Redox Regulation of SH2-Domain-Containing Protein Tyrosine Phosphatases by Two Backdoor Cysteines
Redox Regulation of SH2-Domain-Containing Protein Tyrosine Phosphatases by Two Backdoor Cysteines
复制标题
DOI：
10.1021/bi801973z
复制
发表时间：
2009-02-17
期刊：
影响因子：
2.9
通讯作者：
Rudolph, Johannes
中科院分区：
文献类型：
--
作者：
Chen, Cheng-Yu;Willard, Devina;Rudolph, Johannes
蛋白质酪氨酸磷酸酶（PTPs）是已知的磷酸化，定位和蛋白质-蛋白质相互作用的调节。最近，氧化还原依赖性失活已成为响应细胞刺激而减弱PTP活性的关键因素。串联Src同源2结构域的PTP（SHP）属于非受体PTP家族，其活性可通过体内可逆氧化来调节。在此，我们研究了体外可逆氧化的动力学和机制的细节SHP-1和SHP-2。我们已经证实了SHP的活性位点半胱氨酸对氧化失活的敏感性，其氧化速率常数与其他PTP相似（2-10 M-1 s（-1））。SHP-1和SHP-2都可以被还原剂DTT和谷胱甘肽还原和再活化，而只有SHP-2的催化结构域被硫氧还蛋白再活化。SHP的可逆氧化态的稳定化通过不同于其他PTP的新机制进行，其中氧化在催化半胱氨酸和附近的“后门”半胱氨酸之间产生二硫化物或与相邻氨基酸的酰胺骨架氮形成亚磺酰胺键。相反，在可逆氧化和失活的SHP中，催化半胱氨酸被再还原，而两个保守的后门半胱氨酸形成分子内二硫化物。这种后门-后门二硫化物的形成取决于活性位点半胱氨酸的存在，并且可以通过活性位点半胱氨酸-后门半胱氨酸中间体进行。两个后门半胱氨酸的去除导致不可逆的氧化失活，表明这两个半胱氨酸对于确保SHP的可逆氧化是必要且足够的。我们的研究结果扩展了PTPs的氧化还原调节用于调节细胞内信号通路的机制。
Protein tyrosine phosphatases (PTPs) are known to be regulated by phosphorylation, localization, and protein-protein interactions. More recently, redox-dependent inactivation has emerged as a critical factor in attenuating PTP activity in response to cellular stimuli. The tandem Src homology 2 domain-containing PTPs (SHPs) belong to the family of nonreceptor PTPs whose activity can be modulated by reversible oxidation in vivo. Herein we have investigated in vitro the kinetic and mechanistic details of reversible oxidation of SHP-1 and SHP-2. We have confirmed the susceptibility of the active site cysteines of SHPs to oxidative inactivation, with rate constants for oxidation similar to other PTPs (2-10 M-1 s(-1)). Both SHP-1 and SHP-2 can be reduced and reactivated with the reductants DTT and gluthathione, whereas only the catalytic domain of SHP-2 is subject to reactivation by thioredoxin. Stabilization of the reversible oxidation state of the SHPs proceeds via a novel mechanism unlike for other PTPs wherein oxidation yields either a disulfide between the catalytic cysteine and a nearby "backdoor" cysteine or a sulfenylamide bond with the amide backbone nitrogen of the adjacent amino acid. Instead, in the reversibly oxidized and inactivated SHPs, the catalytic cysteine is rereduced while two conserved backdoor cysteines form an intramolecular disulfide. Formation of this backdoor-backdoor disulfide is dependent on the presence of the active site cysteine and can proceed via either active site cysteine-backdoor cysteine intermediate. Removal of both backdoor cysteines leads to irreversible oxidative inactivation, demonstrating that these two cysteines are necessary and sufficient for ensuring reversible oxidation of the SHPs. Our results extend the mechanisms by which redox regulation of PTPs is used to modulate intracellular signaling pathways.