Sobieszek Apolinary, Matusovsky Oleg S, Permyakova Tatyana V, Sarg Bettina, Lindner Herbert, Shelud'ko Nikolay S
Institute for Biomedical Aging Research, Life Science Center, Austrian Academy of Sciences, Mitterweg 24, A-6020 Innsbruck, Austria.
Arch Biochem Biophys. 2006 Oct 15;454(2):197-205. doi: 10.1016/j.abb.2006.08.004. Epub 2006 Aug 22.
Myorod, also known as catchin, a newly discovered component of molluscan smooth muscle thick filaments, is an alternative product of the myosin heavy chain gene. It contains a C-terminal rod part that is identical to that part of myosin and a unique N-terminal domain that is very small relative to the myosin head domain. The role of myorod in contraction or relaxation of this muscle type is unknown. In the present study we demonstrated that myorod was phosphorylated not only by a kinase endogenous to molluscan myosin and twitchin but also to vertebrate smooth muscle myosin light chain kinase (MLCK). The rates and maximal levels of phosphorylation were up to threefold higher than those observed by protein kinase A with clear optima at the physiological salt concentrations. Using a mild digestion with chymotrypsin we isolated an 11 kDa phosphopeptide and showed that the phosphorylation site was located at the N-terminal domain of myorod at Thr 141 position. The sequence around this site exhibited a high degree of similarity to that expected for the substrate recognition site of MLCK. The phosphorylation rates strongly depended on the ionic conditions indicating that this site could be readily sterically blocked during myorod polymerization. Another component of the thick filaments involved in regulation of the catch state, twitchin, was phosphorylated by MLCK and exhibited endogenous myorod kinase and MLCK activities. A possible role of these phosphorylation reactions in the regulation of molluscan smooth muscles is discussed.
肌杆蛋白，也被称为捕获蛋白，是软体动物平滑肌粗肌丝新发现的组成部分，是肌球蛋白重链基因的一种替代产物。它包含一个与肌球蛋白该部分相同的C末端杆状部分，以及一个相对于肌球蛋白头部结构域非常小的独特N末端结构域。肌杆蛋白在这种肌肉类型的收缩或舒张中的作用尚不清楚。在本研究中，我们证明肌杆蛋白不仅被软体动物肌球蛋白和抽动蛋白内源性的激酶磷酸化，还被脊椎动物平滑肌肌球蛋白轻链激酶（MLCK）磷酸化。磷酸化的速率和最大水平比蛋白激酶A观察到的高出三倍，在生理盐浓度下有明显的最佳值。通过用胰凝乳蛋白酶进行温和消化，我们分离出了一个11 kDa的磷酸肽，并表明磷酸化位点位于肌杆蛋白N末端结构域的苏氨酸141位置。该位点周围的序列与MLCK底物识别位点预期的序列高度相似。磷酸化速率强烈依赖于离子条件，表明该位点在肌杆蛋白聚合过程中很容易被空间位阻阻断。参与捕获状态调节的粗肌丝的另一个组成部分，抽动蛋白，被MLCK磷酸化，并表现出内源性肌杆蛋白激酶和MLCK活性。讨论了这些磷酸化反应在软体动物平滑肌调节中的可能作用。
