Regulation of Myosin by Phosphorylation of the Heavy Chain

通过重链磷酸化调节肌球蛋白

• 批准号: 8558056
• 负责人: EDWARD D KORN
• 金额: $ 53.32万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8558056
• 关键词: ATP phosphohydrolase; Acanthamoeba; Actins; Affect; Am 80; C-terminal; Data; Dictyostelium; Distant; Down-Regulation; Electron Microscopy; Filament; Head; Length; Light; Mass Spectrum Analysis; Molecular; Mutate; Mutation; Myosin ATPase; Myosin S-1; Myosin Type II; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physarum; Regulation; Research; Serine; Shadowing (Histology); Site; Structure; Tail; Time; inorganic phosphate; mutant; polymerization

The first evidence for the importance of heavy chain phosphorylation was our data in the early 1980s showing that actin-activated MgATPase activity of Acanthamoeba myosin II (AMII) was inhibited by phosphorylation of 1 to 3 serine residues in the 29-residue non-helical tailpiece of each of the two heavy chains. For a period of about 10 years from the mid-1980s to mid-1990s, we had investigated the possible mechanism of this inhibition. The one consistent result was that heteropolymers of unphosphorylated (active) myosin and phosphorylated (inactive) myosin had significantly less actin-activated ATPase activity than equivalent mixtures of homopolymers, i.e. regulation of AMII was at the level of filaments with phosphorylated AMII inactivating unphosphorylated AMII when both are in the same filaments. Interestingly, the phosphorylation sites at the C-terminal end of the tail are 80-nm distant from the ATPase site in the globular separated by a coiled-coil helix. At that time, neither we nor others had any evidence that phosphorylation of the non-helical tailpiece affected the filament structure. We are now reinvestigating this problem by studying the structure and actin-activated ATPase activity of expressed wild-type and mutated AMII. Consistent with earlier results, incubation with kinase incorporates 4 phosphates per heavy chain (HC) into full-length AMII but, contrary to the earlier conclusions, we now find that 1 phosphate is incorporated per HC of heavy meromyosin (HMM), subfragment 1 (S1) and myosin with the heavy chain non-helical tailpiece deleted (NHT). Also, all the myosin mutants have actin-activated MgATPase activity that is inhibited by phosphorylation. Therefore, phosphorylation of the serines in the non-helical tailpiece is not responsible for down-regulation of actin-activated MgATPase activity of AMII, but phosphorylation of a serine in head region is. This serine is being identified by mass spectroscopy and site-specific mutations. The molecular mechanism of down-regulation by serine phosphorylation is also being investigated. Although phosphorylation of the non-helical tailpiece serines does not affect enzymatic activity, we find it does affect the structure of the AMII minifilaments as seen by electron microscopy of rotary shadowed filaments.

重链磷酸化的重要性的第一个证据是我们在20世纪80年代早期的数据，显示肌动蛋白激活的阿米巴肌球蛋白II（AMII）的MgATP酶活性被两条重链中每一条的29个残基非螺旋尾片段中的1至3个丝氨酸残基的磷酸化抑制。从20世纪80年代中期到90年代中期的大约10年时间里，我们研究了这种抑制的可能机制。一个一致的结果是，非磷酸化（活性）肌球蛋白和磷酸化（非活性）肌球蛋白的杂聚物具有显着低于肌动蛋白激活的ATP酶活性比等效的均聚物的混合物，即调节AMII是在与磷酸化AMII失活未磷酸化AMII丝的水平时，两者都在相同的丝。有趣的是，在尾部的C-末端的磷酸化位点是80 nm的距离在球状的ATP酶位点由卷曲螺旋螺旋分开。当时，我们和其他人都没有任何证据表明非螺旋尾片段的磷酸化影响了细丝结构。 我们现在正在重新调查这个问题，通过研究表达的野生型和突变的AMII的结构和肌动蛋白激活的ATP酶活性。 与早期的结果一致，与激酶孵育将每个重链（HC）的4个磷酸盐掺入全长AMII中，但与早期的结论相反，我们现在发现，每个重酶解肌球蛋白（HMM）、亚片段1（S1）和重链非螺旋尾片段缺失的肌球蛋白（NHT）的HC掺入1个磷酸盐。 此外，所有的肌球蛋白突变体具有肌动蛋白激活的MgATP酶活性，其被磷酸化抑制。 因此，非螺旋尾段中丝氨酸的磷酸化不是AMII肌动蛋白激活的MgATP酶活性下调的原因，但头部区域中丝氨酸的磷酸化是。 这种丝氨酸正在通过质谱和位点特异性突变来鉴定。 丝氨酸磷酸化下调的分子机制也正在研究中。 虽然磷酸化的非螺旋尾部丝氨酸不影响酶的活性，我们发现它确实影响的AMII微丝的结构，如通过旋转阴影丝的电子显微镜观察。