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 t his complex with alkaline phosphatase, the Rho A and Rho GDI component s were released to their free forms. Following migration in 2D-PAGE an d specific immunodetection, the shape of the spot of Rho GDI was found to depend markedly on whether Rho GDI subjected to electrophoresis wa s present in a Rho A-Rho GDI complex or in a free form. in the first c ase Rho GDI focused as an elongated spot between pI 5.2 and pI 4.6 whe reas in the later case it focused at a pI of 5.0-5.2 as a round spot A ctivation of neutrophils by anaphylatoxin C5a in a [(32)Pi] supplement ed medium resulted in radiolabeling of Rho GDI. In vitro incubation of Rho GDI with a neutrophil homogenate in the presence of [gamma(3)P] A TP led also to radiolabeling of Rho GDI. Taken together these results suggest that Rho GDI in the Rho A-Rho GDI complex is phosphorylated an d that the stability of the complex depends on the phosphorylation sta te of Rho GDI. (C) 1996 Academic Press, Inc.