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PMID: 3032987 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

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

The Journal of cell biology ·Vol. 104 ·No. 5 ·1987-05-00 ·Pages 1309-23

Griffith LM, Downs SM, Spudich JA

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 approximately 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 approximately 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 approximately 0.3 mol Pi/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 Pi/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 less than or equal to 0.01 mol Pi/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to greater than or equal to 0.96 mol Pi/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 approximately 1.4 micron/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.

MeSH Terms
Alkaline Phosphatase/metabolism Animals Dictyostelium/enzymology,growth & development Kinetics Molecular Weight Muscles/metabolism Myosin-Light-Chain Kinase/isolation & purification,metabolism Myosin-Light-Chain Phosphatase Myosins/physiology Phosphoprotein Phosphatases/isolation & purification,metabolism Phosphorylation Rabbits
Chemicals
Myosin-Light-Chain Kinase Alkaline Phosphatase Phosphoprotein Phosphatases Myosin-Light-Chain Phosphatase Myosins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Griffith L M
Downs S M
Spudich J A
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.104.5.1309
Published
1987-05-00
Pages
1309-23
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2114470
Subset
IM
Grants
NIGMS NIH HHS · 5-F32-GM07781 · United States
NCI NIH HHS · 5-T32-CA09151 · United States
NIGMS NIH HHS · GM-25240 · United States
External Links
PubMed source DOI: 10.1083/jcb.104.5.1309 PMC Full Text View journal details
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