Phosphorylation of ^1,4,5IP₃R and GAP1^IP4BP in intact platelets by cAMP-PK and cGMP-PK.

(A) [³²P] labeled platelets were incubated with either vehicle (CON) or PGI₂ (0.5 μmol/L) or BiMPS (500 μmol/L), and type III ^1,4,5IP₃R immunoprecipitated with CT3. (B) Experiments were carried out as in (A), and immunoprecipitations were carried out using CT1, CT2, and CT3. (C) Experiments were carried out as above with additional stimulation of cGMP-PK using 8pCPT. Immunoprecipitations were carried out using R26 (for type I receptor), R45 (for type III receptor), and anti-GAP1^IP4BP antibody. Western blotting analysis of immunoprecipitates of GAP1^IP4BP is shown. (D) Lysates from experiments involving GAP1^IP4BP were subjected to analysis of the phosphorylation status of VASP using Western blot analysis. Activation of the kinases is associated with an increase of the upper 50-kd form of VASP. In control cells, nonphosphorylated VASP migrates as a 46-kd protein.