G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D -: Dependence on plasma membrane translocation and protein kinase Cε
G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D -: Dependence on plasma membrane translocation and protein kinase Cε
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DOI：
10.1074/jbc.m403265200
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发表时间：
2004-08-13
影响因子：
4.8
通讯作者：
Rozengurt, E
中科院分区：
文献类型：
--
作者：
Rey, O;Reeve, JR;Rozengurt, E
蛋白激酶D （PKD）是一种丝氨酸/苏氨酸蛋白激酶，由G蛋白偶联受体（GPCR）激动剂激活，其机制尚未完全确定，包括其可逆的质膜易位和通过蛋白激酶C （PKC）依赖途径激活环磷酸化。为了更好地了解GPCR刺激下PKD活化的调节机制，我们研究了其快速质膜易位在其激活环磷酸化中的作用，并鉴定了体内介导该事件的内源性PKC同工酶。我们发现PKD突变体的激活环对二酰基甘油和酚酯的亲和力降低，仅在其质膜结合时被磷酸化。我们还发现PKD的激活环磷酸化和快速质膜解离可以通过阻止PKCepsilon的质膜易位，通过消除其与活化的C激酶受体的相互作用，或者通过抑制PKCepsilon的表达来抑制PKD的激活环磷酸化和快速质膜解离。因此，本研究表明，PKD在GPCR刺激下的质膜易位对于PKD激活环的pkcepsilon介导的磷酸化是必要的，并且这一事件需要两种激酶易位到质膜上。基于这些和先前的结果，我们提出了一个gpcr介导的PKD调控模型，该模型整合了其分布、催化活性和多位点磷酸化的变化。
Protein kinase D (PKD) is a serine/threonine protein kinase activated by G protein-coupled receptor ( GPCR) agonists through an incompletely characterized mechanism that includes its reversible plasma membrane translocation and activation loop phosphorylation via a protein kinase C (PKC)-dependent pathway. To gain a better understanding of the mechanism regulating the activation of PKD in response to GPCR stimulation, we investigated the role of its rapid plasma membrane translocation on its activation loop phosphorylation and identified the endogenous PKC isozyme that mediates that event in vivo. We had found that the activation loop of a PKD mutant, with reduced affinity for diacylglycerol and phorbol esters, was only phosphorylated upon its plasma membrane association. We also found that the activation loop phosphorylation and rapid plasma membrane dissociation of PKD were inhibited either by preventing the plasma membrane translocation of PKCepsilon, through abolition of its interaction with receptor for activated C kinase, or by suppressing the expression of PKCepsilon via specific small interfering RNAs. Thus, this study demonstrates that the plasma membrane translocation of PKD, in response to GPCR stimulation, is necessary for the PKCepsilon-mediated phosphorylation of the activation loop of PKD and that this event requires the translocation of both kinases to the plasma membrane. Based on these and previous results, we propose a model of GPCR-mediated PKD regulation that integrates its changes in distribution, catalytic activity, and multisite phosphorylation.