Fig. S6.

CDK5 mediates its role in cell migration in part through phosphorylation of GIV S1674. (A) Schematic showing role of CDK5 in activating GIV’s GEF function. In control cells (Upper), EGF stimulation leads to activation of CDK5, which in turn activates the GEF function of GIV by phosphorylating S1674. In cells treated with Roscovitine (Middle), a CDK5-specific inhibitor, S1674, is not phosphorylated and GIV-GEF remains “off.” Cells harboring a phosphomimetic GIV (74D) (Lower) bypass the need for CDK5-mediated activation and are in a constitutively GEF “on” mode. (B) Phosphomimetic mutation at S1674 can compensate for CDK5 inactivation in MDA-MB231 cell migration in response to EGF. Chemotaxis of MDA-MB231 (vector, WT, and 74D) cells toward EGF supplemented serum-free medium was evaluated using a Transwell migration assay with or without Roscovitine treatment. Cells were allowed to migrate for 6 h and were fixed and stained with Toluidine Blue. Representative images are shown. (Magnification: B, 10×.)