细胞中Ca(2+)/钙调蛋白结合及肌球蛋白轻链激酶激活的定量测量。
Quantitative measurements of Ca(2+)/calmodulin binding and activation of myosin light chain kinase in cells.
作者信息
Geguchadze Ramaz, Zhi Gang, Lau Kim S, Isotani Eiji, Persechini Anthony, Kamm Kristine E, Stull James T
机构信息
Department of Physiology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9040, USA.
出版信息
FEBS Lett. 2004 Jan 16;557(1-3):121-4. doi: 10.1016/s0014-5793(03)01456-x.
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+) increased MLCK activation and RLC phosphorylation coincidently. There was no evidence for CaM binding but not activating MLCK at low Ca(2+). At saturating Ca(2+) MLCK was not fully activated probably due to limited availability of cellular Ca(2+)/CaM.
钙（Ca²⁺）/钙调蛋白（CaM）依赖性肌球蛋白轻链激酶（MLCK）介导的肌球蛋白 II 调节轻链（RLC）磷酸化与许多细胞肌动蛋白细胞骨架功能有关。我们使用荧光生物传感器 MLCK 对 MLCK 激活进行了定量检测，结果表明，在体外，激酶活性的 Ca²⁺依赖性增加与荧光共振能量转移（FRET）的降低同时发生。在稳定转染 CaM 传感器 MLCK 的细胞中，细胞内 Ca²⁺浓度（[Ca²⁺]i）升高会同时增加 MLCK 激活和 RLC 磷酸化。没有证据表明在低[Ca²⁺]i 时 CaM 与 MLCK 结合但不激活它。在饱和[Ca²⁺]i 时，MLCK 未被完全激活，这可能是由于细胞内 Ca²⁺/CaM 的可用性有限。
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