Regulation of protein tyrosine phosphatase 1B in intact cells by S-nitrosothiols
Regulation of protein tyrosine phosphatase 1B in intact cells by S-nitrosothiols
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DOI：
10.1016/s0003-9861(02)00696-3
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发表时间：
2003-02-15
影响因子：
3.9
通讯作者：
Whorton, AR
中科院分区：
文献类型：
--
作者：
Li, S;Whorton, AR
蛋白质酪氨酸磷酸酶 (PTPases) 含有活性位点半胱氨酸，氧化时会导致磷酸酶活性丧失和磷蛋白积累。例如，EGF 刺激后产生的氧化剂会抑制 PTP1B 并增强 EGF 受体磷酸化。由于 NO 衍生物质也会修饰反应性硫醇，因此我们推测 NO 会可逆地抑制 PTP1B。在我们的研究中，我们将 A431 或 Jurkat 细胞暴露于 NO 供体并测量 PTP1B 活性或使用 3-马来酰亚胺基丙酰生物胞素 (MPB) 来测量硫醇氧化还原状态。亚硝基硫醇通过在培养基中添加半胱氨酸大大增强的机制导致 PTP1B 的快速抑制。使用免疫沉淀 PTP1B 分析硫醇氧化状态显示修饰与活性丧失一致。在完整细胞中或在细胞裂解液中添加 DTT 后，酶抑制和修饰都是可逆的。虽然 DTT 可以逆转氧化，但抗坏血酸却不能，这表明混合二硫化物（可能是谷胱甘肽化）的形成而不是 S-亚硝基化导致了 PTP1B 抑制。重要的是，即使在没有外源添加 EGF 的情况下，亚硝基硫醇对 PTP1B 的抑制也会导致 EGF 受体磷酸化。这些发现表明 NO 在调节信号通路中发挥着重要作用，因为抑制 PTP 酶可能会增强或延长磷蛋白的活性。 (C) 2002 年爱思唯尔科学（美国）。版权所有。
Protein tyrosine phosphatases (PTPases) contain an active site cysteine which when oxidized leads to loss of phosphatase activity and accumulation of phosphoproteins. For example, oxidants produced following EGF stimulation inhibit PTP1B and enhance EGF receptor phosphorylation. Because NO-derived species also modify reactive thiols, we postulated that NO would reversibly inhibit PTP1B. In our studies we exposed A431 or Jurkat cells to NO donors and measured PTP1B activity or used 3-maleimidylpropionylbiocytin (MPB) to measure thiol redox status. Nitrosothiols led to a rapid inhibition of PTP1B through a mechanism that was greatly enhanced by addition of cysteine to the medium. Analysis of thiol oxidation status using immunoprecipitated PTP1B showed modification consistent with loss of activity. Both enzyme inhibition and modification were reversible in intact cells or after addition of DTT to cell lysates. While DTT reversed oxidation, ascorbate did not, suggesting that formation of a mixed disulfide (possibly glutathionylation) rather than S-nitrosylation accounts for PTP1B inhibition. Importantly, PTP1B inhibition by nitrosothiols led to EGF receptor phosphorylation even in the absence of exogenously added EGF. These findings suggest an important role for NO in modulating signaling pathways since inhibition of PTPases could potentially enhance or prolong activity of phosphoproteins. (C) 2002 Elsevier Science (USA). All rights reserved.