Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse

Eiji Isotani, Gang Zhi, Kim S. Lau, Jian Huang, Yusuke Mizuno, Anthony Persechini, Ramaz Geguchadze, Kristine E. Kamm, James T. Stull

Research output: Contribution to journalArticle

89 Citations (Scopus)

Abstract

Ca2+/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) initiates smooth muscle contraction and regulates actomyosin-based cytoskeletal functions in nonmuscle cells. The net extent of RLC phosphorylation is controlled by MLCK activity relative to myosin light chain phosphatase activity. We have constructed a CaM-sensor MLCK where Ca2+-dependent CaM binding increases the catalytic activity of the kinase domain, whereas coincident binding to the biosensor domain decreases fluorescence resonance energy transfer between two fluorescent proteins. We have created transgenic mice expressing this construct specifically in smooth muscle cells to perform real-time evaluations of the relationship between smooth muscle contractility and MLCK activation in intact tissues and organs. Measurements in intact bladder smooth muscle demonstrate that MLCK activation increases rapidly during KCI-induced contractions but is not maximal, consistent with a limiting amount of cellular CaM. Carbachol treatment produces the same amount of force development and RLC phosphorylation, with much smaller increases in [Ca2+]i and MLCK activation. A Rho kinase inhibitor suppresses RLC phosphorylation and force but not MLCK activation in carbachol-treated tissues. These observations are consistent with a model in which the magnitude of an agonist-mediated smooth muscle contraction depends on a rapid but limited Ca2+/CaM-dependent activation of MLCK and Rho kinase-mediated inhibition of myosin light chain phosphatase activity. These studies demonstrate the feasibility of producing transgenic biosensor mice for investigations of signaling processes in intact systems.

Original languageEnglish (US)
Pages (from-to)6279-6284
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number16
DOIs
StatePublished - Apr 20 2004

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Myosin-Light-Chain Kinase
Biosensing Techniques
Calmodulin
Smooth Muscle
Muscles
Myosin-Light-Chain Phosphatase
Phosphorylation
Smooth Muscle Myosins
rho-Associated Kinases
Carbachol
Muscle Contraction
Light
Transgenic Mice
Actomyosin
Myosin Light Chains
Fluorescence Resonance Energy Transfer
Feasibility Studies
Smooth Muscle Myocytes
Urinary Bladder
Phosphotransferases

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse. / Isotani, Eiji; Zhi, Gang; Lau, Kim S.; Huang, Jian; Mizuno, Yusuke; Persechini, Anthony; Geguchadze, Ramaz; Kamm, Kristine E.; Stull, James T.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 101, No. 16, 20.04.2004, p. 6279-6284.

Research output: Contribution to journalArticle

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