Ito M, Feng J, Tsujino S, Inagaki N, Inagaki M, Tanaka J, Ichikawa K, Hartshorne D J, Nakano T
First Department of Internal Medicine, Mie University School of Medicine, Tsu, Japan.
Biochemistry. 1997 Jun 17;36(24):7607-14. doi: 10.1021/bi9702647.
The 130 kDa myosin-binding subunit (MBS) of smooth muscle myosin phosphatase was detected in cytoskeletal, cytosolic, and membrane fractions of T24 cells. Also, MBS was distributed between cytoplasm and plasmalemma in mitotic REF52 cells. These observations prompted this study of the interaction(s) of phospholipids with myosin phosphatase. Using a sedimentation assay, gizzard myosin phosphatase bound to vesicles of acidic phospholipids, i.e. phosphatidylserine (PS), phosphatidylinositol, and phosphatidic acid (PA). Neutral phospholipids did not bind. Binding of PS to myosin phosphatase also was demonstrated by electrophoresis under nondenaturing conditions. Preferential binding of PA, compared to that of the other acidic phospholipids, was indicated. Interaction of acidic phospholipids with myosin phosphatase inhibited phosphatase activity toward phosphorylated myosin. The extent of PS binding with myosin phosphatase decreased on increasing ionic strength and Mg2+ concentration. MBS (M130/M133) and M20 were phosphorylated by protein kinase A to 3 and 1 mol of P/(mol of subunit), respectively. Phosphorylation of the holoenzyme decreased phospholipid binding with recovery of phosphatase activity. Using limited proteolysis of the holoenzyme and various mutants, it was shown that phospholipid binding was associated with the C-terminal part of MBS, Ser 667-Ile 1004, and M20. The phosphorylation site involved in regulation of phospholipid binding is within the C-terminal MBS sequence. These results suggest that myosin phosphatase may interact with membranes and that phosphorylation by protein kinase A could modify this interaction. This mechanism could be important in localization of myosin phosphatase and in targeting substrates at different loci.
在T24细胞的细胞骨架、胞质和膜组分中检测到平滑肌肌球蛋白磷酸酶的130 kDa肌球蛋白结合亚基（MBS）。此外，在有丝分裂的REF52细胞中，MBS分布于细胞质和质膜之间。这些观察结果促使开展了本项关于磷脂与肌球蛋白磷酸酶相互作用的研究。使用沉降分析法，砂囊肌球蛋白磷酸酶可与酸性磷脂囊泡结合，即磷脂酰丝氨酸（PS）、磷脂酰肌醇和磷脂酸（PA）。中性磷脂则不结合。在非变性条件下进行的电泳也证明了PS与肌球蛋白磷酸酶的结合。结果表明，与其他酸性磷脂相比，PA具有优先结合性。酸性磷脂与肌球蛋白磷酸酶的相互作用抑制了磷酸酶对磷酸化肌球蛋白的活性。随着离子强度和Mg2+浓度的增加，PS与肌球蛋白磷酸酶的结合程度降低。MBS（M130/M133）和M20分别被蛋白激酶A磷酸化为3和1摩尔磷/（摩尔亚基）。全酶的磷酸化降低了磷脂结合并恢复了磷酸酶活性。通过对全酶和各种突变体进行有限的蛋白水解，结果表明磷脂结合与MBS的C末端部分（Ser 667 - Ile 1004）和M20相关。参与磷脂结合调节的磷酸化位点位于MBS的C末端序列内。这些结果表明，肌球蛋白磷酸酶可能与膜相互作用，并且蛋白激酶A的磷酸化可能会改变这种相互作用。这种机制可能在肌球蛋白磷酸酶的定位以及在不同位点靶向底物方面具有重要意义。
