title: Quantitative measurements of Ca(2+)/calmodulin binding and activation of myosin light chain kinase in cells.
abstract: Myosin II regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) is implicated in many cellular actin cytoskeletal functions. We examined MLCK activation quantitatively with a fluorescent biosensor MLCK where Ca(2+)-dependent increases in kinase activity were coincident with decreases in fluorescence resonance energy transfer (FRET) in vitro. In cells stably transfected with CaM sensor MLCK, increasing [Ca(2+)](i) increased MLCK activation and RLC phosphorylation coincidently. There was no evidence for CaM binding but not activating MLCK at low [Ca(2+)](i). At saturating [Ca(2+)](i) MLCK was not fully activated probably due to limited availability of cellular Ca(2+)/CaM.
authors.affiliation: Department of Physiology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9040, USA. ramaz.geguchadze@utsouthwestern.edu
authors.forename: Ramaz
authors.initials: R
authors.lastname: Geguchadze
authors.forename: Gang
authors.initials: G
authors.lastname: Zhi
authors.forename: Kim S
authors.initials: KS
authors.lastname: Lau
authors.forename: Eiji
authors.initials: E
authors.lastname: Isotani
authors.forename: Anthony
authors.initials: A
authors.lastname: Persechini
authors.forename: Kristine E
authors.initials: KE
authors.lastname: Kamm
authors.forename: James T
authors.initials: JT
authors.lastname: Stull
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007668:Kidney; D007700:Kinetics; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D009219:Myosin-Light-Chain Kinase; D002118:Calcium