Protein Kinase D Regulates RhoA Activity via Rhotekin Phosphorylation*
Protein Kinase D Regulates RhoA Activity via Rhotekin Phosphorylation*
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DOI：
10.1074/jbc.m112.339564
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发表时间：
2012-01
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影响因子：
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通讯作者：
G. Pusapati;T. Eiseler;A. Rykx;S. Vandoninck;R. Derua;E. Waelkens;J. Van Lint;G. von Wichert;T. Seufferlein
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文献类型：
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作者：
G. Pusapati;T. Eiseler;A. Rykx;S. Vandoninck;R. Derua;E. Waelkens;J. Van Lint;G. von Wichert;T. Seufferlein
背景：调节肌动蛋白细胞骨架重组的蛋白激酶D（PKD）的底物在很大程度上是未知的。结果：Rhotekin是一种新型PKD底物，通过增强膜结合来调节RhoA活性。结论：PKD介导的rhotekin磷酸化是RhoA激活和肌动蛋白应力纤维形成的一种新机制。意义：本研究有助于我们理解PKD调控RhoA活化和肌动蛋白细胞骨架重组的机制。丝氨酸/苏氨酸激酶的蛋白激酶D（PKD）家族的成员是促进肿瘤的佛波酯、G蛋白偶联受体和活化的蛋白激酶C同种型（PKC）的主要靶标。PKD通过各种底物的磷酸化发挥其功能的细胞过程的扩展列表包括增殖、凋亡、迁移、血管生成和囊泡运输。因此，鉴定新的PKD底物对于理解该激酶家族在信号转导中的重要作用是必要的。在这里，我们表明，rhotekin，RhoA GTdR的效应，是PKD的一种新的底物。我们确定了Rhotekin中的Ser-435为PKD体内靶向的潜在位点。一个磷酸模拟S435 E rhotekin突变体的表达导致内源性活性RhoA GTdR水平的增加。PKD 2对rhotekin的磷酸化调节RhoA在质膜中的锚定。因此，S435 E rhotekin突变体在血清饥饿的成纤维细胞中表达时显示出增强的应力纤维形成。因此，我们的数据确定了PKD作为RhoA活性和肌动蛋白应力纤维形成的调节剂，通过磷酸化rhotekin的一种新的作用。
Background: The substrates of protein kinase D (PKD) that regulate actin cytoskeletal reorganization are largely unknown. Results: Rhotekin is a novel PKD substrate that regulates RhoA activity by enhancing its membrane association. Conclusion: PKD-mediated rhotekin phosphorylation demonstrates a novel mechanism of RhoA activation and actin stress fiber formation. Significance: This study contributes to our understanding of the mechanism of RhoA activation and actin cytoskeletal reorganization regulated by PKD. The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin.