Department of Molecular Physiology and Biophysics, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
J Muscle Res Cell Motil. 2011 Mar;31(5-6):349-58. doi: 10.1007/s10974-011-9240-2. Epub 2011 Feb 1.
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.
麻痹贻贝前贻贝收肌（ABRM）用作一个简单的系统来测试激酶是否存在拉伸依赖性激活，如已经提出的肌联蛋白和微管结合蛋白 twitchin。ABRM 是一种平滑肌，表现出捕获，即当细胞内 Ca(++)浓度降低到亚最大水平时，高力维持和对拉伸的阻力的条件，当刺激时。在捕获状态下，twitchin 未磷酸化，肌肉通过可能是在厚和薄丝之间的 twitchin 介导的系绳维持力，而无需肌球蛋白交联桥循环。在捕获中，长度的微小变化导致力的巨大变化。添加的肽的磷酸化状态，一种用于软体动物 twitchin 激酶的良好底物，序列 KKRAARATSNVFA 被用作激酶激活的测量。我们发现，在捕获中的 ABRM 10%拉伸时，添加的肽的磷酸化增加了约两倍。增加的磷酸化是由于激酶的激活而不是磷酸酶的抑制。当 twitchin 被磷酸化并且不存在捕获力时，肽的磷酸化程度降低。然而，当肌肉在 pCa 5 中被激活并且不存在捕获状态时，肽的磷酸化也会大大增加。力敏激酶活性被 ML-9 和 ML-7 降低，它们是 twitchin 激酶的抑制剂，但不是 Rho 激酶抑制剂 Y-27632。肌球蛋白轻链没有可检测到的磷酸化，但 twitchin 的磷酸化随着拉伸而增加一小部分，但显著程度。有可能是 twitchin 感觉到力输出导致力敏感的 twitchin 激酶活性，导致 twitchin 在除了负责捕获松弛的部位之外的部位发生自动磷酸化。
