Regulation of Myosin by Phosphorylation of the Heavy Chain

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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.