Regulation of Myosin by Phosphorylation of the Heavy Chain

重链磷酸化的重要性的第一个证据是我们在20世纪80年代初的数据显示，棘阿米巴肌球蛋白II(AMII)的肌动蛋白激活的镁ATPase活性被两个重链的29个残基的非螺旋尾部的1到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，我们花了大约10年的时间研究了这种抑制的可能机制。一个一致的结果是，未磷酸化(活性)的肌球蛋白和磷酸化(非活性)的肌球蛋白杂化聚合物的肌动蛋白激活的ATPase活性显著低于同等的均聚物混合物，即当两者处于同一细丝中时，对AMII的调节处于细丝水平，而磷酸化的AMII失活未磷酸化的AMII。有趣的是，尾巴C末端的磷酸化位点与球状结构中的ATPase位点相距80 nm，中间由螺旋螺旋隔开。当时，无论是我们还是其他人，都没有任何证据表明非螺旋尾段的磷酸化影响了细丝结构。 我们现在正在通过研究表达的野生型和突变型AMII的结构和肌动蛋白激活的ATPase活性来重新研究这个问题。与早期的结果一致，与激酶孵育在全长AMII中每个重链(HC)结合了4个磷酸盐，但与先前的结论相反，我们现在发现每个HC中有1个磷酸被结合到重肌球蛋白(HMM)、亚片段1(S1)和肌球蛋白中，而重链非螺旋尾段缺失(NHT)。此外，所有肌球蛋白突变体都具有肌动蛋白激活的镁ATPase活性，该活性被磷酸化抑制。因此，非螺旋尾段丝氨酸的磷酸化不是AMII肌动蛋白激活的镁ATPase活性下调的原因，但头部丝氨酸的磷酸化是下调AMII活性的原因。这种丝氨酸正在通过质谱学和定点突变进行鉴定。丝氨酸磷酸化下调的分子机制也在研究中。尽管非螺旋尾段丝氨酸的磷酸化不影响酶的活性，但我们发现它确实影响了AMII微丝的结构，正如旋转阴影细丝的电子显微镜所见。

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.