This phosphorylation did not change RhoA GTPase activity or binding to GTP, but led to the exit of phosphorylated RhoA from the plasma membranes and an increased presence of the RhoARhoGDI complex in the cytosol. Increased cellular cAMP levels and PKA activity resulted in morphological changes consistent with RhoA inhibition. It was therefore suggested that PKAmediated phosphorylation of RhoA inhibits Rho activity by promoting formation of a RhoA-RhoGDI complex. Similarly, PKA-mediated phosphorylation and a resultant increase in complex formation with RhoGDI was observed with both RhoA and Cdc42 in studies of NSC-718781 rodent brain. It is not clear whether Rac1 is a phosphorylation target for PKA, but Kwon et al. FTY720 demonstrated phosphorylation of Rac1 on Ser-71 by Akt in human melanoma cells. This Akt-mediated Rac1 phosphorylation resulted in an approximately 50% reduction in GTP binding by Rac1, but did not change GTPase activity. In the case of Cdc42, tyrosine phosphorylation at position 64 was observed following treatment with epidermal growth factor, and this was mediated by Src in COS-7 cells. Tyrosine-64 was identified as the major phosphorylation site in these experiments, but tyrosine phosphorylation on Y64 was not required for Cdc42 activation. Tyrosine phosphorylation on Y64 of Cdc42 also did not affect its binding with several target/effector proteins including PAK, ACK2, MRCK, WASP or IQGAP �C but increased association with RhoGDI was noted. Since Cdc42-RhoGDI interactions are involved in Cdc42-induced cellular transformation, it was suggested that phosphorylation of Cdc42 led to alteration of its targeting via RhoGDI. The pattern that emerges from this earlier work is that protein phosphorylation may serve a specific role in signal modulation of Rho family GTPases by altering binding interactions with upstream regulators, with GTP, and with RhoGDI. Tyrosine phosphorylation of Rac1 has not been explored to date, although we have demonstrated that tyrosine phosphorylation of bPIX is associated with increased binding to Rac1 in vitro, and augmentation of cell spreading. Given that human Rac1 and Cdc42 share high homology and have the identical amino acid sequence at residues 61�C70, site-directed mutagenesis was used here to investigate the impact of Tyr-64 phosphorylation on cell spreading and the interaction of Rac1 with regulatory and effector proteins. Rac1-Y64F was used to obviate phosphorylation at this site, while Rac1-Y64D was employed to mimic the constitutively phosphorylated state. Strikingly, expression of the Rac1-Y64D mutant greatly inhibited cell spreading and decreased Rac1 binding to PAK. Expression of the Rac1-Y64F mutant facilitated cell spreading, while it increased Rac1 binding to GTP and to Rac1-associated GEFs, and decreased binding to RhoGDI.