Specific translocation of protein kinase Cα to the plasma membrane requires both Ca2+ and PIP2 recognition by its C2 domain
Specific translocation of protein kinase Cα to the plasma membrane requires both Ca2+ and PIP2 recognition by its C2 domain
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DOI：
10.1091/mbc.e05-06-0499
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发表时间：
2006-01-01
影响因子：
3.3
通讯作者：
Falke, JJ
中科院分区：
文献类型：
--
作者：
Evans, JH;Murray, D;Falke, JJ
蛋白激酶C α（PKC α）的C2结构域控制该激酶在细胞质Ca 2+信号期间从细胞质到质膜的易位。本研究使用荧光融合蛋白的细胞内共成像和体外FRET膜结合试验，以进一步研究这种易位的性质。我们发现，Ca 2+激活的PKC α和其分离的C2结构域仅定位于质膜在体内和质膜脂质，磷脂酰肌醇-4，5-二磷酸（PIP 2），显着增强了Ca 2+触发的结合的C2结构域的膜在体外。类似地，将PKC α Ca 2+结合环（CBL）和PIP 2结合位点（β链3-4）置换到不同的C2结构域中的杂合构建体表现出天然Ca 2+触发的对质膜的靶向并识别PIP 2。相反，含有CBL但缺乏PIP 2位点的杂交体主要易位到反式高尔基体网络（TGN）并且不能识别PIP 2。类似地，在PIP 2位点具有突变的PKC α C2结构域主要靶向TGN并且不能识别PIP 2。总体而言，这些发现表明CBL对于Ca 2+触发的膜结合是必需的，但对于特异性质膜靶向是不够的。相反，靶向特异性由β链3-4上的碱性残基提供，其结合质膜PIP 2。
The C2 domain of protein kinase C alpha (PKC alpha) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKC alpha and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKC alpha Ca2+-binding loops (CBLs) and PIP2 binding site (beta-strands 3-4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKC alpha C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on beta-strands 3-4, which bind to plasma membrane PIP2.