On average, there was one cycle of brief rapid extension followed by quick CPI-613 Dehydrogenase inhibitor retraction every 2.5 min in the cells expressing Rac1-Y64D. This pattern of extension and retraction of the leading membrane edge was also observed in the EGFP-transfected control cells within the first 5 min of plating, but this Semaxanib behavior quickly disappeared and stable lamellipodia were then established. In contrast, EGFP-Rac1- Y64D-transfected cells continued to exhibit this pattern of rapid and extension and retraction with little spreading at 60 minutes following plating. In Rac1-Y64F-transfected cells, stable lamellipodial extension began immediately after cell attachment to matrix. Statistical analysis of multiple cells and kymographs for Rac1-WT, -64F, or -64D transfectants are shown in Figure 3B, and additional images are shown for each cell population in Figure S2. These findings suggest a role for tyrosine phosphorylation in the modulation of Rac1 function during the stabilization of cytoskeletal infrastructure and/or transmembrane adhesions during cell spreading and movement. To further elucidate the molecular mechanisms of Rac1-Y64F enhancement of cell spreading, lamellipodial stability, and the targeting of Rac1 to focal adhesions, we studied Rac1 activity in MEF that were transfected with EGFP-Rac1-WT, or mutations that included Q61L, T17N, Y64D, or Y64F. Rac1 activity was analyzed in aliquots of lysates from transfected MEF by a GST affinity pull-down assay using the Rac-binding domain of PAK and 15 mg of protein from each sample were blotted with anti Rac1 antibody for total Rac1 as a loading control. MEF expressed fairly comparable amounts of the respective EGFPtagged Rac1 proteins, wild type or mutants, except for EGFPRac1- Y64D, which appeared to be quickly degraded. When 800 mg of total protein lysates were reacted in vitro with 50 mg of GST-PBD produced in E. coli, the controls were as expected: more amount of the constitutively active Rac1-61L was pulled down as compared to the wild type, and no dominant negative Rac1-17N was pulled down. Interestingly, more of the EGFP-Rac1-Y64F was pulled down by GST-PBD than the wild type Rac1, indicating more GTP binding and higher Rac1 activity in the presence of the Y64F mutation. The degraded EGFP-Rac1-Y64D product did not bind to GST-PBD indicating a decrease in both GTP binding and Rac1 activity. Densitometry data from four sets of these experiments are shown in Figure 5B. GTP-bound active Rac1 in the pull downs was normalized to the amount found by Western blot in total lysates. A 3.5 fold increase in Rac1 activity was observed for the EGFP-Rac1-Y64F mutant compared to the wild type. Interestingly, in the background of the constitutively active 61L mutation, 64F and 64D mutations also affected Rac1 activity. Unlike the single mutation EGFP-Rac1-Y64D which was degraded in transfected cells, EGFP-Rac1-61L/64D was more stable. EGFP-Rac1-61L/64D had lower Rac1 activity than either the 61L or 61L/64F mutants.