Melowic Heather R, Stahelin Robert V, Blatner Nichole R, Tian Wen, Hayashi Keitaro, Altman Amnon, Cho Wonhwa
Department of Chemistry, University of Illinois, Chicago, Illinois 60607, USA.
J Biol Chem. 2007 Jul 20;282(29):21467-76. doi: 10.1074/jbc.M700119200. Epub 2007 Jun 4.
Protein kinase C (PKC) is a novel PKC that plays a key role in T lymphocyte activation. PKC has been shown to be specifically recruited to the immunological synapse in response to T cell receptor activation. To understand the basis of its unique subcellular localization properties, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKC. PKC showed phosphatidylserine selectivity in membrane binding and kinase action, which contributes to its translocation to the phosphatidylserine-rich plasma membrane in HEK293 cells. Unlike any other PKCs characterized so far, the isolated C1B domain of PKC had much higher affinity for DAG-containing membranes than the C1A domain. Also, the mutational analysis indicates that the C1B domain plays a predominant role in the DAG-induced membrane binding and activation of PKC. Furthermore, the Ca(2+)-independent C2 domain of PKC has significant affinity for anionic membranes, and the truncation of the C2 domain greatly enhanced the membrane affinity and enzyme activity of PKC. In addition, membrane binding properties of Y90E and Y90F mutants indicate that phosphorylation of Tyr(90) of the C2 domain enhances the affinity of PKC for model and cell membranes. Collectively, these results show that PKC has a unique membrane binding and activation mechanism that may account for its subcellular targeting properties.
蛋白激酶C（PKC）是一种新型PKC，在T淋巴细胞激活中起关键作用。已证明PKC可响应T细胞受体激活而特异性募集至免疫突触。为了解其独特亚细胞定位特性的基础，我们研究了PKC在体外和细胞中由sn-1,2-二酰甘油（DAG）介导的膜结合机制。PKC在膜结合和激酶作用方面表现出对磷脂酰丝氨酸的选择性，这有助于其在HEK293细胞中转运至富含磷脂酰丝氨酸的质膜。与迄今为止所表征的任何其他PKC不同，PKC分离的C1B结构域对含DAG的膜的亲和力远高于C1A结构域。此外，突变分析表明C1B结构域在DAG诱导的PKC膜结合和激活中起主要作用。此外，PKC的钙非依赖性C2结构域对阴离子膜具有显著亲和力，C2结构域的截短极大地增强了PKC的膜亲和力和酶活性。此外，Y90E和Y90F突变体的膜结合特性表明，C2结构域Tyr(90)的磷酸化增强了PKC对模型膜和细胞膜的亲和力。总体而言，这些结果表明PKC具有独特的膜结合和激活机制，这可能解释了其亚细胞靶向特性。
