Purification of a vasodilator-regulated phosphoprotein from human platelets.
Cyclic-nucleotide-elevating vasodilators such as prostaglandin E1, prostacyclin, sodium nitroprusside and endothelium-derived relaxing factor inhibit both contraction of vascular smooth muscle cells and the aggregation of platelets at an early step of the activation cascade. Previous studies from this laboratory [Waldmann, R., Nieberding, M. and Walter, U. (1987) Eur. J. Biochem. 167, 441-448) established that in human platelets cyclic-nucleotide-elevating vasodilators stimulated a pattern of protein phosphorylation which was mediated by both cAMP- and cGMP-dependent protein kinases. Of particular interest was a membrane-bound 50-kDa protein whose phosphorylation was increased both by cAMP- and cGMP-elevating vasodilators in intact platelets and by endogenous cAMP- and cGMP-dependent protein kinase in platelet membranes. Since the molecular mechanism of action of cyclic-nucleotide-elevating vasodilators is unknown, this 50-kDa phosphoprotein from human platelets was purified to apparent homogeneity by salt extraction, anion, cation and dye-ligand chromatography. The purified protein migrated as a 46-kDa protein in SDS/PAGE, was an excellent substrate for both cAMP- and cGMP-dependent protein kinases and migrated in SDS/PAGE as a 50-kDa protein after phosphorylation by these protein kinases. Analysis by limited proteolysis, tryptic fingerprinting and of phosphoamino acids established that the purified protein is identical with the 50-kDa protein phosphorylated by both cAMP- and cGMP-dependent protein kinases in platelet membranes and in response to cAMP- and cGMP-elevating vasodilators with intact platelets. Evidence is presented that the purified protein contains at least two phosphorylation sites, each of which is preferentially phosphorylated by either cAMP- or cGMP-dependent protein kinase. The availability of this vasodilator-regulated phosphoprotein as a purified protein should now allow new approaches for investigating the function of this protein and its possible role in the mechanism of action of cyclic-nucleotide-elevating vasodilators.
Halbrügge
M
M
Labor für Klinische Biochemie, Universität Würzburg, Federal Republic of Germany.
Walter
U
U
D001792:Blood Platelets / Q000032:analysis*; D002118:Calcium / Q000378:metabolism; D002845:Chromatography; D006801:Humans; D007951:Leukemia, Myeloid / Q000097:blood; D008970:Molecular Weight; D009712:Nucleotides, Cyclic / Q000502:physiology*; D010750:Phosphoproteins / Q000302:isolation & purification*; D011494:Protein Kinases / Q000502:physiology; D014665:Vasodilator Agents / Q000494:pharmacology*
D009712:Nucleotides, Cyclic; D010750:Phosphoproteins; D014665:Vasodilator Agents; D011494:Protein Kinases; D002118:Calcium