Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes.
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.
Forget
Marie-Annick
MA
Laboratoire de médecine moléculaire, Hôpital Sainte-Justine-Université du Québec à Montréal, P.O. Box 8888, Centre-ville station, Montréal, Québec, Canada H3C 3P8.
Desrosiers
Richard R
RR
Gingras
Denis
D
Béliveau
Richard
R
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D020730:Guanine Nucleotide Dissociation Inhibitors; D011994:Recombinant Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein