Regulation of Myosin by Phosphorylation of the Heavy Chain

第一个证明重链磷酸化重要性的证据是我们在20世纪80年代早期的数据，该数据表明，肌动蛋白激活的棘阿米巴肌球蛋白II （AMII）的MgATPase活性被两条重链各29个残基的非螺旋尾部1至3个丝氨酸残基的磷酸化所抑制。从20世纪80年代中期到90年代中期，我们对这种抑制的可能机制进行了大约10年的研究。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）肌球蛋白的异聚物比同等的同聚物混合物具有明显更低的肌动蛋白激活的atp酶活性，即AMII的调节是在纤维水平上，磷酸化的AMII使未磷酸化的AMII失活，当两者处于同一纤维中时。有趣的是，尾部c末端的磷酸化位点距离球体中被螺旋状螺旋分隔的atp酶位点80纳米远。当时，我们和其他人都没有任何证据表明非螺旋尾部的磷酸化会影响丝状结构。

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.