A temporal Ca2+ desensitization of myosin light chain kinase in phasic smooth muscles induced by CaMKKβ/PP2A pathways

Am J Physiol Cell Physiol. 2021 Sep 1;321(3):C549-C558. doi: 10.1152/ajpcell.00136.2021. Epub 2021 Jun 9.


Cell signaling pathways regulating myosin regulatory light chain (LC20) phosphorylation contribute to determining contractile responses in smooth muscles. Following excitation and contraction, phasic smooth muscles, such as the digestive tract and urinary bladder, undergo relaxation due to a decline of cellular Ca2+ concentration and decreased Ca2+ sensitivity of LC20 phosphorylation, named Ca2+ desensitization. Here, we determined the mechanisms underlying the temporal Ca2+ desensitization of LC20 phosphorylation in phasic smooth muscles using permeabilized strips of the mouse ileum and urinary bladder. Upon stimulation with pCa6.0 at 20°C, contraction and LC20 phosphorylation peaked within 30 s and then declined to about 50% of the peak force at 2 min after stimulation. During the relaxation phase after the contraction, LC20 kinase [myosin light chain kinase (MLCK)] was inactivated, but no fluctuation in LC20 phosphatase activity occurred, suggesting that MLCK inactivation is a cause of the Ca2+-induced Ca2+ desensitization of LC20 phosphorylation. MLCK inactivation was associated with phosphorylation at the calmodulin-binding domain of the kinase. Treatment with STO-609 and TIM-063 antagonists for Ca2+/calmodulin (CaM)-dependent protein kinase kinase-β (CaMKKβ) attenuated both the phasic response of the contraction and MLCK phosphorylation, whereas neither CaM kinase II, AMP-activated protein kinase, nor p21-activated kinase induced MLCK inactivation in phasic smooth muscles. Conversely, protein phosphatase 2A inhibition amplified the phasic response. Signaling pathways through CaMKKβ and protein phosphatase 2A may contribute to regulating the phasic response of smooth muscle contraction.

Keywords: digestive tract; kinase cascade; myography; signal transduction; smooth muscle.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / genetics
  • AMP-Activated Protein Kinases / metabolism
  • Animals
  • Benzimidazoles / pharmacology
  • Calcium / metabolism
  • Calcium Signaling
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase / genetics*
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase / metabolism
  • Female
  • Gene Expression Regulation
  • Ileum / metabolism
  • Mice
  • Muscle Contraction / drug effects
  • Muscle Contraction / physiology
  • Muscle, Smooth, Vascular / drug effects
  • Muscle, Smooth, Vascular / metabolism*
  • Myosin Light Chains / genetics*
  • Myosin Light Chains / metabolism
  • Myosin-Light-Chain Kinase / genetics*
  • Myosin-Light-Chain Kinase / metabolism
  • Naphthalimides / pharmacology
  • Phosphorylation
  • Protein Phosphatase 2 / genetics*
  • Protein Phosphatase 2 / metabolism
  • Tissue Culture Techniques
  • Urinary Bladder / metabolism
  • p21-Activated Kinases / genetics
  • p21-Activated Kinases / metabolism


  • Benzimidazoles
  • Myosin Light Chains
  • Naphthalimides
  • STO 609
  • p21-Activated Kinases
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase
  • Myosin-Light-Chain Kinase
  • AMP-Activated Protein Kinases
  • Protein Phosphatase 2
  • Calcium