Structural requirements for localization and activation of protein kinase Cμ (PKCμ) at the Golgi compartment
Structural requirements for localization and activation of protein kinase Cμ (PKCμ) at the Golgi compartment
复制标题
DOI：
10.1083/jcb.200110047
复制
发表时间：
2002-01-07
影响因子：
7.8
通讯作者：
Johannes, FJ
中科院分区：
文献类型：
--
作者：
Hausser, A;Link, G;Johannes, FJ
在这里，我们描述了高尔基体定位和一个顺序的，定位依赖激活过程的蛋白激酶C（PKC）亩涉及自动和转磷酸化的结构要求。通过共聚焦显微镜分析表达各种PKCmu-绿色荧光蛋白融合蛋白的HeLa细胞与高尔基体区室特异性标记物p24和p230共染色的高尔基体区室定位的结构基础。删除的NH 2-末端疏水或半胱氨酸区域，但不是普列克底物蛋白同源性或酸性结构域，PKCmu完全废除高尔基本地化的PKCmu。由于NH 2-末端PKCmu片段与p24共定位，该区域的PKCmu是必不可少的，足以介导与高尔基体膜的关联。光漂白研究后的荧光恢复证实了组成，胞质PKC μ的快速招聘，并与稳定的协会，独立于激活环磷酸化的高尔基室。高尔基复合体靶向不需要激酶活性，从显微镜和细胞分级分离研究中可以看出，激酶死亡的PKCmu仅位于细胞内膜。我们提出了一个顺序激活过程的PKCmu，其中高尔基体区室招聘之前，是必不可少的激活环磷酸化（丝氨酸738/742）的transacting激酶，其次是自动和转磷酸化的NH 2-末端丝氨酸（S）的监管结构域。PKC μ活化环磷酸化对于底物磷酸化是不可缺少的，因此PKC μ在高尔基体区室的功能是不可缺少的。
We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC)mu involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKCmu-green fluorescent protein fusion proteins costained with the Golgi compartment-specific markers p24 and p230. Deletions of either the NH2-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKCmu completely abrogated Golgi localization of PKCmu. As an NH2-terminal PKCmu fragment was colocalized with p24, this region of PKCmu is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKCmu to, and stable association with, the Golgi compartment independent of activation loop phosphorylation. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinase-dead PKCmu found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKCmu, in which Golgi compartment recruitment precedes and is essential for activation loop phoshorylation (serines 738/742) by a transacting kinase, followed by auto- and transphosphorylation of NH2-terminal serine(s) in the regulatory domain. PKCmu activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKCmu function at the Golgi compartment.