Related Experiment Videos
Myosin light chain kinase phosphorylation: regulation of the Ca2+ sensitivity of contractile elements
J T Stull1, M G Tansey, R A Word
1University of Texas Southwestern Medical Center, Department of Physiology, Dallas 75235.
Abstract:
Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C and the multifunctional calmodulin-dependent protein kinase II. Since phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labelled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A, perhaps by calmodulin-dependent protein kinase II, may play a role in reported desensitization of contractile elements in smooth muscle to activation by Ca2+.
Insights
Smooth muscle contraction is regulated by myosin light chain kinase (MLCK) phosphorylation. Site A phosphorylation on MLCK correlates with muscle contraction, suggesting a role in desensitization to calcium.
Area of Science:
- Biochemistry
- Molecular Biology
- Physiology
Background:
- Myosin light chain kinase (MLCK) regulates smooth muscle contractility.
- MLCK can be phosphorylated by several protein kinases, including cyclic AMP-dependent protein kinase, protein kinase C, and calmodulin-dependent protein kinase II.
- Phosphorylation at a specific site (site A) desensitizes MLCK to Ca2+/calmodulin activation.
Purpose of the Study:
- To investigate the role of MLCK phosphorylation in regulating smooth muscle contractility in bovine tracheal smooth muscle.
- To determine which specific kinases phosphorylate MLCK in response to contractile stimuli.
- To examine the correlation between site A phosphorylation and smooth muscle contraction.
Main Methods:
- Utilized 32P-labelled bovine tracheal smooth muscle.
- Treated tissues with contractile agents (carbachol, KCl) and non-contractile agents (isoproterenol, phorbol 12,13-dibutyrate).
- Identified and quantified phosphopeptides of MLCK using phosphopeptide mapping.
Main Results:
- Carbachol, KCl, isoproterenol, and phorbol 12,13-dibutyrate increased MLCK phosphorylation.
- Six primary phosphopeptides (A-F) of MLCK were identified.
- Site A phosphorylation occurred significantly with carbachol and KCl, correlating with smooth muscle contraction and increased Ca2+/calmodulin activation requirements.
Conclusions:
- Cyclic AMP-dependent protein kinase and protein kinase C do not mediate smooth muscle contractility changes via site A phosphorylation of MLCK.
- Phosphorylation of MLCK at site A, potentially by calmodulin-dependent protein kinase II, may contribute to the desensitization of smooth muscle contractile elements to Ca2+ activation.