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 phosphorylated^2–6. 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 kinase^7,8. 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 chain^9,10. 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 state^5,11. Phosphorylation of human platelet myosin⁴, guinea pig vas deferens³ and chicken gizzard^12,13 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.