Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

Yusuke Mizuno, Eiji Isotani, Jian Huang, Hailei Ding, James T. Stull, Kristine E. Kamm

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Ca2+/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca2+ concentration ([Ca2+]i). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca 2+]i with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rhokinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume295
Issue number2
DOIs
StatePublished - Aug 2008

Fingerprint

Myosin-Light-Chain Phosphatase
Myosin-Light-Chain Kinase
Smooth Muscle
Phosphorylation
Calcium
Carbachol
Calmodulin
Smooth Muscle Myosins
Light
Photophobia
rho-Associated Kinases
Myosin Light Chains
Phosphoric Monoester Hydrolases
Urinary Bladder
Phosphotransferases

Keywords

  • Bladder
  • Calmodulin
  • Myosin light chain phosphatase
  • Myosin regulatory light chain
  • Rho kinase

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

Cite this

Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo. / Mizuno, Yusuke; Isotani, Eiji; Huang, Jian; Ding, Hailei; Stull, James T.; Kamm, Kristine E.

In: American Journal of Physiology - Cell Physiology, Vol. 295, No. 2, 08.2008.

Research output: Contribution to journalArticle

@article{26d51d9907ef42d5bd82b18d9517803a,
title = "Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo",
abstract = "Ca2+/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca2+ concentration ([Ca2+]i). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca 2+]i with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20{\%} activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rhokinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.",
keywords = "Bladder, Calmodulin, Myosin light chain phosphatase, Myosin regulatory light chain, Rho kinase",
author = "Yusuke Mizuno and Eiji Isotani and Jian Huang and Hailei Ding and Stull, {James T.} and Kamm, {Kristine E.}",
year = "2008",
month = "8",
doi = "10.1152/ajpcell.90645.2007",
language = "English (US)",
volume = "295",
journal = "American Journal of Physiology - Heart and Circulatory Physiology",
issn = "0363-6135",
publisher = "American Physiological Society",
number = "2",

}

TY - JOUR

T1 - Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

AU - Mizuno, Yusuke

AU - Isotani, Eiji

AU - Huang, Jian

AU - Ding, Hailei

AU - Stull, James T.

AU - Kamm, Kristine E.

PY - 2008/8

Y1 - 2008/8

N2 - Ca2+/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca2+ concentration ([Ca2+]i). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca 2+]i with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rhokinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.

AB - Ca2+/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca2+ concentration ([Ca2+]i). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca 2+]i with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rhokinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.

KW - Bladder

KW - Calmodulin

KW - Myosin light chain phosphatase

KW - Myosin regulatory light chain

KW - Rho kinase

UR - http://www.scopus.com/inward/record.url?scp=55949091220&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=55949091220&partnerID=8YFLogxK

U2 - 10.1152/ajpcell.90645.2007

DO - 10.1152/ajpcell.90645.2007

M3 - Article

VL - 295

JO - American Journal of Physiology - Heart and Circulatory Physiology

JF - American Journal of Physiology - Heart and Circulatory Physiology

SN - 0363-6135

IS - 2

ER -