Quantitative measurements of Ca(2+)/calmodulin binding and activation of myosin light chain kinase in cells.
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.
Geguchadze
Ramaz
R
Department of Physiology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9040, USA. ramaz.geguchadze@utsouthwestern.edu
Zhi
Gang
G
Lau
Kim S
KS
Isotani
Eiji
E
Persechini
Anthony
A
Kamm
Kristine E
KE
Stull
James T
JT
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
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D009219:Myosin-Light-Chain Kinase; D002118:Calcium