Tiam1 cooperates with PKCζ to regulate S1P-induced Rac activation and chemotaxis. (A) WT and Tiam1^−/− T cells were either nonstimulated or stimulated with 150 nM S1P for the indicated time periods. Subsequently PKCζ phosphorylation status was determined. (Top panel) Phosphorylated PKCζ. (Middle panel) Total PKCζ. (Bottom panel) Tiam1. Sizes are indicated in kilodaltons. (B) WT T cells were either nontreated or treated with 2 μM PKCζ inhibitor for 1 hour and subsequently stimulated with 150 nM S1P for the indicated time periods. Subsequently, Rac activity status was determined. (Top panel) GTP-bound Rac. (Bottom panel) Total Rac. Sizes are indicated in kilodaltons. (C) Quantification of the Rac activation as shown in panel B. Rac activity value in nonstimulated WT T cells is set to 1. (D) Chemotaxis of WT and Tiam1^−/− lymphocytes either treated or untreated with 2 μM PKCζ inhibitor was measured in response to 15 nM S1P after 1 hour. The results are presented as the percentage of the input cells and are based on 3 independent experiments. Error bars indicate SD; p, P value.