PKC ε Phosphorylates and Mediates the Cell Membrane Localization of RhoA.
PKC ε Phosphorylates and Mediates the Cell Membrane Localization of RhoA.
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DOI：
10.1155/2013/329063
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Pan Q
中科院分区：
文献类型：
--
作者：
Su T;Straight S;Bao L;Xie X;Lehner CL;Cavey GS;Teknos TN;Pan Q
蛋白激酶Cε(PKCε)通过RhoA信号调节细胞的侵袭和运动。在本研究中，定义了PKCε和RhoA之间的多方面相互作用。磷酸肽图显示，PKCε在T127和S188处使RhoA磷酸化。在没有三磷酸腺苷的情况下，重组PKCε可与重组RhoA结合，这表明PKCε与RhoA之间的结合不需要活性的ε构象。用时移荧光显微镜观察，PKCε的激活导致PKCε和RhoA从细胞质到细胞膜的戏剧性的协调移位。化学计量学FRET分析表明，PKCε与RhoA之间的分子相互作用是一个双相事件，在整个过程中(12.5min)，在细胞质中出现一个初始峰值，并在细胞膜上逐渐延长。这些结果表明，PKCε-RhoA复合体在细胞质中组装，随后募集到细胞膜上。K437R)PKCε能将RhoA募集到细胞膜上，表明ε与RhoA的结合位于活性催化部位附近，可能不依赖于ε-RhoA的磷酸化事件。这项工作首次证明了PKCε能够磷酸化并调节RhoA的细胞膜转位。
Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA.