HUMAN CYCLIC GMP-DEPENDENT PROTEIN-KINASE IP OVEREXPRESSION INCREASESPHOSPHORYLATION OF AN ENDOGENOUS FOCAL CONTACT-ASSOCIATED VASODILATOR-STIMULATED PHOSPHOPROTEIN WITHOUT ALTERING THE THROMBIN-EVOKED CALCIUM RESPONSE

The role of the cGMP-dependent protein kinase (cGK) and one of its maj or substrates, the vasodilator-stimulated phosphoprotein (VASP), in th e regulation of a receptor-evoked calcium response was investigated. T he human type 1 beta cGK was stably transfected in human embryonic kid ney 293 cells and Swiss mouse 3T6 fibroblasts, which contained signifi cant or no detectable levels of the focal adhesion protein VASP, respe ctively. Western blot analysis and protein kinase activity measurement s demonstrated an 8-fold overexpression of cGK-1 beta in 293 cells (7- fold in 3T6 cells), representing an intracellular cGK concentration of 0.33 mu M. In experiments with intact 293 cells expressing cGK-1 beta , beta-phenyl-1,N-2-etheno-cGMP and 8-(p-chlorophenylthio)-cGMP were c apable of converting up to 30-40% of the 46-kDa VASP to its 50-kDa pho spho- form, equivalent to results observed with cGMP analogs that caus e a marked inhibition of the stimulated Ca2+ transient in intact human platelets. In contrast to platelets, preincubation of fura-2-loaded 2 93 and 3T6 cells with 8-(p-chlorophenylthio)-cGMP did not significantl y inhibit thrombin-evoked calcium transients, although sufficient cGK- mediated VASP phosphorylation was clearly detectable under these condi tions in cGK-1 beta-expressing 293 cells. These results demonstrate th at cGK inhibition of agonist-evoked calcium mobilization is not a mech anism common to all cell types and that VASP phosphorylation may not b e an essential or sufficient component of the cGK effect on calcium le vels. In contrast, the observed VASP phosphorylation mediated by recom binant human cGK-1 beta in intact 293 cells does support the hypothesi s that focal adhesions and their associated proteins are important cel lular sites of cGK action.