for a time sufficient to induce reversible oxidation of PTP1B in the cell;
• isolating anaerobically PTP1B in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a PTP1B active site invariant cysteine;
• and
• determining under reducing conditions a level of dephosphorylation of a detectably labeled PTP1B substrate by PTP1B, wherein PTP1B comprises a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS:
• 2, 4, 6, 8, 10, and 12, and wherein detectable substrate dephosphorylation indicates that an active PTP1B is present, and therefrom identifying a PTP1B that is reversibly oxidized in a cell.

A method for identifying a TC45 protein tyrosine phosphatase (TC45) that is reversibly oxidized in a cell, comprising:

• contacting a biological sample comprising a cell that comprises TC45 with a stimulus under conditions and for a time sufficient to induce reversible oxidation of TC45 in the cell;
• isolating anaerobically TC45 in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a TC45 active site invariant cysteine;
• and
• determining under reducing conditions a level of dephosphorylation of a detectably labeled TC45 substrate by TC45, wherein TC45 comprises a polypeptide comprising an amino acid sequence set forth in NM—080422, and wherein detectable substrate dephosphorylation indicates that an active TC45 is present, and therefrom identifying a TC45 that is reversibly oxidized in a cell.

A method for identifying a SHP-2 protein tyrosine phosphatase (SHP-2) that is reversibly modified by a PTP active site-binding agent in a cell, comprising:

• contacting a PTP active site-binding agent that is capable of reversibly modifying a sulfhydryl group of a SHP-2 active site invariant cysteine with a biological sample comprising a cell that comprises SHP-2;
• isolating SHP-2 in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a SHP-2 active site invariant cysteine;
• and
• determining, under conditions that are capable of reversing a reversible modification of a sulfhydryl group of a SHP-2 active site invariant cysteine, a level of dephosphorylation of a detectably labeled SHP-2 substrate by SHP-2, wherein SHP-2 comprises a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS:
• 14, 16, 26, 28, 30, and 32, wherein detectable substrate dephosphorylation indicates that an active SHP-2 is present, and therefrom identifying a SHP-2 that is reversibly modified by a PTP active site-binding agent in a cell.

A method for identifying a PTP1B protein tyrosine phosphatase (PTP1B) that is reversibly modified by a PTP active site-binding agent in a cell, comprising:

• contacting a PTP active site-binding agent that is capable of reversibly modifying a sulfhydryl group of a PTP1B active site invariant cysteine with a biological sample comprising a cell that comprises PTP1B;
• isolating PTP1B in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a PTP1B active site invariant cysteine;
• and
• determining, under conditions that are capable of reversing a reversible modification of a sulfhydryl group of a PTP1B active site invariant cysteine, a level of dephosphorylation of a detectably labeled PTP1B substrate by PTP1B, wherein PTP1B comprises a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS:
• 2, 4, 6, 8, 10, and 12, and wherein detectable substrate dephosphorylation indicates that an active PTP1B is present, and therefrom identifying a PTP1B that is reversibly modified by a PTP active site-binding agent in a cell.

A method for identifying a TC45 protein tyrosine phosphatase (TC45) that is reversibly modified by a PTP active site-binding agent in a cell, comprising:

• contacting a PTP active site-binding agent that is capable of reversibly modifying a sulfhydryl group of a TC45 active site invariant cysteine with a biological sample comprising a cell that comprises TC45;
• isolating TC45 in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a TC45 active site invariant cysteine;
• and
• determining, under conditions that are capable of reversing a reversible modification of a sulfhydryl group of a TC45 active site invariant cysteine, a level of dephosphorylation of a detectably labeled TC45 substrate by TC45, wherein TC45 comprises a polypeptide comprising an amino acid sequence set forth in NM—080422, and wherein detectable substrate dephosphorylation indicates that an active TC45 is present, and therefrom identifying a TC45 that is reversibly modified by a PTP active site-binding agent in a cell.

A method for identifying a SHP-2 protein tyrosine phosphatase (SHP-2) that is a reversibly modified component of an inducible biological signaling pathway in a cell, comprising:

• contacting a biological sample comprising a cell that comprises SHP-2 with a stimulus that induces a biological signaling pathway under conditions and for a time sufficient to induce the biological signaling pathway and thereby reversibly protect a SHP-2 active site invariant cysteine from modification;
• isolating the SHP-2 in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a SHP-2 active site invariant cysteine;
• and
• determining, under conditions that reverse the reversible protection of the SHP-2 active site invariant cysteine from modification, a level of dephosphorylation of a detectably labeled SHP-2 substrate by SHP-2, wherein SHP-2 comprises a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS:
• 14, 16, 26, 28, 30, and 32, and wherein detectable substrate dephosphorylation indicates that an active SHP-2 is present, and therefrom identifying a SHP-2 that is a reversibly modified component of an inducible biological signaling pathway in a cell.

A method for identifying a PTP1B protein tyrosine phosphatase (PTP1B) that is a reversibly modified component of an inducible biological signaling pathway in a cell, comprising:

• contacting a biological sample comprising a cell that comprises PTP1B with a stimulus that induces a biological signaling pathway under conditions and for a time sufficient to induce the biological signaling pathway and thereby reversibly protect a PTP1B active site invariant cysteine from modification;
• isolating the PTP1B in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a PTP1B active site invariant cysteine;
• and
• determining, under conditions that reverse the reversible protection of the PTP1B active site invariant cysteine from modification, a level of dephosphorylation of a detectably labeled PTP1B substrate by PTP1B, wherein PTP1B comprises a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS:
• 2, 4, 6, 8, 10, and 12, and wherein detectable substrate dephosphorylation indicates that an active PTP1B is present, and therefrom identifying a PTP1B that is a reversibly modified component of an inducible biological signaling pathway in a cell.

A method for identifying a TC45 protein tyrosine phosphatase (TC45) that is a reversibly modified component of an inducible biological signaling pathway in a cell, comprising:

• contacting a biological sample comprising a cell that comprises TC45 with a stimulus that induces a biological signaling pathway under conditions and for a time sufficient to induce the biological signaling pathway and thereby reversibly protect a TC45 active site invariant cysteine from modification;
• isolating the TC45 in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a TC45 active site invariant cysteine;
• and
• determining, under conditions that reverse the reversible protection of the TC45 active site invariant cysteine from modification, a level of dephosphorylation of a detectably labeled TC45 substrate by TC45, wherein TC45 comprises a polypeptide comprising an amino acid sequence set forth in NM—080422, and wherein detectable substrate dephosphorylation indicates that an active TC45 is present, and therefrom identifying a TC45 that is a reversibly modified component of an inducible biological signaling pathway in a cell.

A method for identifying an agent that alters an inducible biological signaling pathway, comprising:

• (a) identifying a SHP-2 protein tyrosine phosphatase (SHP-2) that is reversibly oxidized in a cell according to a method comprising:
• (i) contacting a first biological sample comprising a cell that comprises SHP-2 with a stimulus under conditions and for a time sufficient to induce reversible oxidation of SHP-2 in the cell;
• (ii) isolating SHP-2 in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a SHP-2 active site invariant cysteine;
• (iii) determining under reducing conditions a level of dephosphorylation of a detectably labeled SHP-2 substrate by SHP-2, wherein detectable substrate dephosphorylation indicates that an active SHP-2 is present, and therefrom identifying a SHP-2 that is reversibly oxidized in a cell;
• (b) contacting, in the presence and absence of a candidate agent, a second biological sample comprising a cell that comprises SHP-2 that is reversibly oxidized as identified according to the method of (a) with the stimulus under conditions and for a time sufficient to induce reversible oxidation of SHP-2;
• (c) isolating SHP-2 in the presence of a sulfhydryl-reactive agent that is capable of covalently modifying a sulfhydryl group of a SHP-2 active site invariant cysteine;
• and
• (d) determining under reducing conditions a level of dephosphorylation of a detectably labeled SHP-2 substrate by SHP-2, wherein SHP-2 comprises a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS:
• 14, 16, 26, 28, 30, and 32,
• wherein a level of substrate dephosphorylation that is decreased when the second sample is contacted with the stimulus in the presence of the candidate agent relative to the level of substrate dephosphorylation when the sample is contacted with the stimulus in the absence of the agent indicates that the agent is an inhibitor of an inducible biological signaling pathway,
• wherein a level of substrate dephosphorylation that is increased when the sample is contacted with the stimulus in the presence of the candidate agent relative to the level of substrate dephosphorylation when the sample is contacted with the stimulus in the absence of the agent indicates that the agent is a potentiator of an inducible biological signaling pathway.

A method for identifying an agent that alters an inducible biological signaling pathway, comprising:

• (a) identifying a PTP1B protein tyrosine phosphatase (PTP1B) that is reversibly oxidized in a cell according to a method comprising:
• (i) contacting a first biological sample comprising a cell that comprises PTP1B with a stimulus under conditions and for a time sufficient to induce reversible oxidation of PTP1B in the cell;
• (ii) isolating PTP1B in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a PTP1B active site invariant cysteine;
• (iii) determining under reducing conditions a level of dephosphorylation of a detectably labeled PTP1B substrate by PTP1B, wherein detectable substrate dephosphorylation indicates that an active PTP1B is present, and therefrom identifying a PTP1B that is reversibly oxidized in a cell;
• (b) contacting, in the presence and absence of a candidate agent, a second biological sample comprising a cell that comprises PTP1B that is reversibly oxidized as identified according to the method of (a) with the stimulus under conditions and for a time sufficient to induce reversible oxidation of PTP1B;
• (c) isolating PTP1B in the presence of a sulfhydryl-reactive agent that is capable of covalently modifying a sulfhydryl group of a PTP1B active site invariant cysteine;
• and
• (d) determining under reducing conditions a level of dephosphorylation of a detectably labeled PTP1B substrate by PTP1B, wherein PTP1B comprises a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS:
• 2, 4, 6, 8, 10, and 12,
• wherein a level of substrate dephosphorylation that is decreased when the second sample is contacted with the stimulus in the presence of the candidate agent relative to the level of substrate dephosphorylation when the sample is contacted with the stimulus in the absence of the agent indicates that the agent is an inhibitor of an inducible biological signaling pathway,
• and wherein a level of substrate dephosphorylation that is increased when the sample is contacted with the stimulus in the presence of the candidate agent relative to the level of substrate dephosphorylation when the sample is contacted with the stimulus in the absence of the agent indicates that the agent is a potentiator of an inducible biological signaling pathway.

A method for identifying an agent that alters an inducible biological signaling pathway, comprising:

• (a) identifying a TC45 protein tyrosine phosphatase (TC45) that is reversibly oxidized in a cell according to a method comprising:
• (i) contacting a first biological sample comprising a cell that comprises TC45 with a stimulus under conditions and for a time sufficient to induce reversible oxidation of TC45 in the cell;
• (ii) isolating TC45 in the presence of a sulfhydryl-reactive agent that is capable of irreversibly modifying a sulfhydryl group of a TC45 active site invariant cysteine;
• (iii) determining under reducing conditions a level of dephosphorylation of a detectably labeled TC45 substrate by TC45, wherein detectable substrate dephosphorylation indicates that an active TC45 is present, and therefrom identifying a TC45 that is reversibly oxidized in a cell;
• (b) contacting, in the presence and absence of a candidate agent, a second biological sample comprising a cell that comprises TC45 that is reversibly oxidized as identified according to the method of (a) with the stimulus under conditions and for a time sufficient to induce reversible oxidation of TC45;
• (c) isolating TC45 in the presence of a sulfhydryl-reactive agent that is capable of covalently modifying a sulfhydryl group of a TC45 active site invariant cysteine;
• and
• (d) determining under reducing conditions a level of dephosphorylation of a detectably labeled TC45 substrate by TC45, wherein TC45 comprises a polypeptide comprising an amino acid sequence set forth in NM—080422,
• wherein a level of substrate dephosphorylation that is decreased when the second sample is contacted with the stimulus in the presence of the candidate agent relative to the level of substrate dephosphorylation when the sample is contacted with the stimulus in the absence of the agent indicates that the agent is an inhibitor of an inducible biological signaling pathway,
• and wherein a level of substrate dephosphorylation that is increased when the sample is contacted with the stimulus in the presence of the candidate agent relative to the level of substrate dephosphorylation when the sample is contacted with the stimulus in the absence of the agent indicates that the agent is a potentiator of an inducible biological signaling pathway.