The data presented here suggest structure-function relationships within the Rac1 Switch II domain that may be altered by tyrosine phosphorylation and change Rac1-mediated cytoskeletal dynamics during cell spreading. Tyrosine 64 is located in the Switch II domain of Rac1 – one of two regions that are distinguished by conformational differences between the GDP-bound and the GTP-bound states. The conformational sensitivity of this location and its susceptibility to phosphorylation strongly suggest its importance in regulating Rac1 function. Modeling and experiments by others implicate it directly in the regulation of Rac1 activation by nucleotide exchange, and in similar events for Ras. Our data indicate that Y64 phosphorylation provides a Afatinib negative input on GTP-binding and cell spreading. Interestingly, X-ray crystallography of the Cdc42- RhoGDI complex shows that the Y64 of Cdc42 is in close proximity to lysine residues at positions 43 and 52 of RhoGDI. It is believed that the negative charge induced by tyrosine phosphorylation at Y64 could stabilize the interaction with these two positively charged basic residues on RhoGDI. This hypothesis is supported by our finding that the Rac1-Y64F mutation weakened Rac1 interaction with RhoGDI. Experimental probing for potential interactions between tyrosine phosphorylation and either constitutive or dominant negative changes in Rac1 activation indicate that there may be separable and combinatorial actions of these two signaling mechanisms with regard to GTP loading, Rac1 targeting to focal adhesions, and cell spreading. The 3-fold increase in GTP loading on Rac1-Y64F as compared with the wild type may be due in part to the fact that the Rac1-Y64F mutant binds more efficiently with Rac1-associated GEFs. Thus, we note an 89% increase in binding with b-PIX, and a more difficult to quantify increase in binding to Tiam1 for Rac1-Y64F as compared with Rac1-WT. Further, the Rac1-Y64D mutant that mimics a FTY720 constitutively phosphorylated state appears to exert a downward regulatory effect on GTP loading, focal adhesion targeting, and cell spreading efficiency in both constitutively active and dominant negative Rac1 mutants. Taken together with the RhoGDI data, these findings depict a pattern of negative regulation that is a precedented theme in signaling regulation by tyrosine kinases and has been demonstrated in the context of FAK interactions with endophilin A, Src, and MMP-14. In the case under study here, down-regulation of Rac1 function by FAK and Src may directly oppose and thereby modulate the largely positive effects that these two nonreceptor tyrosine kinases have on lamellipodial extension by other means, e.g. via bPIX phosphorylation and its subsequent increased activation of Rac1. We have previously demonstrated that Rac1 can localize to focal adhesions and focal complexes at the leading edges of actively evolving membrane ruffles and lamellipodia.