Myoblast myosin phosphorylation is a prerequisite for actin-activation.

Abstract

CYTOPLASMIC myosins are found in most non-muscle cellular systems studied to date1. In vertebrates these myosins are composed of two heavy chains of 200,000 molecular weight (MW) and four light chains, two each of 20,000 and 15,000 MW. A number of cytoplasmic myosins, as well as skeletal, smooth, and cardiac muscle myosin, can be enzymatically phosphorylated2–6. That is, those light chains of MWs 18,000–20,000, the so-called P-light chains6, can undergo phosphorylation catalysed by a myosin light chain kinase7,8. In this reaction the terminal phosphate of ATP is transferred to a specific amino acid residue on the myosin P-light chain, up to one mole of phosphate per mole of light chain9,10. A myosin light chain phosphatase that catalyses the removal of the covalently-linked phosphate group from the P-light chain, returns the myosin to the non-phosphorylated state5,11. Phosphorylation of human platelet myosin4, guinea pig vas deferens3 and chicken gizzard12,13 smooth muscle myosin results in an increase in the actin-activated ATPase activities of these myosins. Although the phosphorylation of the P-light chain of rabbit skeletal muscle myosin has been described10, no functional change in this myosin's activity due to phosphorylation has been demonstrated11. To study the role of myosin phosphorylation during muscle cell differentiation, we investigated precursor cells to skeletal muscle, namely proliferative myoblasts. The Yaffe L-5 810 line of cloned proliferative rat myoblasts is an actively dividing cell line in culture which under the appropriate conditions can form myofibrils. We now report: (1) that myosin isolated from proliferative myoblasts contains light chains of 20,000 and 15,000 MW, (2) the 20,000 MW light chain can be phosphorylated by an endogenous myosin light chain kinase14, (3) the proliferative myoblast myosin light chain kinase does not require calcium ions for activity, and (4) phosphorylation is a pre-requisite for actin activation of the myosin's ATPase activity.