Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: Effects of reversible phosphorylation on myosin structure and function

L. M. Griffith, S. M. Downs, J. A. Spudich

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82 Scopus citations

Abstract

We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of ~1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of ~30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has ~0.3 mol P(i)/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol P(i)/mol 18,000-D light chain. MLCP, which was partially purified, selectively removes phosphate from the 18,000-D light chain but not from the heavy chain of Dictyostelium myosin. Phosphatase-treated Dictyostelium myosin has ≤0.01 mol P(i)/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to ≥0.96 mol P(i)/mol 18,000-D light chain by incubation with MLCK and ATP. We found myosin thick filament assembly to be independent of the extent of 18,000-D light-chain phosphorylation when measured as a function of ionic strength. However, actin-activated Mg2+-ATPase activity of Dictyostelium myosin was found to be directly related to the extent of phosphorylation of the 18,000-D light chain. MLCK-treated myosin moved in an in vitro motility assay (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.), 305:31-35) at ~1.4 μm/s whereas phosphatase-treated myosin moved only slowly or not at all. The effects of phosphatase treatment on the movement were fully reversed by subsequent treatment with MLCK.

Original languageEnglish (US)
Pages (from-to)1309-1323
Number of pages15
JournalJournal of Cell Biology
Volume104
Issue number5
DOIs
StatePublished - 1987

ASJC Scopus subject areas

  • Cell Biology

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