In human platelets, thrombin not only stimulates the phosphorylation of pleckstrin (P47) and of myosin P-light chains, but also induces the dephosphorylation of an 18-19 kDa phosphoprotein (P18) [Imaoka, Lynham and Haslam (1983) J. Biol. Chem. 258, 11404-11414]. We have now studied this protein in detail. The thrombin-induced dephosphorylation reaction did not begin until the phosphorylation of myosin P-light chains and the secretion of dense-granule 5-hydroxytryptamine were nearly complete, but did parallel the later stages of platelet aggregation. Experiments with ionophore A23187 and phorbol 12-myristate 13-acetate indicated that dephosphorylation of P18 was stimulated by Ca2+, but not by protein kinase C. Two-dimensional analysis of platelet proteins, using non-equilibrium pH gradient electrophoresis followed by SDS/PAGE, showed that thrombin decreased the amount of phosphorylated P18 in platelets by up to 70% and slightly increased the amount of a more basic unlabelled protein that was present in 3-fold excess of P18 in unstimulated platelets. These two proteins were identified as the phosphorylated and non-phosphorylated forms of the pH-sensitive actin-depolymerizing protein, cofilin, by sequencing of peptide fragments and immunoblotting with a monoclonal antibody specific for cofilin. The molar concentration of cofilin in platelets was approx. 10% that of actin. Platelet cofilin was phosphorylated exclusively on serine. Experiments with electropermeabilized platelets showed that dephosphorylation of cofilin could be stimulated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) in the absence of Ca2+ or by a free Ca2+ concentration of 10 microM. This GTP[S]-induced dephosphorylation reaction was inhibited by 1-naphthyl phosphate, but not by okadaic acid. Our results add cofilin to the actin-binding proteins that may regulate the platelet cytoskeleton, and suggest that platelet cofilin can be activated by dephosphorylation reactions initiated either by a GTP-binding protein or Ca2+.
Research Article| July 01 1994
Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+
M M L Davidson;
Biochem J (1994) 301 (1): 41–47.
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M M L Davidson, R J Haslam; Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+. Biochem J 1 July 1994; 301 (1): 41–47. doi: https://doi.org/10.1042/bj3010041
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