Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells.
Open Access
- 15 December 1994
- journal article
- Published by Rockefeller University Press in The Journal of cell biology
- Vol. 127 (6), 1945-1955
- https://doi.org/10.1083/jcb.127.6.1945
Abstract
In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.Keywords
This publication has 68 references indexed in Scilit:
- CONTROL OF NONMUSCLE MYOSINS BY PHOSPHORYLATIONAnnual Review of Biochemistry, 1992
- Functional implications of the unusual amino acid sequence of the regulatory light chain ofAcanthamoeba castellanii myosin-IIJournal of Muscle Research and Cell Motility, 1991
- Actomyosin Organization in Mitotic Dictyostelium AmoebaeaAnnals of the New York Academy of Sciences, 1990
- Domains, motions and regulation in the myosin headJournal of Muscle Research and Cell Motility, 1988
- Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding.The Journal of cell biology, 1987
- Myosin specific phosphatases isolated fromDictyostelium discoideumJournal of Muscle Research and Cell Motility, 1986
- Site-directed mutagenesis of the regulatory light-chain Ca2+/Mg2+ binding site and its role in hybrid myosinsNature, 1986
- Hybrid formation between scallop myofibrils and foreign regulatory light-chainsJournal of Molecular Biology, 1980
- Regulatory light chains in myosinsJournal of Molecular Biology, 1976
- Biochemical and structural studies of actomyosin-like proteins from non-muscle cells: Isolation and characterization of myosin from amoebae of Dictyostelium discoideumJournal of Molecular Biology, 1974