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J Muscle Res Cell Motil. 2011 Mar;31(5-6):349-58. doi: 10.1007/s10974-011-9240-2. Epub 2011 Feb 1.

A force-activated kinase in a catch smooth muscle.

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  • 1Department of Molecular Physiology and Biophysics, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA. thomas.butler@jefferson.edu

Abstract

Permeabilized anterior byssus retractor muscles (ABRM) from Mytilus edulis were used as a simple system to test whether there is a stretch dependent activation of a kinase as has been postulated for titin and the mini-titin twitchin. The ABRM is a smooth muscle that shows catch, a condition of high force maintenance and resistance to stretch following stimulation when the intracellular Ca(++) concentration has diminished to sub-maximum levels. In the catch state twitchin is unphosphorylated, and the muscle maintains force without myosin crossbridge cycling through what is likely a twitchin mediated tether between thick and thin filaments. In catch, a small change in length results in a large change in force. The phosphorylation state of an added peptide, a good substrate for molluscan twitchin kinase, with the sequence KKRAARATSNVFA was used as a measure of kinase activation. We find that there is about a two-fold increase in phosphorylation of the added peptide with a 10% stretch of the ABRM in catch. The increased phosphorylation is due to activation of a kinase rather than to an inhibition of a phosphatase. The extent of phosphorylation of the peptide is decreased when twitchin is phosphorylated and catch force is not present. However, there is also a large increase in peptide phosphorylation when the muscle is activated in pCa 5, and the catch state does not exist. The force-sensitive kinase activity is decreased by ML-9 and ML-7 which are inhibitors of twitchin kinase, but not by the Rho kinase inhibitor Y-27632. There is no detectable phosphorylation of myosin light chains, but the phosphorylation of twitchin increases by a small, but significant extent with stretch. It is possible that twitchin senses force output resulting in a force-sensitive twitchin kinase activity that results in autophosphorylation of twitchin on site(s) other than those responsible for relaxation of catch.

PMID:
21286791
[PubMed - indexed for MEDLINE]
PMCID:
PMC3051186
Free PMC Article
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