Regulation of Myosin by Phosphorylation of the Heavy Chain

1)去年，我们报道了在阿米巴肌球蛋白II的非螺旋尾部丝氨酸的磷酸化修饰其细丝结构。磷酸化肌球蛋白的双极、反平行微丝在微丝两端的头部之间具有比非磷酸化肌球蛋白更长的裸露区，并且每一端的头部比非磷酸化肌球蛋白更紧密地聚集在一起。这些结果有显着的影响，为三种哺乳动物的非肌肉肌球蛋白II的聚合和丝结构的调节，因为它们的非螺旋尾段和相邻的卷曲螺旋区有多个丝氨酸和苏氨酸磷酸化，在体外和体内。因此，我们已经开始研究磷酸化对三种哺乳动物匪II的临界浓度（与细丝平衡的单体浓度）和细丝结构的影响。今年我们获得了非磷酸化重组肌球蛋白的对照数据。 分别在Sf 9细胞中表达具有N-末端FLAG标签的NMIIA、NMIIB和NMIIC的重链以及调节轻链和必需轻链（RLC和ELC），并通过亲和层析将蛋白质纯化至电泳均一性。在10 mM MOPS，pH 7.0，150 mM NaCl，0.1 mM EGTA，2 mM MgCl 2中于0 ℃聚合过夜后，加入和不加入1 mM ATP，以及在调节轻链磷酸化之前和之后，通过光散射和超离心测定其临界浓度。三种重组肌球蛋白在所有条件下的临界浓度均显著不同于先前报道的从胸腺和肠刷状缘纯化的肌球蛋白的值。报告我们成果的文件正在编写中。 2)有各种证据表明匪II与生物膜的功能相关，但有证据支持和反对匪II与膜脂质的直接相关。关于NMIIs与脂质体的结合，有最少但阳性的数据，但没有使用纯NMIIA、NMIIB和NMIIC的数据，也没有关于这些肌球蛋白与磷脂结合的分子基础的数据。我们发现纯的重组全长NMIIA、NMIIB和NMIIC与100%磷脂酰丝氨酸脂质体结合，但不与100%磷脂酰胆碱脂质体结合。与含有各种浓度的磷脂酰丝氨酸（PS）或磷脂酰肌醇-4，5-二磷酸（PIP 2）的磷脂酰胆碱脂质体的结合与脂质体的净负电荷成比例，对PIP 2与PS没有特异性。 与以前的建议相反，从肌球蛋白棒与脂质体的关联的研究中，我们发现，删除非螺旋尾片段不影响重组匪II与脂质体的结合。正在编写一份报告这些结果和相关结果的文件。

1) Last year, we reported that phosphorylation of the serines in the non-helical tailpiece of Acanthamoeba myosin II modifies its filament structure. The bipolar, antiparallel minifilaments of phosphorylated myosin have longer bare zones than non-phosphorylated myosin between the heads at the opposite ends of the filaments, and the heads at each end are more tightly clustered than in non-phosphorylated myosin. These results have significant implications for the regulation of the polymerization and filament structure of the three mammalian non-muscle myosin IIs as their non-helical tailpiece and adjacent coiled-coil region have multiple serines and threonines that are phosphorylated both in vitro and in vivo. Therefore, we have initiated studies on the effect of phosphorylation on the critical concentrations (the monomer concentration in equilibrium with filaments) and filament structures of the three mammalian NMIIs. This year we obtained the control data for non-phosphorylated recombinant myosins. The heavy chains of NMIIA, NMIIB and NMIIC with N-terminal FLAG tags were separately expressed in Sf9 cells together with the regulatory and essential light chains (RLC and ELC), and the proteins were purified to electrophoretic homogeneity by affinity chromatography. Their critical concentrations were determined by light scattering and ultracentrifugation after polymerization overnight at 0 degrees in 10 mM MOPS, pH 7.0, 150 mM NaCl, 0.1 mM EGTA, 2 mM MgCl2, with and without addition of 1 mM ATP, and before and after phosphorylation of the regulatory light chain. The critical concentrations under all conditions were significantly different for the three recombinant myosins than the values reported previously for myosins purified from thymus and intestinal brush border. A paper reporting our results is in preparation. 2) There is a variety of evidence for the functional association of NMIIs with biological membranes, but there is evidence both for and against the direct association of NMIIs with the membrane lipids. There are minimal, but positive, data for association of NMIIs with liposomes, but there are no data using pure NMIIA, NMIIB and NMIIC, and no data on the molecular basis of the binding of these myosins to phospholipids. We found that pure, recombinant full length NMIIA, NMIIB and NMIIC bind to 100% phosphatidylserine liposomes but not to 100% phosphatidylcholine liposomes. Binding to phosphatidylcholine liposomes containing various concentrations of either phosphatidylserine (PS) or phosphatidylinositol-4,5-diphosphate (PIP2) was proportional to the net negative charge of the liposomes with no specificity for PIP2 vs. PS. Contrary to previous proposals, from studies of the association of myosin rods with liposomes, we found that deletion of the non-helical tailpiece did not affect binding of the recombinant NMIIs to liposomes. A paper reporting these and related results is in preparation.