CAMP-DEPENDENT AND CGMP-DEPENDENT PROTEIN-KINASE PHOSPHORYLATION SITES OF THE FOCAL ADHESION VASODILATOR-STIMULATED PHOSPHOPROTEIN (VASP) IN-VITRO AND IN INTACT HUMAN PLATELETS

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cAMP-dependent- (cAK) and cGMP-dependent protein kinase (cGK) in h uman platelets and other cardiovascular cells. To identify the VASP ph osphorylation sites, purified VASP was phosphorylated by either protei n kinase and subjected to trypsin, V8 and Lys-C proteolysis, The phosp horylated proteolytic fragments obtained were separated by reversed ph ase high performance liquid chromatography. Sequence analysis of the p hosphorylated peptides and P-32 measurement of the released P-32-label ed amino acids revealed three phosphorylation sites: a serine 1-contai ning site (LRKVSXQEEA), a serine 2-containing site (HIERRVSNAG), and a threonine-containing site (MNAVLARRRKATQVGE). Additional experiments with purified VASP demonstrated that both cAK and cGK phosphorylated s erine 2 rapidly and the threonine residue slowly, whereas cGK phosphor ylated the serine 1 residue more rapidly than the cAK. These differenc es in the phosphorylation rates of VASP by the two protein kinases wer e also observed with synthetic peptides corresponding to the sequences of the three identified phosphorylation sites. These experiments also established the synthetic peptide serine 1 as one of the best in vitr o cGK substrates and the serine 2-containing site as the site responsi ble for the phosphorylation-induced mobility shift of VASP in sodium d odecyl sulfate-polyacrylamide gel electrophoresis. Experiments with P- 32-labeled platelets provided evidence that VASP is phosphorylated at the same three identified sites also in intact cells and that selectiv e activation of cAK or cGK primarily increased the phosphorylation of both serine 2 and serine 1 but not threonine. Our results demonstrated overlapping substrate specificities of cAK and cGK in vitro and in in tact cells. However, important quantitative and qualitative difference s between cAK- and cGK-mediated phosphorylation of the focal adhesion protein VASP in human platelets were also observed, suggesting distinc t functions of the two types of cyclic nucleotide-mediated VASP phosph orylation.