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Evidence for cAMP-dependent Platelet Ectoprotein Kinase Activity That Phosphorylates Platelet Glycoprotein IV (CD36)
Ist Teil von
The Journal of biological chemistry, 1996-10, Vol.271 (40), p.24776-24780
Ort / Verlag
United States: American Society for Biochemistry and Molecular Biology
Erscheinungsjahr
1996
Quelle
Free E-Journal (出版社公開部分のみ)
Beschreibungen/Notizen
The dephosphorylating enzyme alkaline phosphatase, by removing phosphate groups from the external platelet membrane proteins,
modulates platelet activation (Hatmi, M., Haye, B., Gavaret, J. M., Vargaftig, B. B., and Jacquemin, C. (1991) Br. J. Pharmacol. 104, 554-558). This observation, together with findings reported by others (Ehrlich, Y. H., Davis, T. B., Bock, E., Kornecki,
E., and Lenox, R. H. (1986) Nature 320, 67-70; Dusenbery, K. E., Mendiola, J. R., and Skubitz, K. M. (1988) Biochem. Biophys. Res. Commun. 153, 7-13), indicate the existence of ectoprotein kinase activity on the blood platelet surface.
In this study, we demonstrate that washed human platelets phosphorylate the synthetic heptapeptide kemptide in a cAMP-dependent
mode. The intensity of the phosphorylation was concentration-dependent for kemptide. In addition, incubation of platelets
with [γ- 32 P]ATP resulted in a rapid incorporation of [ 32 P] phosphate into proteins at the outer membrane surface that was sensitive to alkaline phosphatase treatment. When cAMP was
added to the medium, major phosphorylation of an 88-kDa ectoprotein occurred. Its isoelectric point determined by isoelectric
focusing SDS-polyacrylamide gel electrophoresis was around pH 6.2. Phosphorylations of this 88-kDa polypeptide and of the
exogenous kemptide substrate were both prevented by the specific protein kinase A inhibitor peptide. When platelets were preincubated
with [ 32 P]inorganic phosphate to label intracellular proteins, the protein phosphorylation pattern was different from that obtained
with [γ- 32 P]ATP, indicating that the latter occurred at the outer surface of the cells. Prostacyclin, which induces the increase of
intracellular cAMP levels and, consequently, its liberation into the extracellular medium, increased phosphorylation of both
kemptide and platelet 88-kDa polypeptide. The major protein of 88-kDa, which was phosphorylated in the presence of cAMP and
external [γ- 32 P]ATP, was identified by immunoprecipitation to GPIV (CD36), one of thrombospondin and collagen binding sites on platelets.
The phosphorylation of CD36 also occurred in platelet-rich plasma, suggesting a physiological role for this ectoenzyme.
In the present study, we clearly demonstrate the presence of an ectoprotein kinase A activity at the surface of intact human
platelets, and we revealed its principal endogenous substrate as being CD36.