RRC ID 28967
Author Ostrow BD, Chen P, Chisholm RL.
Title Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells.
Journal J Cell Biol
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.
Volume 127(6 Pt 2)
Pages 1945-55
Published 1994-12-1
DOI 10.1083/jcb.127.6.1945
PMID 7806572
PMC PMC2120314
MeSH Actins / pharmacology Adenosine Triphosphatases / metabolism Amino Acid Sequence Animals Base Sequence Cell Compartmentation Cell Division / physiology* Dictyostelium / cytology Dictyostelium / genetics Dictyostelium / growth & development* Enzyme Activation Fluorescent Antibody Technique Genetic Complementation Test Molecular Sequence Data Mutagenesis, Site-Directed Mutation* Myosin-Light-Chain Kinase / metabolism Myosins / biosynthesis* Myosins / drug effects Myosins / genetics Myosins / isolation & purification Phosphorylation Recombinant Proteins / biosynthesis Serine / metabolism Structure-Activity Relationship
IF 8.811
Times Cited 85
Cellular slime molds S00033