Sathyamoorthy V, Atkinson M A, Bowers B, Korn E D
Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1990 Apr 17;29(15):3793-7. doi: 10.1021/bi00467a028.
The actin-activated Mg2(+)-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serines in its 29-residue, nonhelical, COOH-terminal tailpiece, i.e., serines-1489, -1494, and -1499 or, in reverse order, residues 11, 16, and 21 from the COOH terminus. To investigate the essential requirements for regulation, myosin II filaments in the presence of F-actin were digested by arginine-specific submaxillary gland protease. Two-dimensional peptide mapping of purified, cleaved myosin II showed that the two most terminal phosphorylation sites, serines-1494 and -1499, had been removed. Cleaved dephosphorylated myosin II retained full actin-activated Mg2(+)-ATPase activity (with no change in Vmax or Kapp) and the ability to form filaments similar to those of the native enzyme. However, higher Mg2+ concentrations were required for both filament formation and maximal ATPase activity. The one remaining regulatory serine in the cleaved myosin II was phosphorylatable by myosin II heavy-chain kinase, and phosphorylation inactivated the actin-activated Mg2(+)-ATPase activity, as in the case of the native myosin II. Also as in the case of the native myosin II, phosphorylated cleaved myosin II inhibited the actin-activated Mg2(+)-ATPase activity of dephosphorylated cleaved myosin II when the two were copolymerized. These results suggest that at least 18 of the 29 residues in the nonhelical tailpiece of the heavy chain are not required for either actin-activated Mg2(+)-ATPase activity or filament formation and that phosphorylation of Ser-1489 is sufficient to regulate the actin-activated Mg2(+)-ATPase activity of myosin II.
来自卡氏棘阿米巴的肌球蛋白II的肌动蛋白激活的Mg2(+)-ATP酶活性受其29个残基的非螺旋COOH末端尾段中3个丝氨酸的磷酸化调节，即丝氨酸-1489、-1494和-1499，或者按相反顺序，从COOH末端起的第11、16和21位残基。为了研究调节的基本要求，在F-肌动蛋白存在下，用精氨酸特异性颌下腺蛋白酶消化肌球蛋白II丝。对纯化的、切割后的肌球蛋白II进行二维肽图谱分析表明，两个最末端的磷酸化位点，丝氨酸-1494和-1499，已被去除。切割后的去磷酸化肌球蛋白II保留了完全的肌动蛋白激活的Mg2(+)-ATP酶活性（Vmax或Kapp无变化）以及形成与天然酶相似的丝的能力。然而，丝形成和最大ATP酶活性都需要更高的Mg2+浓度。切割后的肌球蛋白II中剩余的一个调节性丝氨酸可被肌球蛋白II重链激酶磷酸化，并且磷酸化使肌动蛋白激活的Mg2(+)-ATP酶活性失活，就像天然肌球蛋白II的情况一样。同样与天然肌球蛋白II的情况一样，当两者共聚时，磷酸化的切割后的肌球蛋白II抑制去磷酸化的切割后的肌球蛋白II的肌动蛋白激活的Mg2(+)-ATP酶活性。这些结果表明，重链非螺旋尾段中的29个残基中至少有18个对于肌动蛋白激活的Mg2(+)-ATP酶活性或丝形成不是必需的，并且丝氨酸-1489的磷酸化足以调节肌球蛋白II的肌动蛋白激活的Mg2(+)-ATP酶活性。
