Interaction of protein kinase C isozymes with Rho GTPases
Interaction of protein kinase C isozymes with Rho GTPases
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DOI：
10.1021/bi001654n
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发表时间：
2001-04-10
期刊：
影响因子：
2.9
通讯作者：
Stubbs, CD
中科院分区：
文献类型：
--
作者：
Slater, SJ;Seiz, JL;Stubbs, CD
证据提供了蛋白激酶C（PKC）α，β I，β II，γ，δ，β和ζ与小GTP酶的Rho家族成员之间的直接蛋白质-蛋白质相互作用。以前的研究，基于免疫沉淀的方法，提供了与直接相互作用一致的证据，但这仍有待证明。在这里提出的研究中，在体外试验，仅由纯化的蛋白质和必要的PKC激活剂和辅因子，用于确定Rho GTPases对不同的PKC亚型的活动的影响。结果发现，PKC α的活性被RhoA和Cdc 42有效地增强，并在较小程度上被Rad增强，而对PKC β I、-β II、-γ、-δ、-β和-zeta的活性的影响大大降低。这些结果表明PKC α和每种Rho GTP酶之间存在直接相互作用。然而，对于每种Rho GTdR，Rho GTdR对PKC α活性的增强作用的浓度依赖性不同，并且按以下顺序：RhoA > Cdc 42> Rac 1。PKC α以佛波酯和Ca 2+依赖的方式被激活。这通过在Ca 2+存在下对活性的佛波酯浓度要求的显著降低来反映，对于每种Rho GTdR，在低纳摩尔佛波酯浓度范围内诱导Ca 2+。还发现PKC α的活性依赖于Rho GTP酶的GTP或GDP结合状态的性质，这表明相互作用可能受PKC α和Rho GTP酶的构象变化的调节。这种相互作用可能导致这两个信号元件调节的不同途径之间的显著串扰。
Evidence is provided for direct protein-protein interactions between protein kinase C (PKC) alpha, betaI, beta II, gamma, delta, epsilon, and zeta and members of the Rho family of small GTPases. Previous investigations, based on the immunoprecipitation approach, have provided evidence consistent with a direct interaction, but this remained to be proven. In the study presented here, an in vitro assay, consisting only of purified proteins and the requisite PKC activators and cofactors, was used to determine the effects of Rho GTPases on the activities of the different PKC isoforms. It was found that the activity of PKC alpha was potently enhanced by RhoA and Cdc42 and to a lesser extent by Rad, whereas the effects on the activities of PKC betaI, -beta II, -gamma, -delta, -epsilon, and -zeta were much reduced. These results indicate a direct interaction between PKC alpha and each of the Rho GTPases. However, the Rho GTPase concentration dependencies for the potentiating effects on PKC alpha activity differed for each Rho GTPase and were in the following order: RhoA > Cdc42 > Rac1. PKC alpha was activated in a phorbol ester- and Ca2+-dependent manner. This was reflected by a substantial decrease in the phorbol ester concentration requirements for activity in the presence of Ca2+ which for each Rho GTPase was induced within a low nanomolar phorbol ester concentration range. The activity of PKC alpha also was found to be dependent on the nature of the GTP- or GDP-bound state of the Rho GTPases, suggesting that the interaction may be regulated by conformational changes in both PKC alpha and Rho GTPases. Such an interaction could result in significant cross-talk between the distinct pathways regulated by these two signaling elements.