The carboxyl terminus of protein kinase C provides a switch to regulate its interaction with the phosphoinositide-dependent kinase, PDK-1
The carboxyl terminus of protein kinase C provides a switch to regulate its interaction with the phosphoinositide-dependent kinase, PDK-1
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DOI：
10.1074/jbc.m101357200
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发表时间：
2001-06-01
影响因子：
4.8
通讯作者：
Newton, AC
中科院分区：
文献类型：
--
作者：
Gao, TY;Toker, A;Newton, AC
蛋白激酶C家族成员的功能取决于两种紧密耦合的磷酸化机制：磷酸肌醇依赖性激酶PDK-1对活化环的磷酸化，随后在COOH末端的两个位置，即转角基序和疏水基序处发生自磷酸化。在此，我们阐述了PDK-1调节蛋白激酶C PII的分子机制，免疫共沉淀研究表明，PDK-1协会优先与其底物，非磷酸化蛋白激酶C，通过直接的机制。蛋白激酶C的暴露的COOH末端为PDK-1提供了主要的相互作用位点，羧基末端的构建体的共表达有效地破坏了体内的相互作用。这种相互作用的破坏促进了蛋白激酶C的自磷酸化，表明PDK-1与羧基末端的结合保护其免于自磷酸化，对COOH末端构建体的研究表明，PDK-1对磷酸化COOH末端的内在亲和力比对未磷酸化COOH末端的内在亲和力大一个数量级以上，这与PDK-1不能有效结合磷酸化蛋白激酶C的发现形成对比。然而，磷酸化物质的有效结合可由蛋白激酶C的活化构象诱导。这表明羧基末端在自磷酸化后被掩蔽，该过程可通过伴随活化的构象变化逆转。我们的数据表明了一种模型，其中PDK-1提供了蛋白激酶C的两个调节点：1）活化环的磷酸化，其由PDK-1的内在活性调节，和2）羧基末端的磷酸化，其由PDK-1的释放调节以允许自磷酸化。
The function of protein kinase C family members depends on two tightly coupled phosphorylation mechanisms: phosphorylation of the activation loop by the phosphoinositide-dependent kinase, PDK-1, followed by autophosphorylation at two positions in the COOH terminus, the turn motif, and the hydrophobic motif, Here we address the molecular mechanisms underlying the regulation of protein kinase C PII by PDK-1, Co-immunoprecipitation studies reveal that PDK-1 associates preferentially with its substrate, unphosphorylated protein kinase C, by a direct mechanism. The exposed COOH terminus of protein kinase C provides the primary interaction site for PDK-1, with co-expression of constructs of the carboxyl terminus effectively disrupting the interaction in vivo, Disruption of this interaction promotes the autophosphorylation of protein kinase C, suggesting that the binding of PDK-1 to the carboxyl terminus protects it from autophosphorylation, Studies with constructs of the COOH terminus reveal that the intrinsic affinity of PDK-1 for phosphorylated COOH terminus is over an order of magnitude greater than that for unphosphorylated COOH terminus, contrasting with the finding that PDK-1 does not bind phosphorylated protein kinase C effectively. However, effective binding of the phosphorylated species can be induced by the activated conformation of protein kinase C, This suggests that the carboxyl terminus becomes masked following autophosphorylation, a process that can be reversed by the conformational changes accompanying activation, Our data suggest a model in which PDK-1 provides two points of regulation of protein kinase C: 1) phosphorylation of the activation loop, which is regulated by the intrinsic activity of PDK-1, and 2) phosphorylation of the carboxyl terminus, which is regulated by the release of PDK-1 to allow autophosphorylation.