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

被引:143
作者
Forget, MA [1 ]
Desrosiers, RR [1 ]
Gingras, D [1 ]
Béliveau, R [1 ]
机构
[1] Univ Quebec, Hop St Justine, Mol Med Lab, Montreal, PQ H3C 3P8, Canada
关键词
ionic strength; rho; small GTP-binding proteins; GDI;
D O I
10.1042/0264-6021:3610243
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:243 / 254
页数:12
相关论文
共 56 条
[1]   RhoA prenylation is required for promotion of cell growth and transformation and cytoskeleton organization but not for induction of serum response element transcription [J].
Allal, C ;
Favre, G ;
Couderc, B ;
Salicio, S ;
Sixou, S ;
Hamilton, AD ;
Sebti, SM ;
Lajoie-Mazenc, I ;
Pradines, A .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (40) :31001-31008
[2]  
Ausubel F. M., 1999, SHORT PROTOCOLS MOL
[3]   CARBOXYL METHYLATION OF THE LOW-MOLECULAR-WEIGHT GTP-BINDING PROTEIN G25K - REGULATION OF CARBOXYL METHYLATION BY RHOGDI [J].
BACKLUND, PS .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1993, 196 (02) :534-542
[4]   PHOSPHORYLATION OF 2 SMALL GTP-BINDING PROTEINS OF THE RAB FAMILY BY P34CDC2 [J].
BAILLY, E ;
MCCAFFREY, M ;
TOUCHOT, N ;
ZAHRAOUI, A ;
GOUD, B ;
BORNENS, M .
NATURE, 1991, 350 (6320) :715-718
[5]   Regulation of Rho protein binding to membranes by rhoGDI:: inhibition of releasing activity by physiological ionic conditions [J].
Bilodeau, D ;
Lamy, S ;
Desrosiers, RR ;
Gingras, D ;
Béliveau, R .
BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE, 1999, 77 (01) :59-69
[6]   Regulation of cell function by Rho family GTPases [J].
Bokoch, GM .
IMMUNOLOGIC RESEARCH, 2000, 21 (2-3) :139-148
[7]   Phosphorylation of Rho GDI stabilizes the Rho A-Rho GDI complex in neutrophil cytosol [J].
Bourmeyster, N ;
Vignais, PV .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1996, 218 (01) :54-60
[8]  
Chardin P, 1999, Prog Mol Subcell Biol, V22, P39
[9]  
CHUANG TH, 1993, J BIOL CHEM, V268, P26206
[10]   cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKα [J].
Dong, JM ;
Leung, T ;
Manser, E ;
Lim, L .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (35) :22554-22562