Complex regulation of PKCβ2 and PDK-1/AKT by ROCK2 in diabetic heart.
Complex regulation of PKCβ2 and PDK-1/AKT by ROCK2 in diabetic heart.
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DOI：
10.1371/journal.pone.0086520
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Macleod KM
中科院分区：
文献类型：
--
作者：
Lin G;Brownsey RW;Macleod KM
RhoA/ROCK通路在糖尿病心肌病中的作用部分是通过促进PKCβ2的持续激活来实现的，但它们相互作用的细节尚不清楚。本研究的目的是研究糖尿病大鼠心脏中ROCK的过度激活是否导致PKCβ-2的直接磷酸化和激活，以及它们之间的相互作用是否会影响PDK-1/AKT信号转导。用Western blotting和免疫共沉淀法研究ROCK对12~14周糖尿病大鼠全心和心肌细胞PKC、β-2及相关激酶的调节作用。体外检测蛋白激酶Cβ2的直接磷酸化。用siRNA沉默的方法证实ROCK2在高糖培养的血管平滑肌细胞PKCβ2磷酸化中的作用。此外，应用激光共聚焦显微镜观察了ROCK抑制对心肌细胞GLUT4转位的影响。糖尿病大鼠心肌组织中ROCK2表达增加，PKCβ2表达及磷酸化水平升高。相互免疫沉淀证实了两种蛋白激酶之间的物理相互作用，而ROCK2在体外直接磷酸化了T641处的PKCβ2。血管平滑肌细胞ROCK2siRNA或糖尿病心脏ROCK抑制PKCβ2T641的磷酸化，与PKCβ2活性减弱有关。PKCAK2与β-1及其靶蛋白AKT形成复合体，ROCK抑制可上调PDK1和AKT的磷酸化，增加葡萄糖转运蛋白4(GLUT4)向质膜的转位。本研究表明，ROCK-2的过度激活可能通过多种机制参与糖尿病心肌病的发生，包括直接磷酸化和激活PKC-β-2，以及干扰PDK-1介导的磷酸化、激活和GLUT4转位。提示ROCK2是糖尿病心肌病变发生发展过程中的一个关键节点，可能是改善糖尿病心功能的有效靶点。
The RhoA/ROCK pathway contributes to diabetic cardiomyopathy in part by promoting the sustained activation of PKCβ2 but the details of their interaction are unclear. The purpose of this study was to investigate if over-activation of ROCK in the diabetic heart leads to direct phosphorylation and activation of PKCβ2, and to determine if their interaction affects PDK-1/Akt signaling. Regulation by ROCK of PKCβ2 and related kinases was investigated by Western blotting and co-immunoprecipitation in whole hearts and isolated cardiomyocytes from 12 to 14-week diabetic rats. Direct ROCK2 phosphorylation of PKCβ2 was examined in vitro. siRNA silencing was used to confirm role of ROCK2 in PKCβ2 phosphorylation in vascular smooth muscle cells cultured in high glucose. Furthermore, the effect of ROCK inhibition on GLUT4 translocation was determined in isolated cardiomyocytes by confocal microscopy. Expression of ROCK2 and expression and phosphorylation of PKCβ2 were increased in diabetic hearts. A physical interaction between the two kinases was demonstrated by reciprocal immunoprecipitation, while ROCK2 directly phosphorylated PKCβ2 at T641 in vitro. ROCK2 siRNA in vascular smooth muscle cells or inhibition of ROCK in diabetic hearts reduced PKCβ2 T641 phosphorylation, and this was associated with attenuation of PKCβ2 activity. PKCβ2 also formed a complex with PDK-1 and its target AKT, and ROCK inhibition resulted in upregulation of the phosphorylation of PDK-1 and AKT, and increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane in diabetic hearts. This study demonstrates that over-activation of ROCK2 contributes to diabetic cardiomyopathy by multiple mechanisms, including direct phosphorylation and activation of PKCβ2 and interference with the PDK-1-mediated phosphorylation and activation of AKT and translocation of GLUT4. This suggests that ROCK2 is a critical node in the development of diabetic cardiomyopathy and may be an effective target to improve cardiac function in diabetes.
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