A low M(r) GTP-binding protein, Rap1, in human platelets: localization, translocation and phosphorylation by cyclic AMP-dependent protein kinase.
Subcellular fractions were prepared from human platelet membranes by sucrose density gradient centrifugation and the localization of a low M(r) GTP-binding protein, rap1 protein (Rap1) was analysed by immunoblotting using a specific antibody. Rap1, which has been purified from human platelets, was found to be located in plasma membrane and alpha-granule fractions in resting platelets. Treatment of isolated alpha-granules with pronase led to proteolysis of Rap1, indicating that this protein is exposed to the cytoplasmic face of the granules. Degranulation of alpha-granules consists of translocation and subsequent fusion of the granules with the open canalicular system. Activation of this process by thrombin induced the redistribution of Rap1 on the alpha-granules to plasma membranes. On the other hand, Rap1 is known to be phosphorylated by cyclic AMP-dependent protein kinase (A-kinase) in vitro and in vivo. In intact human platelets, phosphorylation of Rap1 by A-kinase in response to prostaglandin E1 (PGE1) was observed only in Rap1 localized in plasma membranes and not on alpha-granules, although Rap1 was phosphorylated in a cell-free system when plasma membranes and alpha-granule membranes were exposed to A-kinase as substrates. These results strongly suggest that Rap1 in plasma membranes and the protein on alpha-granules are regulated by different mechanisms, and have different functions.
Nagata
K
K
Department of Biochemistry, Gifu University School of Medicine, Japan.
Nozawa
Y
Y
D001792:Blood Platelets / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D015539:Platelet Activation; D014178:Translocation, Genetic; D020668:rap GTP-Binding Proteins
D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins