Quantitative measurements of Ca2+/calmodulin binding and activation of myosin light chain kinase in cells

FEBS Letters

Zhi, Gang¹  Isotani, Eiji¹  Kamm, Kristine E¹  Persechini, Anthony²  Geguchadze, Ramaz¹  Lau, Kim S¹  Stull, James T¹ 

[1] Department of Physiology, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040, USA;Division of Molecular Biology and Biochemistry, University of Missouri at Kansas City, 5007 Rockhill Road, Room B412, Kansas City, MO 64110-2499, USA

关键词: Myosin light chain kinase;    Calmodulin;    Calcium;    Phosphorylation;    Fluorescence resonance energy transfer;    CaM;    calmodulin;    DIFP;    diisopropylfluorophosphate;    DTT;    dithiothreitol;    E64;    trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane;    FRET;    fluorescence resonance energy transfer;    MOPS;    3-(N-morpholino)-propanesulfonic acid;    RLC;    regulatory light chain;    MLCK;    myosin light chain kinase;    EYFP;    enhanced yellow fluorescent protein;    ECFP;    enhanced cyan fluorescent protein;    GFP;    green fluorescent protein;    BFP;    blue fluorescent protein;   

DOI  :  10.1016/S0014-5793(03)01456-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Myosin II regulatory light chain (RLC) phosphorylation by Ca²⁺/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 Ca²⁺-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 [Ca²⁺] _i increased MLCK activation and RLC phosphorylation coincidently. There was no evidence for CaM binding but not activating MLCK at low [Ca²⁺] _i . At saturating [Ca²⁺] _i MLCK was not fully activated probably due to limited availability of cellular Ca²⁺/CaM.

【 授权许可】

Unknown