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Quantitative measurements of Ca 2+ /calmodulin binding and activation of myosin light chain kinase in cells
Author(s) -
Geguchadze Ramaz,
Zhi Gang,
Lau Kim S,
Isotani Eiji,
Persechini Anthony,
Kamm Kristine E,
Stull James T
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)01456-x
Subject(s) - myosin light chain kinase , calmodulin , myosin , phosphorylation , chemistry , förster resonance energy transfer , kinase , biophysics , microbiology and biotechnology , actin , cytoskeleton , biochemistry , immunoglobulin light chain , protein kinase a , fluorescence , biology , enzyme , cell , physics , quantum mechanics , antibody , immunology
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.