Phosphorylation of Rho GDI stabilizes the Rho A-Rho GDI complex in neutrophil cytosol.
The GDP dissociation inhibitor Rho GDI from bovine neutrophil cytosol was purified in association with prenylated Rho A. Upon treatment of this complex with alkaline phosphatase, the Rho A and Rho GDI components were released to their free forms. Following migration in 2D-PAGE and specific immunodetection, the shape of the spot of Rho GDI was found to depend markedly on whether Rho GDI subjected to electrophoresis was present in a Rho A-Rho GDI complex or in a free form. In the first case Rho GDI focused as an elongated spot between pI 5.2 and pI 4.6 whereas in the later case it focused at a pI of 5.0-5.2 as a round spot. Activation of neutrophils by anaphylatoxin C5a in a [32Pi] supplemented medium resulted in radiolabeling of Rho GDI. In vitro incubation of Rho GDI with a neutrophil homogenate in the presence of [gamma 32P] ATP led also to radiolabeling of Rho GDI. Taken together these results suggest that Rho GDI in the Rho A-Rho GDI complex is phosphorylated and that the stability of the complex depends on the phosphorylation state of Rho GDI.
Bourmeyster
N
N
Laboratoire de Biochimie, URA 1130 CNRS, Départment de Biologie Moléculaire et Structurale, CEA/Grenoble, France.
Vignais
P V
PV
D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D010766:Phosphorylation; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein
D020730:Guanine Nucleotide Dissociation Inhibitors; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D009243:NAD; D000469:Alkaline Phosphatase; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein