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 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′-[γ-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 of GDP—GTP cycling.

Serine Phosphorylation Negatively Regulates RhoA in Vivo

“…Although PKA phosphorylation is linked to Rap activation (17,18), evidence indicates that it negatively regulates RhoA function. RhoA phosphorylation promotes formation of RhoA⅐RhoGDI complexes (13,19) and enhances the ability of RhoGDI to extract RhoA from membranes (13,20). In support of enhanced RhoGDI binding, the ability of RhoA to cycle from membranes has been linked to cAMP and cGMP signaling within cells (21,22).…”

von Hippel‐Lindau tumor suppressor protein stimulation by thrombin involves RhoA activation

“…47 PKA can inhibit RhoA by phosphorylation, which induces its release from membranes as we have previously shown. 26 However, in the presence of H89, a PKA inhibitor that should protect RhoA against PKA inactivation, thrombin-induced pVHL expression is not increased (data not shown). This could be explained by the fact that GTP-bound RhoA is already strongly stimulated upon thrombin treatment, as seen in Figure 6a.…”

“…Like Ras and the G ␣ subunits of Gproteins, the function of Rho proteins is regulated by GTP/GDP switches that cycle between active GTP-bound and inactive GDPbound forms. 25,26 Constitutively activated RhoA increases the invasive properties and metastatic potential of tumor cells. 27 Among targets of RhoA, Rho kinase is the most characterized since several inhibitors are available.…”

Phosphorylation of phospholipase D1 and the modulation of its interaction with RhoA by cAMP-dependent protein kinase

“…Most PLD enzymes are membrane-associated proteins, although cytosolic PLD has been reported in certain cells (Siddiqi et al, 1995;Singer et al, 1997) and the membrane-bound active form of RhoA was extractable by PKA-mediated phosphorylation (Lang et al, 1996;Kwak and Uhlinger, 2000;Forget et al, 2002). We examined whether cAMP-induced phosphorylation could affect the association of PLD with the membrane.…”

“…However, no prior evidence of the direct phosphorPhosphorylation of phospholipase D1 and the m odulation of its interaction with RhoA by cAMP-dependent protein kinase ylation of PLD by PKA in vivo and in vitro has been presented. Moreover, direct involvement of the phosphorylations of RhoA and/or PLD in the inhibition of their association is not clearly understood although RhoA phosphorylation by PKA was shown to enhance or inhibit the association between RhoA and its binding proteins, such as Rho GDP dissociation inhibitor (RhoGDI) and Rho kinase (Lang et al, 1996;Dong et al, 1998;Forget et al, 2002;Ellerbroek et al, 2003;Qiao et al, 2003).…”