Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation
Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation
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DOI：
10.1074/jbc.m109395200
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发表时间：
2001-12-28
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;Sinnett-Smith, J;Rozengurt, E
蛋白激酶D(PKD)/蛋白激酶CMU是一种丝氨酸/苏氨酸蛋白激酶，由生长因子、抗原受体结合和G蛋白偶联受体(GPCR)激动剂通过磷酸化依赖的机制激活，需要蛋白激酶C(PKC)的活性。为了探讨GPCR信号转导的动力学机制，用荧光蛋白标记的PKD成像方法分析了PKD在活细胞内的分布，并用免疫细胞化学方法分析了PKD在固定细胞中的分布。我们发现，在成纤维细胞和上皮细胞中，PKD都穿梭于细胞质和细胞核之间。用激活PKD的有丝分裂原GPCR激动剂刺激细胞，可诱导一过性的PKD核积聚，这一过程可通过抑制PKC活性来阻止。PKD的核输入需要它的cys2结构域与核输入受体结合，而它的核输出则需要它的pleckstrin同源结构域和一个依赖CRM1的核输出途径。因此，这项研究描述了信号分子对有丝分裂原GPCR激动剂的调控核运输，并将PKD定位为丝氨酸激酶，其激酶活性和细胞内定位由PKC协调。
Protein kinase D (PKD)/protein kinase Cmu is a serine/ threonine protein kinase activated by growth factors, antigen-receptor engagement, and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires protein kinase C (PKC) activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular distribution of PKD was analyzed in live cells by imaging fluorescent protein-tagged PKD and in fixed cells by immunocytochemistry. We found that PKD shuttled between the cytoplasm and the nucleus in both fibroblasts and epithelial cells. Cell stimulation with mitogenic GPCR agonists that activate PKD induced a transient nuclear accumulation of PKD that was prevented by inhibiting PKC activity. The nuclear import of PKD requires its cys2 domain in conjunction with a nuclear import receptor, while its nuclear export requires its pleckstrin homology domain and a competent Crm1-dependent nuclear export pathway. This study thus characterizes the regulated nuclear transport of a signaling molecule in response to mitogenic GPCR agonists and positions PKD as a serine kinase whose kinase activity and intracellular localization is coordinated by PKC.