Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes

Abstract

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 Mg(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), 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 Mg(2+) and PIP(2). 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 of GDP-GTP cycling.

Chemotherapy Resistance in Diffuse-Type Gastric Adenocarcinoma Is Mediated by RhoA Activation in Cancer Stem-Like Cells

“…Given there is no reliable antibody to detect active RhoA in human tissues, we used an antibody for phosphorylated RhoA. Phosphorylation of RhoA significantly increases its interaction with Rho GDP dissociation inhibitor (RhoGDI) thus keeping RhoA in its inactive state (29). Thus phosphorylated RhoA is inversely associated with RhoA activity.…”

An Adenosine-Mediated Signaling Pathway Suppresses Prenylation of the GTPase Rap1B and Promotes Cell Scattering

“…In addition to the A2BR-mediated pathway described here, it is likely that additional receptor-mediated pathways regulate the prenylation and localization of other GTPases that have phosphorylation sites in their PBRs, including K-Ras4B (43), RhoA (44), Cdc42 (isoform 1) (44), and Rnd3 (also known as RhoE) (45). SmgGDS-607 associates with many of these PBRcontaining small GTPases before they are prenylated (15), and phosphorylation of the PBRs of these small GTPases might diminish their association with SmgGDS-607 and could alter their prenylation and trafficking.…”

Tyrosine Phosphorylation of Rac1: A Role in Regulation of Cell Spreading

“…It was therefore suggested that PKA-mediated phosphorylation of RhoA inhibits Rho activity by promoting formation of a RhoA-RhoGDI complex. Similarly, PKA-mediated phosphorylation and a resultant increase in complex formation with RhoGDI was observed with both RhoA and Cdc42 in studies of rodent brain [17]. It is not clear whether Rac1 is a phosphorylation target for PKA, but Kwon et al demonstrated phosphorylation of Rac1 on Ser-71 by Akt in human melanoma cells [18].…”