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M Meinecke, J Geiger, E Butt, M Sandberg, T Jahnsen, T Chakraborty, U Walter, T Jarchau and SM Lohmann
Medizinische Universitatsklinik, Klinische Biochemie und Pathobiochemie, Wurzburg, Germany.
The role of the cGMP-dependent protein kinase (cGK) and one of its major substrates, the vasodilator-stimulated phosphoprotein (VASP), in the regulation of a receptor-evoked calcium response was investigated. The human type I beta cGK was stably transfected in human embryonic kidney 293 cells and Swiss mouse 3T6 fibroblasts, which contained significant or no detectable levels of the focal adhesion protein VASP, respectively. Western blot analysis and protein kinase activity measurements demonstrated an 8-fold overexpression of cGK-I beta in 293 cells (7-fold in 3T6 cells), representing an intracellular cGK concentration of 0.33 microM. In experiments with intact 293 cells expressing cGK-I beta, beta-phenyl-1,N2-etheno-cGMP and 8-(p- chlorophenylthio)-cGMP were capable of converting up to 30-40% of the 46-kDa VASP to its 50-kDa phospho- form, equivalent to results observed with cGMP analogs that cause a marked inhibition of the stimulated Ca2+ transient in intact human platelets. In contrast to platelets, preincubation of fura-2-loaded 293 and 3T6 cells with 8-(p- chlorophenylthio)-cGMP did not significantly inhibit thrombin-evoked calcium transients, although sufficient cGK-mediated VASP phosphorylation was clearly detectable under these conditions in cGK-I beta-expressing 293 cells. These results demonstrate that cGK inhibition of agonist-evoked calcium mobilization is not a mechanism common to all cell types and that VASP phosphorylation may not be an essential or sufficient component of the cGK effect on calcium levels. In contrast, the observed VASP phosphorylation mediated by recombinant human cGK-I beta in intact 293 cells does support the hypothesis that focal adhesions and their associated proteins are important cellular sites of cGK action.
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