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

详细信息    查看全文

• 作者：Geguchadze ; Ramaz ; Zhi ; Gang ; Lau ; Kim S. ; Isotani ; Eiji ; Persechini ; Anthony ; Kamm ; Kristine E. ; et. al.
• 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 ; fluorescenc
• 刊名：FEBS Letters
• 出版年：2004
• 出版时间：January 16, 2004
• 年：2004
• 卷：557
• 期：1-3
• 页码：121-124
• 全文大小：117 K

文摘

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.