The development and functional analysis of a monoclonal antibody (16C2) are reported; the antibody recognizes vasodilator-stimulated phosphoprotein (VASP; an established substrate of both cAMP-and cGMP-dependent protein kinase) only when serine 239 is phosphorylated. VASP serine 239 represents one of the best characterized cGMP-dependent protein kinase phosphorylation sites in vitro and in intact cells. Experiments with purified, recombinant human VASP and various VASP constructs with mutated

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... osphorylation sites (S157A, S239A, T278A) and experiments with intact cells (human/rat platelets and other cells) treated with cyclic nucleotide-elevating agents demonstrated the specificity of the monoclonal antibody 16C2. Quantitative analysis of the VASP shift from 46 to 50 kDa (indicating VASP serine 157 phosphorylation) and the appearance of VASP detected by the 16C2 monoclonal antibody (VASP serine 239 phosphorylation) in human platelets stimulated by selective protein kinase activators confirmed that serine 239 is the VASP phosphorylation site preferred by cGMP-dependent protein kinase in intact cells. Immunofluorescence experiments with human platelets treated with cGMP analogs showed that the 16C2 monoclonal antibody also detects VASP serine 239 phosphorylation in situ at established intracellular localization sites. Analysis of VASP serine 239 phosphorylation by the 16C2 antibody appears to be the best method presently available to measure cGMP-dependent protein kinase activation in intact cells. Also, the 16C2 antibody promises to be an excellent tool for the evaluation of VASP function in intact cells. Although cGMP-dependent protein kinases (cGPKs) 1 have been recognized as important components of major signal transduction pathways (1-3), quantitative analysis of cGPK activation in intact cells has been very difficult (1-4). This is because of the relatively low expression of cGPK in most cell types compared with the relatively high expression of its closest functional homolog, the cAMP-dependent protein kinase (cAPK), and the scarcity of specific cGPK substrates. Unfortunately, the mediating role of cGPK for a given effect/function is often implied or excluded by the use of cGPK activators and/or inhibitors alone, which is clearly insufficient to establish or rule out functional roles of cGPKs (1-4). One of the few established cGPK substrates is the 46-kDa/50-kDa vasodilator-stimulated phosphoprotein (VASP), which was initially discovered and characterized as a substrate of both cAPK and cGPK in human platelets (5-8). VASP phosphorylation in response to cyclic nucleotide-regulating vasodilators (i.e. cAMP-elevating prostaglandins and cGMP-elevating nitric oxide donors) closely correlates with platelet inhibition and in particular with the inhibition of fibrinogen binding to the integrin ␣ IIb ␤ 3 of human platelets (9 -11). Molecular cloning of human, canine, and mouse VASP predicted highly homologous proteins and revealed a proline-rich protein that is organized into three structural segments of different sequence complexity (12, 13). VASP is the founding member of a new family of proline-rich proteins, which includes Enabled (Ena), a dose-dependent suppressor of Drosophila Abl-and Disabled-dependent phenotypes, its mammalian homolog Mena, and the Ena-VASP-like protein Evl (14 -16). These proteins all share an overall domain organization consisting of highly homologous NH 2 -terminal and COOHterminal domains (Ena-VASP homology domains 1 and 2, EVH1 and EVH2), which are separated by a proline-rich central domain of low complexity (12) (13) (14) (15) (16) . In platelets and many other cells including vascular smooth muscle cells, endothelial cells, and fibroblasts, VASP has been found to be associated with stress fibers, focal adhesions, cell-cell contacts, and highly dynamic membrane regions (16, 17) . VASP colocalizes with profilins and binds directly to their poly(L-proline) binding site (18), binds to and colocalizes with zyxin and vinculin (16, 19) , and also directly binds to Listeria monocytogenes surface protein ActA, which is essential for the actin polymerization-based intracellular motility of this pathogen (20). Functional evidence indicates that VASP is a crucial factor involved in the enhancement of spatially confined actin filament formation (16, 20, 21) . Three distinct phosphorylation sites were biochemically identified in VASP (serine 157, serine 239, and threonine 278) which are used in vitro and in intact human platelets by both cAPK and cGPK and by the serine/threonine protein phosphatases 2A and 2B with overlapping selectivity (8, 22) . Phosphorylation of serine 157, the site preferred by the cAPK, leads to a marked shift in apparent molecular mass of VASP in SDS-PAGE from 46 kDa to 50 kDa (6, 8).