Heierhorst J, Probst W C, Kohanski R A, Buku A, Weiss K R
Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, USA.
Eur J Biochem. 1995 Oct 15;233(2):426-31. doi: 10.1111/j.1432-1033.1995.426_2.x.
The unusually large (approximately 600 to > 3000 kDa) myosin-associated proteins of the titin/twitchin superfamily are considered to be important cytoskeletal rulers for thick filament assembly in muscle. This function is maintained by approximately 60-240 modular fibronectin-type-III and immunoglobulin-C2 repeats in these proteins which further contain a protein serine/threonine kinase domain of unknown function. In this study, the bacterially expressed kinase domain of Aplysia twitchin was used in order to identify a potential physiological substrate. Addition of the recombinant kinase to Aplysia actomyosin preparations resulted in the specific phosphorylation of the 19-kDa myosin regulatory light chains. The twitchin kinase phosphorylated purified light chains on Thr15 in a region which shared a high degree of similarity with the phosphorylation site for vertebrate smooth muscle myosin light chain kinase. Peptide analogs of the twitchin substrate sequence and the similar sequence in vertebrate smooth muscle myosin light chains were phosphorylated with good kinetic properties. These data reveal the first potential substrate for any of the giant protein kinases and support a dual role of twitchin in molluscan muscle as a cytoskeletal protein as well as a myosin light chain kinase.
肌联蛋白/肌动蛋白超家族中异常大（约600至>3000 kDa）的肌球蛋白相关蛋白被认为是肌肉中粗肌丝组装的重要细胞骨架标尺。这些蛋白中约60 - 240个模块化的纤连蛋白III型和免疫球蛋白C2重复序列维持了这一功能，这些重复序列还包含一个功能未知的蛋白丝氨酸/苏氨酸激酶结构域。在本研究中，使用了经细菌表达的海兔肌动蛋白激酶结构域来鉴定潜在的生理底物。将重组激酶添加到海兔肌动球蛋白制剂中导致19 kDa的肌球蛋白调节轻链发生特异性磷酸化。肌动蛋白激酶在Thr15位点磷酸化纯化的轻链，该区域与脊椎动物平滑肌肌球蛋白轻链激酶的磷酸化位点具有高度相似性。肌动蛋白底物序列的肽类似物以及脊椎动物平滑肌肌球蛋白轻链中的相似序列以良好的动力学特性被磷酸化。这些数据揭示了任何一种巨型蛋白激酶的首个潜在底物，并支持肌动蛋白在软体动物肌肉中作为细胞骨架蛋白以及肌球蛋白轻链激酶的双重作用。
