Chemical Probe-Based Approach to Delineate the Underlying Mechanism for Nitric Oxide-Mediated Inactivation of Protein Tyrosine Phosphatases in Regulation of Insulin Signaling

• Main Authors: Ming-Fo Hsu, 徐銘佛
• Other Authors: 孟子青
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/28894300698434482551

博士 === 臺灣大學 === 生化科學研究所 === 98 === Enzymes in the protein tyrosine phosphatase (PTP) superfamily are involved in the regulation of many aspects of biological processes. The catalytic activity of PTPs is mediated by an invariant Cys residue, which has a remarkably low pKa, therefore being able to carry out nucleophilic attack on a substrate, leading to Tyr dephosphorylation. Dysfunction of PTPs results in aberrant tyrosine phosphorylation signaling which has been linked to the etiology of several human diseases, including cancer and diabetes. However, a high efficient and convenient tool for tagging PTPs as a whole is still lacking at the present time. Here, we first established a novel strategy that tags PTPs in a complicated proteome by using a commercial Iodoacetyl-PEO-Biotin probe (the PEO probe) under acidic conditions. We proposed that a low pH condition might facilitate the specificity of labeling reaction towards the active site Cys of PTPs, due to its low pKa character. Indeed, we found that the purified human PTP1B, a prototype of PTP enzymes, could be efficiently alkylated by the PEO probe at as low as pH 6.0 even in a protein mixture with excess amounts of BSA and Catalase. We then applied this probe to tag and isolate endogenous PTPs, and the results show that a number of PTPs expressed in Caco-2 or EA.hy926 cells were appeared in the pull-down fraction. In the second part of our study, the analytic pulldown platform was used to tag endogenous PTPs involved in the regulation of sodium butyrate (NaB)-induced Caco-2 cell differentiation. Our results demonstrated a novel role of TC-PTP as a negative regulator in the control of differentiation in Caco-2 cells. We further observed that a high level of ectopically expressed TC-PTP prevented Caco-2 cells from the entry of differentiation in NaB-treated cells. In the next phase of study, our strategy was applied for the characterization of S-nitrosylation on PTPs. We showed that, not only purified PTP1B but also endogenous PTPs were susceptible to nitric oxide (NO)-mediated S-nitrosylation and inactivation. Our data indicated that multiple cellular PTPs are likely S-nitrosylated at the active site Cys residue concomitantly with a burst of intrinsic NO production. We also observed a critical role of NO in insulin responsiveness, as evidenced by an NO-dependent increase of tyrosine phosphorylation levels of the insulin receptor and its downstream effectors IRS-1 and PKB/AKT. Employing the chemical probe-based approach, we demonstrated that NO mediates the inhibition of insulin receptor PTPs, leading to the enhancement of insulin responsiveness.