Regulation of Myosin by Phosphorylation of the Heavy Chain

1)去年，带有n端FLAG标签的NM2A、NM2B和NM2C重链在Sf9细胞中分别与调控轻链和必需轻链（RLC和ELC）共表达。通过亲和层析纯化蛋白，使其达到电泳均匀性。在10 mM MOPS、pH 7.0、150 mM NaCl、0.1 mM EGTA、2 mM MgCl2、添加和不添加1 mM ATP以及RLC磷酸化前后，通过光散射确定聚合所需的最小浓度作为聚合后肌球蛋白浓度的函数。所有三种NM2s （rlc -磷酸化和rlc -未磷酸化）聚合所需的最低蛋白质浓度在ATP存在时比在ATP不存在时更高，但差异远远小于先前发表的从胸腺和肠刷状边界纯化的NM2s的结果。

1) Last year, the heavy chains of NM2A, NM2B and NM2C with N-terminal FLAG tags were separately co-expressed in Sf9 cells with the regulatory and essential light chains (RLC and ELC). The proteins were purified to electrophoretic homogeneity by affinity chromatography. The minimal concentrations required for polymerization were determined by light scattering as a function of myosin concentration after polymerization overnight at 0 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 RLC. The minimal protein concentration required for polymerization of all three NM2s, both RLC-phosphorylated and RLC-unphosphorylated, were higher in the presence of ATP than in its absence, but the differences were very much less than expected from previous published results for NM2s purified from thymus and intestinal brush border. 1) Last year, the heavy chains of NM2A, NM2B and NM2C with N-terminal FLAG tags were separately co-expressed in Sf9 cells with the regulatory and essential light chains (RLC and ELC). The proteins were purified to electrophoretic homogeneity by affinity chromatography. The minimal concentrations required for polymerization were determined by measuring light scattering as a function of myosin concentration after polymerization overnight at 0 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 RLC. The minimal protein concentration required for polymerization of all three NM2s, both RLC-phosphorylated and RLC-unphosphorylated, were higher in the presence of ATP than in its absence, but the differences were very much less than expected from previously published results for NM2s purified from thymus and intestinal brush border. This year, we confirmed previous reports that addition of 1 mM ATP substantially reduces the light scattering of polymerized RLC-unphosphorylated NM2s, and that this is reversed by phosphorylation of the RLC by myosin light chain kinase. It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments upon addition of ATP and filament repolymerization upon RLC-phosphorylation. However, the difference in the extent of polymerization of unphosphorylated and RLC-phosphorylated NM2s in the presence of ATP is much too small to explain the observed changes in light scattering. We found that the changes in light scattering are due to previously unknown differences in light scattering of equal concentrations of polymerized unphosphorylated NM2s in the presence and absence of ATP. This decrease in light scattering of polymerized RLC-unphosphorylated NM2 filaments upon addition of ATP results from a previously undescribed ATP-induced decrease in filament size, and the formation of tetrameric and hexameric myosin oligomers. ATP has little, if any, effect on the size of filaments of RLC-phosphorylated NM2s, which is similar to that of unphosphorylated filaments in the absence of ATP. 2) There is multiple evidence for the functional association of NM2s with biological membranes with evidence both for and against the direct association of NM2s with the membrane lipids. There are minimal, but positive, data for association of NM2s with liposomes, but there are no data using pure NM2A, NM2B and NM2C, 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 (PS) liposomes but not to 100% phosphatidylcholine (PC) liposomes. We find that binding to PC liposomes containing various concentrations of either PS, phosphatidylinositol-4,5-diphosphate (PIP2), or phosphatidylinositol-3,4,5-triphosphate (PIP3) is proportional to the net negative charge of the liposomes with no specificity for PIP2, PIP3 or PS. Contrary to previous proposals, from studies of the association of myosin rods with liposomes, we find that deletion of the non-helical tailpiece does not affect binding of the three recombinant NM2s to liposomes. A paper reporting these and related results has been submitted.