Rab6 is phosphorylated in thrombin-activated platelets by a protein kinase C-dependent mechanism: effects on GTP/GDP binding and cellular distribution.
Rab6 is phosphorylated in thrombin-activated platelets by a protein kinase C-dependent mechanism: effects on GTP/GDP binding and cellular distribution.
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DOI：
10.1042/bj3420353
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发表时间：
1999-09
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影响因子：
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通讯作者：
Michael L. Fitzgerald;Guy L. Reed
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文献类型：
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作者：
Michael L. Fitzgerald;Guy L. Reed
在血小板和其他分泌细胞中，蛋白激酶C（PKC）在由生理性细胞外信号刺激的胞吐中起作用，尽管其与分泌机制的联系知之甚少。我们研究了Rab 6，一种与血小板α颗粒分离的GTP结合蛋白，是否可能参与连接这些过程。我们发现Rab 6包含两个进化上保守的PKC磷酸化位点。在用[（32）P]P（i）代谢标记的血小板中，Rab 6磷酸化被佛波酯或凝血酶诱导。这种磷酸化被特异性PKC抑制剂（Ro-31-8220）阻断，但不被p38丝裂原活化蛋白激酶抑制剂（PD-169316）阻断。血小板的生理刺激引起的PKC依赖性易位Rab 6从血小板颗粒组分，几乎加倍的分数Rab 6在胞质溶胶。人Rab 6亚型（Rab 6C），优先在人血小板RNA中表达的克隆和其磷酸化的PKC的特点。Rab 6C掺入的[（32）P]P（i）最多为每摩尔活性蛋白2摩尔。Rab 6C结合GDP和GTP的K（d）值分别为113+/-12和119+/-27 nM，水解GTP的速率为100+/-15 micromol GTP/mol Rab 6C/min。Rab 6C的PKC磷酸化使对GTP的亲和力增加3倍，尽管对GDP的影响较小（1.6倍）。磷酸化不改变GT3活性。总之，血小板的凝血酶活化导致Rab 6的PKC依赖性磷酸化和Rab 6向胞质溶胶的易位。我们认为，PKC磷酸化可能是一个重要的机制，通过这种机制，Rab在囊泡运输和分泌的功能相互作用可以改变响应外部刺激。
In platelets and other secretory cells, protein kinase C (PKC) plays a role in exocytosis stimulated by physiological extracellular signals, although its linkage to the secretory machinery is poorly understood. We investigated whether Rab6, a GTP-binding protein that fractionates with platelet alpha-granules, may be involved in linking these processes. We found that Rab6 contains two PKC consensus phosphorylation sites that are evolutionarily conserved. In platelets metabolically labelled with [(32)P]P(i), Rab6 phosphorylation was induced by phorbol esters or by thrombin. This phosphorylation was blocked by a specific PKC inhibitor (Ro-31-8220), but not by a p38 mitogen-activated protein kinase inhibitor (PD-169316). Physiological stimulation of platelets caused a PKC-dependent translocation of Rab6 from platelet particulate fractions, nearly doubling the fraction of Rab6 in the cytosol. A human Rab6 isoform (Rab6C) that is preferentially expressed in human platelet RNA was cloned and its phosphorylation by PKC was characterized. Rab6C incorporated up to 2 mol of [(32)P]P(i) per mol of active protein. Rab6C bound GDP and GTP with K(d) values of 113+/-12 and 119+/-27 nM respectively, and hydrolysed GTP at a rate of 100+/-15 micromol of GTP/mol of Rab6C per min. PKC phosphorylation of Rab6C increased the affinity for GTP by 3-fold, although it had lesser effects on GDP (1.6-fold). Phosphorylation did not alter the GTPase activity. In summary, thrombin activation of platelets leads to PKC-dependent phosphorylation of Rab6 and a translocation of Rab6 to the cytosol. We suggest that PKC phosphorylation may be an important mechanism through which Rab functional interactions in vesicle trafficking and secretion can be altered in response to an external stimulus.