Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes
Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes
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DOI：
10.1042/0264-6021:3610243
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发表时间：
2002-01-15
影响因子：
4.1
通讯作者：
Béliveau, R
中科院分区：
文献类型：
--
作者：
Forget, MA;Desrosiers, RR;Béliveau, R
Rho GDP解离抑制剂（RhoGDI）通过从膜提取Rho小GTP酶（如Cdc 42和RhoA）来调节它们的活化-失活循环。为了研究Mg 2+、磷脂酰肌醇4，5-二磷酸（PIP 2）、离子强度和磷酸化对RhoGDI与Cdc 42和RhoA相互作用的影响，我们开发了一种新的、有效的和可靠的利用酵母酿酒酵母生产异戊二烯化Rho蛋白的方法。先前已经报道，分离的膜的蛋白激酶A（PKA）处理增加了RhoGDI从膜中提取RhoA [Lang，Gesbert，Delespine-Carmagnat，Stancou，Pouchelet和Bertoglio（1996）EMBO J.16，510-519]。在本研究中，我们使用了体外亲和层析系统，以显示磷酸化的RhoA和Cdc 42显着增加其与RhoGDI的离子强度的生理条件下的相互作用。这种增加与加载到Rho蛋白上的核苷酸（GDP或鸟苷5 '-[γ-硫代]三磷酸）以及Mg 2+和PIP 2无关。此外，大鼠脑细胞膜的碱性磷酸酶的去磷酸化显着减少提取RhoA和Cdc 42的RhoGDI。随后通过PKA的再磷酸化恢复了提取水平，表明该过程是可逆的。这些结果清楚地表明，Cdc 42和RhoA的磷酸化状态调节它们与RhoGDI的相互作用，因此，它们从大鼠脑细胞膜的提取。因此，我们认为磷酸化是Cdc 42和RhoA活性的调节机制，其独立于GDP GTP循环。
The Rho GDP dissociation inhibitor (RhoGDI) regulates the activation-inactivation cycle of Rho small GTPases, such as Cdc42 and RhoA, by extracting them from the membrane. To study the roles of Mg2+, phosphatidylinositol 4,5-bisphosphate (PIP2), ionic strength and phosphorylation on the interactions of RhoGDI with Cdc42 and RhoA, we developed a new, efficient and reliable method to produce prenylated Rho proteins using the yeast Saccharomyces cerevisiae. It has been previously reported that protein kinase A (PKA)-treatment of isolated membranes increased RhoA extraction from membranes by RhoGDI [Lang, Gesbert, Delespine-Carmagnat, Stancou, Pouchelet and Bertoglio (1996) EMBO J. 16, 510-519]. In the present study, we used an in vitro affinity chromatography system to show that phosphorylation of RhoA and Cdc42 significantly increased their interaction with RhoGDI under physiological conditions of ionic strength. This increase was independent of the nucleotide (GDP or guanosine 5'-[gamma-thio]triphosphate) loaded on to the Rho proteins, as well as of Mg2+ and PIP2. Moreover, dephosphorylation of rat brain membranes by alkaline phosphatase significantly decreased the extraction of RhoA and Cdc42 by RhoGDI. Subsequent re-phosphorylation by PKA restored the extraction levels, indicating the reversibility of this process. These results clearly demonstrate that the phosphorylation states of Cdc42 and RhoA regulate their interactions with RhoGDI and, consequently, their extraction from rat brain membranes. We therefore suggest that phosphorylation is a mechanism of regulation of Cdc42 and RhoA activity that is independent or GDP GTP cycling.