软体动物捕获肌抽动蛋白n端区磷酸化特性

D. Funabara, Yuuki Nishimura, S. Kanoh
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引用次数: 2

摘要

软体动物的平滑肌,如双壳类内收肌和贻贝前副套收肌(ABRM),表现出一种称为“捕获”的独特收缩。捕获收缩是通过抽动蛋白磷酸化和去磷酸化来调节的。地中海贻贝Mytilus galloprovincialis的ABRM中的Twitchin被cAMP依赖性蛋白激酶(PKA)磷酸化,PKA磷酸化位点位于Twitchin分子的N-和C-末端区域。D2位点与C末端相邻,参与形成肌球蛋白、肌动蛋白和抽动蛋白复合物,该复合物被认为有助于维持捕获状态下的张力。相反,尽管有报道称其与细丝相互作用,但包括D1位点的区域的分子功能在很大程度上仍未得到研究。在D1位点附近鉴定出另外三个PKA共有序列;然而,尚不清楚这些位点是否能被PKA直接磷酸化。在这里,我们使用重组蛋白变体(TWD1-SSSS、TWD1-AAAS、TWD1-AASA、TWD1-AlAA、TWD1-SAAA和TWD1-AAAA)进行磷酸化测定以鉴定D1位点附近的磷酸化位点。除了TWD1-AAAA(其中所有可磷酸化的丝氨酸残基都被丙氨酸取代)外,所有变体都被PKA磷酸化。这四个磷酸化位点从N末端开始依次命名为D1-1、D1-2、D1-3和D1-4(最初鉴定的D1)。使用PKA与每个TWD1变体的1/12.5重量比的磷酸化测定显示D1-4是最快速磷酸化的,紧随其后的是D1-1。然而,D1-2和D1-3在同等条件下以较低水平磷酸化,并且当PKA与每种TWD1变体以1/100重量比孵育时不磷酸化。此外,我们观察到TWD1-SSSS以逐步的方式被磷酸化。这些发现有助于阐明抽搐蛋白D1区域在捕获收缩调节系统中的功能。
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Phosphorylation Properties of the N-Terminal Region of Twitchin from Molluscan Catch Muscle
Molluscan smooth muscles, such as the bivalve adductor muscles and the mussel anterior byssus retractor muscles (ABRM), exhibit a unique contraction called “catch”. Catch contraction is regulated through twitchin phosphorylation and dephosphorylation. Twitchin from the ABRM of the Mediterranean mussel, Mytilus galloprovincialis, is phosphorylated by cAMP-dependent protein kinase (PKA), and PKA phosphorylation sites are located in both the N- and C-terminal regions of the twitchin molecule. The D2 site, which is adjacently located to the C-terminus, participates in forming a myosin, actin, and twitchin complex that is thought to contribute towards the maintenance of tension in the catch state. In contrast, although it has been reported to interact with thin-filaments, the molecular function of the region including the D1 site has remained largely unstudied. Three additional PKA consensus sequences were identified near the D1 site; however, it was not known if these sites could be directly phosphorylated by PKA. Here, we performed phosphorylation assays to identify phosphorylation sites near the D1 site using recombinant protein variants (TWD1-SSSS, TWD1-AAAS, TWD1-AASA, TWD1-ASAA, TWD1-SAAA, and TWD1-AAAA). All variants, except TWD1-AAAA (where all phosphorylatable serine residues were replaced by alanines), were phosphorylated by PKA. The four phosphorylation sites were named D1-1, D1-2, D1-3, and D1-4 (the originally identified D1) in order from the N-terminus. Phosphorylation assays using a 1/12.5 weight ratio of PKA to each TWD1 variant revealed that D1-4 was the most rapidly phosphorylated, closely followed by D1-1. However, D1-2 and D1-3 were phosphorylated at a lower level under equivalent conditions and were not phosphorylated when PKA was incubated with each TWD1 variant at a 1/100 weight ratio. Furthermore, we observed that TWD1-SSSS was phosphorylated in a stepwise fashion. These findings contribute towards the elucidation of the function of the twitchin D1 region in the regulatory system of catch contraction.
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