Regulation of Myosin by Phosphorylation of the Heavy Chain

AMII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，显示其肌动蛋白激活的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著低于肌动蛋白激活的ATP酶活性的等效混合物的均聚物，即调节AMII似乎是在与磷酸化的AMII灭活未磷酸化的AMII丝的水平，当两者都在相同的丝。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。 我们通过研究重组野生型和突变型AMIIs的细丝结构和肌动蛋白激活的ATP酶活性重新研究了这个问题。 我们现在发现，通过定量质谱，与肌球蛋白II重链激酶孵育磷酸化全长AMII的非螺旋尾段（NHT）中的四个丝氨酸，和全长AMII的马达结构域的环2中的Ser 639，重肌球蛋白（HMM）和亚片段1（S1）。 所有肌球蛋白结构的肌动蛋白激活的ATP酶都被磷酸化抑制，由于HMM和S1缺乏肌球蛋白尾，因此肌动蛋白激活的ATP酶活性的抑制必须由Ser 639的磷酸化引起。 与该结论一致，全长AMII的S639 A突变体不受NHT-丝氨酸磷酸化或其突变为谷氨酸的抑制。 相反，S1和全长AMII的S639 D突变体是无酶活性的，与全长AMII的NHT-丝氨酸的磷酸化状态无关。重组野生型和S639 D S1的肌球蛋白和肌动球蛋白ATP酶循环中的每个步骤的动力学分析表明，Pi从肌动球蛋白ADP-Pi复合物中的释放是S639 D突变主要抑制的步骤。 虽然磷酸化的丝氨酸在非螺旋尾不影响酶的活性，我们发现它确实影响的AMII微丝的结构。 如通过旋转阴影细丝的电子显微镜所见，全长AMII的双极四聚体、六聚体和八聚体微丝（其NHT-丝氨酸磷酸化或突变为谷氨酸）比非磷酸化、非突变的AMII具有更长的裸露区和更紧密的簇头，而不管Ser 639的磷酸化状态如何。 因此，我们已经表明，肌动蛋白激活的ATP酶活性的AMII的调节Ser 639的磷酸化在电机域和AMII的丝结构的修改由磷酸化的一个或多个的四个丝氨酸在NHT。 这些结果具有显着的影响，其NHT-丝氨酸和苏氨酸也可以在体外和体内磷酸化的哺乳动物非肌肉肌球蛋白II的丝结构的调节。

The first evidence for the importance of heavy chain phosphorylation of AMII was our data in the early 1980s showing that its actin-activated MgATPase activity 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 seemed to be 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 have reinvestigated this problem by studying the filament structure and actin-activated ATPase activity of recombinant wild-type and mutant AMIIs. We now find, by quantitative mass spectrometry, that incubation with myosin II heavy chain kinase phosphorylates four serines in the non-helical tailpiece (NHT) of full-length AMII, and Ser639 in loop 2 of the motor domain of full-length AMII, heavy meromyosin (HMM) and sub-fragment 1 (S1). The actin-activated ATPase of all of the myosin constructs is inhibited by phosphorylaion and, since HMM and S1 lack the myosin tail, inhibition of actin-activaed ATPase activity must result from phosphorylation of Ser639. Consistent with this conclusion, the S639A mutant of full-length AMII is not inhibited by phosphorylation of the NHT-serines or their mutation to glutamate. Conversely, S639D mutants of both S1 and full-length AMII are enzymatically inactive irrespective of the phosphorylation state of the NHT-serines of full-length AMII. Kinetic analysis of each step in the myosin and actomyosin ATPase cycles of recombinant wild-type and S639D S1 showed that the release of Pi from the actomyosin ADP-Pi complex is the step predominantly inhibited by the S639D mutation. Although phosphorylation of the serines in the non-helical tailpiece 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, bipolar tetrameric, hexameric and octameric minifilaments of full-length AMII with their NHT-serines either phosphorylated or mutated to glutamate have longer bare zones and more tightly clustered heads than non-phosphorylated, non-mutated AMII irrespective of the phosphorylation state of Ser639. Thus, we have shown tha the actin-activated ATPase activity of AMII is regulated by phosphorylation of Ser639 in the motor domain and the filament structure of AMII is modified by phosphorylation of one or more of the four serines in the NHT. These results have significant implications for the regulation of the filament structure of mammalian non-muscle myosins IIs whose NHT-serines and threonines can also be phosphorylated both in vitro and in vivo.