Constitutive association of cortactin with PAK in resting platelets.

(A) In vitro association of PAK and cortactin. Pull-down experiments were performed from resting platelets on GST-fused proteins bound to Sepharose beads. The left immunoblots (IB) show the pull-down of cortactin on GST-PAK^WT (full length) or GST-PAK^T423E (active mutant). The right blots show the pull-down of PAK on GST-cortactin. Anti-GST immunoblots are shown as controls. (B) In vivo association of cortactin with PAK. (Left) Coprecipitation of cortactin with PAK in resting platelets and dissociation of cortactin after thrombin stimulation. PAK was immunoprecipitated from the detergent-soluble fraction of platelets stimulated with thrombin for 0, 0.2, or 1 minute at 37°C. Associated cortactin was revealed by immunoblotting. (Right) Effect of DNase I on the association of cortactin with PAK in resting platelets. Unstimulated platelet lysates were treated for 45 minutes at 37°C with 0 to 20 μM DNase I, and PAK was immunoprecipitated (IP). Cortactin and actin were revealed by specific immunoblot. Anti-PAK1/2 immunoblot is shown as a control of the amount of immunoprecipitated PAK. (C) Effect of toxin B (1 μg/mL for 2 hours at 37°C) on the association of cortactin with PAK. PAK activity was evaluated as an in vitro autophosphorylation assay, and associated cortactin was revealed by specific immunoblotting. Controls with irrelevant rabbit immunoglobulins are shown in lane C. Results are representative of 2 or 3 separate experiments.