Regulation of Myosin by Phosphorylation of the Heavy Chain

1)去年，NM2A、NM2B和NM2C的重链分别与调控轻链和必需轻链(RLC和ELC)在Sf9细胞中共表达。用亲和层析法纯化蛋白，使其达到电泳均一。通过光散射测定聚合所需的最小浓度，作为在10 mM MOPS、pH 7.0、150 mM NaC、0.1 mM EGTA、2 mM氯化镁、添加和不添加1 mM三磷酸腺苷的情况下以及RLC磷酸化前后在0条件下聚合过夜肌球蛋白浓度的函数。在有ATP存在的情况下，所有三种NM2聚合所需的最低蛋白质浓度，无论是RLC磷酸化的还是RLC非磷酸化的，都高于没有ATP的情况，但与之前发表的从胸腺和肠道刷状缘纯化的NM2的结果相比，差异要小得多。 1)去年，NM2A、NM2B和NM2C的重链分别与调控轻链和必需轻链(RLC和ELC)在Sf9细胞中共表达。用亲和层析法纯化蛋白，使其达到电泳均一。聚合所需的最小浓度通过测量在10 mM MOPS、pH 7.0、150 mM氯化钠、0.1 mM EGTA、2 mM氯化镁、添加和不添加1 mM三磷酸腺苷的情况下，以及RLC磷酸化前后在0条件下聚合后肌球蛋白浓度的函数的光散射来确定。在有ATP存在的情况下，所有三种NM2聚合所需的最低蛋白质浓度，无论是RLC磷酸化的还是RLC非磷酸化的，都高于没有ATP的情况，但与之前发表的从胸腺和肠道刷状缘纯化的NM2的结果相比，差异要小得多。 今年，我们证实了先前的报道，即添加1 mM的ATP大大减少了聚合的RLC非磷酸化的NM2的光散射，并且这一结果被肌球蛋白轻链激酶对RLC的磷酸化所逆转。有人认为，这些变化是由于添加ATP时未磷酸化的NM2细丝大量解聚，以及RLC-磷酸化时细丝重新聚合所致。然而，在ATP存在下，未磷酸化和RLC磷酸化的NM2的聚合程度的差异太小，不能解释观察到的光散射变化。我们发现，光散射的变化是由于先前未知的相同浓度的聚合非磷酸化NM2在存在和不存在ATP的情况下的光散射的差异。当添加ATP时，聚合的RLC非磷酸化NM2细丝的光散射减少是由于先前未描述的由ATP引起的细丝尺寸的减小以及四聚体和六聚体肌球蛋白低聚体的形成。ATP对RLC磷酸化的NM2的细丝尺寸几乎没有影响，这与没有ATP的情况下的未磷酸化NM2的细丝尺寸相似。 2)NM2与生物膜的功能结合有多种证据，支持和反对NM2与膜脂直接结合的证据都有。NM2与脂质体结合的数据很少，但却是肯定的，但没有使用纯NM2A、NM2B和NM2C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、重组的全长NMIIA、NMIIB和NMIIC与100%磷脂酰丝氨酸(PS)脂质体结合，但不与100%磷脂酰胆碱(PC)脂质体结合。我们发现，与含有不同浓度的PS、磷脂酰肌醇-4，5-二磷酸(PIP2)或磷脂酰肌醇-3，4，5-三磷酸(PIP3)的PC脂质体的结合与脂质体的净负电荷成正比，而对PIP2、PIP3或PS没有特异性。与以前的建议相反，从肌球蛋白棒与脂质体结合的研究中，我们发现非螺旋尾段的缺失并不影响三个重组NM2与脂质体的结合。已经提交了一份报告这些和相关结果的文件。

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.