Yamakita Y, Yamashiro S, Matsumura F
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1059.
J Cell Biol. 1994 Jan;124(1-2):129-37. doi: 10.1083/jcb.124.1.129.
Phosphorylation of the regulatory light chain of myosin II (MLC) controls the contractility of actomyosin in nonmuscle and muscle cells. It has been reported that cdc2 phosphorylates MLC in vitro at Ser-1 or Ser-2 and Thr-9 which protein kinase C phosphorylates (Satterwhite, L. L., M. J. Lohka, K. L. Wilson, T. Y. Scherson, L. K. Cisek, J. L. Corden, and T. D. Pollard. 1992 J. Cell Biol. 118:595-605). We have examined in vivo phosphorylation of MLC during mitosis and after the release of mitotic arrest. Phosphate incorporation of MLC in mitotic cells is found to be 6-12 times greater than that in nonmitotic cells. Phosphopeptide maps have revealed that the MLC from mitotic cells is phosphorylated at Ser-1 and/or Ser-2 (Ser-1/2), but not at Thr-9. MLC is also phosphorylated to a much lesser extent at Ser-19 which myosin light chain kinase phosphorylates. On the other hand, MLC of nonmitotic cells is phosphorylated at Ser-19 but not at Ser-1/2. The extent of phosphate incorporation is doubled at 30 min after the release of mitotic arrest when some cells start cytokinesis. Phosphopeptide analyses have revealed that the phosphorylation at Ser-19 is increased 20 times, while the phosphorylation at Ser-1/2 is decreased by half. This high extent of MLC phosphorylation at Ser-19 is maintained for another 30 min and gradually decreased to near the level of interphase cells as cells complete spreading at 180 min. On the other hand, phosphorylation at Ser-1/2 is decreased to 18% at 60 min, and is practically undetectable at 180 min after the release of mitotic arrest. The stoichiometry of MLC phosphorylation has been determined by quantitation of phosphorylated and unphosphorylated forms of MLC separated on 2D gels. The molar ratio of phosphorylated MLC to total MLC is found to be 0.16 +/- 0.06 and 0.31 +/- 0.05 in interphase and mitotic cells, respectively. The ratio is increased to 0.49 +/- 0.05 at 30 min after the release of mitotic arrest. These results suggest that the change in the phosphorylation site from Ser-1/2 to Ser-19 plays an important role in signaling cytokinesis.
肌球蛋白II调节轻链（MLC）的磷酸化控制着非肌肉细胞和肌肉细胞中肌动球蛋白的收缩性。据报道，在体外，cdc2在丝氨酸-1或丝氨酸-2以及苏氨酸-9位点使MLC磷酸化，而蛋白激酶C也可使这些位点磷酸化（萨特怀特，L.L.，M.J.洛卡，K.L.威尔逊，T.Y.舍尔森，L.K.西塞克，J.L.科登，以及T.D.波拉德。1992年《细胞生物学杂志》118:595 - 605）。我们已经研究了有丝分裂期间以及有丝分裂阻滞解除后MLC的体内磷酸化情况。发现有丝分裂细胞中MLC的磷酸掺入量比非有丝分裂细胞高6 - 12倍。磷酸肽图谱显示，有丝分裂细胞中的MLC在丝氨酸-1和/或丝氨酸-2（丝氨酸-1/2）位点磷酸化，但在苏氨酸-9位点未磷酸化。MLC在丝氨酸-19位点也有较低程度的磷酸化，该位点由肌球蛋白轻链激酶磷酸化。另一方面，非有丝分裂细胞中的MLC在丝氨酸-19位点磷酸化，但在丝氨酸-1/2位点未磷酸化。有丝分裂阻滞解除30分钟后，当一些细胞开始胞质分裂时，磷酸掺入量增加了一倍。磷酸肽分析表明，丝氨酸-19位点的磷酸化增加了20倍，而丝氨酸-1/2位点的磷酸化减少了一半。在丝氨酸-19位点的这种高水平的MLC磷酸化在接下来的30分钟内持续存在，并随着细胞在180分钟时完全铺展而逐渐降至接近间期细胞的水平。另一方面，丝氨酸-1/2位点的磷酸化在有丝分裂阻滞解除60分钟后降至18%，在180分钟时几乎检测不到。通过对二维凝胶上分离的MLC磷酸化和未磷酸化形式进行定量，确定了MLC磷酸化的化学计量。发现间期细胞和有丝分裂细胞中磷酸化MLC与总MLC的摩尔比分别为0.16±0.06和0.31±0.05。有丝分裂阻滞解除30分钟后，该比例增加到0.49±0.05。这些结果表明，磷酸化位点从丝氨酸-1/2到丝氨酸-19的变化在胞质分裂信号传导中起重要作用。
