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Activity-based probes for protein tyrosine phosphatases.

ABSTRACT: Protein tyrosine phosphatases (PTPs) are involved in the regulation of many aspects of cellular activity including proliferation, differentiation, metabolism, migration, and survival. Given the large number and complexity of PTPs in cell signaling, new strategies are needed for the integrated analysis of PTPs in the whole proteome. Unfortunately, the activities of many PTPs are tightly regulated by posttranslational mechanisms, limiting the utility of standard genomics and proteomics methods for functional characterization of these enzymes. To facilitate the global analysis of PTPs, we designed and synthesized two activity-based probes that consist of alpha-bromobenzylphosphonate as a PTP-specific trapping device and a linker that connects the trapping device with a biotin tag for visualization and purification. We showed that these probes are active site-directed irreversible inactivators of PTPs and form a covalent adduct with PTPs involving the active site Cys residue. Additionally, we demonstrated that the probes are extremely specific toward PTPs while remaining inert to other proteins, including the whole proteome from Escherichia coli. Consequently, these activity-based PTP probes can be used to profile PTP activity in complex proteomes. The ability to interrogate the entire PTP family on the basis of changes in their activity should greatly accelerate both the assignment of PTP function and the identification of potential therapeutic targets.

SUBMITTER: Kumar S

PROVIDER: S-EPMC419536 | biostudies-literature | 2004 May

REPOSITORIES: biostudies-literature

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Activity-based probes for protein tyrosine phosphatases.

Kumar Sanjai S Zhou Bo B Liang Fubo F Wang Wei-Qing WQ Huang Zhonghui Z Zhang Zhong-Yin ZY

Proceedings of the National Academy of Sciences of the United States of America 20040517 21

Protein tyrosine phosphatases (PTPs) are involved in the regulation of many aspects of cellular activity including proliferation, differentiation, metabolism, migration, and survival. Given the large number and complexity of PTPs in cell signaling, new strategies are needed for the integrated analysis of PTPs in the whole proteome. Unfortunately, the activities of many PTPs are tightly regulated by posttranslational mechanisms, limiting the utility of standard genomics and proteomics methods for ...[more]

PMID: 15148367

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Project description:Protein tyrosine phosphatases (PTPs) play an important role in the regulation of mammalian signal transduction. During some cell signaling processes, the generation of endogenous hydrogen peroxide inactivates selected PTPs via oxidation of the enzyme's catalytic cysteine thiolate group. Importantly, low-molecular weight and protein thiols in the cell have the potential to regenerate the catalytically active PTPs. Here we examined the recovery of catalytic activity from two oxidatively inactivated PTPs (PTP1B and SHP-2) by various low-molecular weight thiols and the enzyme thioredoxin. All monothiols examined regenerated the catalytic activity of oxidized PTP1B, with apparent rate constants that varied by a factor of approximately 8. In general, molecules bearing low-pKa thiol groups were particularly effective. The biological thiol glutathione repaired oxidized PTP1B with an apparent second-order rate constant of 0.023 ± 0.004 M(-1) s(-1), while the dithiol dithiothreitol (DTT) displayed an apparent second-order rate constant of 0.325 ± 0.007 M(-1) s(-1). The enzyme thioredoxin regenerated the catalytic activity of oxidized PTP1B at a substantially faster rate than DTT. Thioredoxin (2 ?M) converted oxidized PTP1B to the active form with an observed rate constant of 1.4 × 10(-3) s(-1). The rates at which these agents regenerated oxidized PTP1B followed the order Trx > DTT > GSHand comparable values observed at 2 ?M Trx, 4 mM DTT, and 60 mM GSH. Various disulfides that are byproducts of the reactivation process did not inactivate native PTP1B at concentrations of 1-20 mM. The common biochemical reducing agent tris(2-carboxyethyl)phosphine regenerates enzymatic activity from oxidized PTP1B somewhat faster than the thiol-based reagents, with a rate constant of 1.5 ± 0.5 M(-1) s(-1). We observed profound kinetic differences between the thiol-dependent regeneration of activity from oxidized PTP1B and SHP-2, highlighting the potential for structural differences in various oxidized PTPs to play a significant role in the rates at which low-molecular weight thiols and thiol-containing enzymes such as thioredoxin and glutaredoxin return catalytic activity to these enzymes during cell signaling events.

Dataset Information

Activity-based probes for protein tyrosine phosphatases.

Publications

Activity-based probes for protein tyrosine phosphatases.

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