Phosphorylation of threonine residues on cloned fragments of the Dictyostelium myosin heavy chain

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Abstract
A tail fragment of Dictyostelium discoideum myosin has been cloned and expressed as a fusion protein with the N‐terminal region of MS‐2 polymerase. The cloned fragment was phosphorylated with myosin heavy chain kinase II from aggregation‐competent D.discoideum cells that specifically phosphorylate threonine residues on the myosin tail. Phosphopeptide maps showed the same site specificity of phosphorylation with the fusion protein as a substrate as with native myosin. An improved assay for the kinase was developed in which the fusion protein is precipitated with a monoclonal antibody that inhibits polymerization of the myosin tails without preventing their phosphorylation. Sites of phosphorylation were tentatively localized to a sequence in the C‐terminal region of the heavy chain where four threonine residues are found.