ANALYSIS AND REGULATION OF VASODILATOR-STIMULATED PHOSPHOPROTEIN SERINE-239 PHOSPHORYLATION IN-VITRO AND IN INTACT-CELLS USING A PHOSPHOSPECIFIC MONOCLONAL-ANTIBODY

The development and functional analysis of a monoclonal antibody (16C2 ) are reported; the antibody recognizes vasodilator-stimulated phospho protein (VASP; an established substrate of both cAMP- and cGMP-depende nt protein kinase) only when serine 239 is phosphorylated. VASP serine 239 represents one of the best characterized cGMP-dependent protein k inase phosphorylation sites in vitro and in intact cells, Experiments with purified, recombinant human VASP and various VASP constructs with mutated phosphorylation sites (S157A, S239A, T278A) and experiments w ith intact cells (human/rat platelets and other cells) treated with cy clic nucleotide-elevating agents demonstrated the specificity of the m onoclonal antibody 16C2. Quantitative analysis of the VASP shift from 46 to 50 kDa (indicating VASP serine 157 phosphorylation) and the appe arance of VASP detected by the 16C2 monoclonal antibody (VASP serine 2 39 phosphorylation) in human platelets stimulated by selective protein kinase activators confirmed that serine 239 is the VASP phosphorylati on site preferred by cGMP-dependent protein kinase in intact cells. Im munofluorescence experiments with human platelets treated with cGMP an alogs showed that the 16C2 monoclonal antibody also detects VASP serin e 239 phosphorylation in situ at established intracellular localizatio n sites. Analysis of VASP serine 239 phosphorylation by the 16C2 antib ody appears to be the best method presently available to measure cGMP- dependent protein kinase activation in intact cells. Also, the 16C2 an tibody promises to be an excellent tool for the evaluation of VASP fun ction in intact cells.