Ikebe M, Reardon S
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
Biochemistry. 1990 Mar 20;29(11):2713-20. doi: 10.1021/bi00463a014.
Bovine platelet myosin is phosphorylated by protein kinase C at multiple sites. Most of the phosphate is incorporated in the 20,000-dalton light chain although some phosphate is incorporated in the heavy chain. Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10 times faster than the phosphorylation of smooth muscle myosin. Platelet myosin light chain is first phosphorylated at a threonine residue followed by a serine residue. Dominant phosphorylation sites of the 20,000-dalton light chain are estimated as serine-1, serine-2, and threonine-9. Prolonged phosphorylation by protein kinase C resulted in an additional phosphorylation site which, on the basis of limited proteolysis, appears to be either serine-19 or threonine-18. Phosphorylation by protein kinase C causes an inhibition of actin-activated ATPase activity of platelet myosin prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. It is shown that platelet myosin also exhibits the 6S to 10S conformation transition as judged by viscosity and gel filtration methods. Mg2(+)-ATPase activity of platelet myosin is paralleled with the 10S-6S transition. Phosphorylation by protein kinase C affects neither the 10S-6S transition nor the myosin filament formation. Therefore, the inhibition of actin-activated ATPase activity of platelet myosin is not due to the change in the myosin conformation.
牛血小板肌球蛋白在多个位点被蛋白激酶C磷酸化。大部分磷酸基团结合在20000道尔顿的轻链上，不过也有一些磷酸基团结合在重链上。血小板肌球蛋白20000道尔顿轻链的磷酸化速度比平滑肌肌球蛋白的磷酸化速度快10倍。血小板肌球蛋白轻链首先在一个苏氨酸残基上被磷酸化，随后是一个丝氨酸残基。20000道尔顿轻链的主要磷酸化位点估计为丝氨酸-1、丝氨酸-2和苏氨酸-9。蛋白激酶C的长时间磷酸化导致了一个额外的磷酸化位点，根据有限的蛋白水解分析，该位点似乎是丝氨酸-19或苏氨酸-18。蛋白激酶C介导的磷酸化会抑制由肌球蛋白轻链激酶预磷酸化的血小板肌球蛋白的肌动蛋白激活的ATP酶活性。ATP酶活性的抑制是由于肌球蛋白对肌动蛋白的亲和力降低，而Vmax没有变化。结果表明，通过粘度和凝胶过滤方法判断，血小板肌球蛋白也呈现6S到10S的构象转变。血小板肌球蛋白的Mg2(+)-ATP酶活性与10S-6S转变平行。蛋白激酶C的磷酸化既不影响10S-6S转变，也不影响肌球蛋白丝的形成。因此，血小板肌球蛋白的肌动蛋白激活的ATP酶活性的抑制不是由于肌球蛋白构象的改变。
