Redox regulation of cAMP-dependent protein kinase signaling - Kinase versus phosphatase inactivation
Redox regulation of cAMP-dependent protein kinase signaling - Kinase versus phosphatase inactivation
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DOI：
10.1074/jbc.m702582200
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发表时间：
2007-07-27
影响因子：
4.8
通讯作者：
Taylor, Susan S.
中科院分区：
文献类型：
--
作者：
Humphries, Kenneth M.;Pennypacker, Juniper K.;Taylor, Susan S.
细胞信号传导途径的许多成分对氧化和还原的调节敏感。之前，我们描述了通过直接氧化激酶激活环中的反应性半胱氨酸来失活 cAMP 依赖性蛋白激酶 (PKA)。在本研究中，我们证明在 HeLa 细胞中 PKA 活性遵循对硫醇氧化的双相反应。在温和的氧化条件下，或短暂接触氧化剂，毛喉素刺激的 PKA 活性会增强。这种增强作用被巯基还原剂阻断，证明了可逆的激活模式。相反，毛喉素刺激的 PKA 活性会受到更严重的氧化条件的抑制。轻度氧化可增强毛喉素、异丙肾上腺素或细胞可渗透类似物 8-溴-cAMP 刺激的 PKA 活性。当细胞在丝氨酸/苏氨酸磷酸酶抑制剂 NaF 存在下裂解时，氧化的 PKA 增强作用减弱。这些结果表明 PKA 抵消磷酸酶的氧化可能受到抑制，从而增强表观激酶活性。使用体内 PKA 活性报告基因，我们证明轻度氧化确实可以通过抑制抵消磷酸酶活性来延长异丙肾上腺素诱导的 PKA 信号。这项研究的结果证明了活细胞中独特的协同机制，PKA 信号通路以明显的双相方式得到增强。
Many components of cellular signaling pathways are sensitive to regulation by oxidation and reduction. Previously, we described the inactivation of cAMP-dependent protein kinase (PKA) by direct oxidation of a reactive cysteine in the activation loop of the kinase. In the present study, we demonstrate that in HeLa cells PKA activity follows a biphasic response to thiol oxidation. Under mild oxidizing conditions, or short exposure to oxidants, forskolin-stimulated PKA activity is enhanced. This enhancement was blocked by sulfhydryl reducing agents, demonstrating a reversible mode of activation. In contrast, forskolin-stimulated PKA activity is inhibited by more severe oxidizing conditions. Mild oxidation enhanced PKA activity stimulated by forskolin, isoproterenol, or the cell-permeable analog, 8-bromo-cAMP. When cells were lysed in the presence of serine/threonine phosphatase inhibitor, NaF, the PKA-enhancing effect of oxidation was blunted. These results suggest oxidation of a PKA-counteracting phosphatase may be inhibited, thus enhancing the apparent kinase activity. Using an in vivo PKA activity reporter, we demonstrated that mild oxidation does indeed prolong the PKA signal induced by isoproterenol by inhibiting counteracting phosphatase activity. The results of this study demonstrate in live cells a unique synergistic mechanism whereby the PKA signaling pathway is enhanced in an apparent biphasic manner.