Figure 1.

PKC phosphorylates cytohesin-2 upon platelet activation. The C-terminus of cytohesin-2 (CYH2) contains the phospho-motif recognized by the pSer PKC substrate antibody (A). X, any amino acid. Hyd, any hydrophobic amino acid. Washed platelets (4 × 10⁸ mL⁻¹) were stimulated with 0.2 U mL⁻¹ α-thrombin (αT) (B), 5 μg mL⁻¹ CRP (D) or 10 μm U46619 (F) and lysed at the indicated time-points. Alternatively, platelets were treated for 15 min with 0.2% DMSO vehicle (Veh) or the PKC inhibitor BIM IX (2 μm) prior to stimulation with the same agonists in the presence of 10 μm ADP (C, E, G). Clarified whole cell lysates (input) and immunoprecipitations (IP) using the pSer PKC substrate antibody were immunoblotted for CYH2. The arrows (➤) indicate the 47 kDa CYH2 band and the asterisk (*) indicates a non-specific band. Results (B–G) are representative of at least three independent experiments.