Matsunaga Yohei, Qadota Hiroshi, Furukawa Miho, Choe Heejoo Helen, Benian Guy M
Department of Pathology, Emory University, Atlanta, GA 30322.
Department of Pathology, Emory University, Atlanta, GA 30322
Mol Biol Cell. 2015 Jun 1;26(11):2096-111. doi: 10.1091/mbc.E14-05-1009. Epub 2015 Apr 7.
In Caenorhabditis elegans, twitchin is a giant polypeptide located in muscle A-bands. The protein kinase of twitchin is autoinhibited by 45 residues upstream (NL) and 60 residues downstream (CRD) of the kinase catalytic core. Molecular dynamics simulation on a twitchin fragment revealed that the NL is released by pulling force. However, it is unclear how the CRD is removed. To identify proteins that may remove the CRD, we performed a yeast two-hybrid screen using twitchin kinase as bait. One interactor is MAK-1, C. elegans orthologue of MAPKAP kinase 2. MAPKAP kinase 2 is phosphorylated and activated by p38 MAP kinase. We demonstrate that the CRD of twitchin is important for binding to MAK-1. mak-1 is expressed in nematode body wall muscle, and antibodies to MAK-1 localize between and around Z-disk analogues and to the edge of A-bands. Whereas unc-22 mutants are completely resistant, mak-1 mutants are partially resistant to nicotine. MAK-1 can phosphorylate twitchin NL-Kin-CRD in vitro. Genetic data suggest the involvement of two other mak-1 paralogues and two orthologues of p38 MAP kinase. These results suggest that MAK-1 is an activator of twitchin kinase and that the p38 MAP kinase pathway may be involved in the regulation of twitchin.
在秀丽隐杆线虫中，抽动蛋白是一种位于肌肉A带的巨大多肽。抽动蛋白的蛋白激酶被激酶催化核心上游45个残基（NL）和下游60个残基（CRD）自动抑制。对抽动蛋白片段的分子动力学模拟表明，NL被拉力释放。然而，目前尚不清楚CRD是如何被去除的。为了鉴定可能去除CRD的蛋白质，我们以抽动蛋白激酶为诱饵进行了酵母双杂交筛选。一个相互作用蛋白是MAK-1，它是丝裂原活化蛋白激酶相关蛋白激酶2（MAPKAP激酶2）的秀丽隐杆线虫直系同源物。MAPKAP激酶2被p38丝裂原活化蛋白激酶磷酸化并激活。我们证明抽动蛋白的CRD对于与MAK-1结合很重要。mak-1在秀丽隐杆线虫体壁肌肉中表达，抗MAK-1抗体定位于Z盘类似物之间和周围以及A带边缘。虽然unc-22突变体完全耐药，但mak-1突变体对尼古丁部分耐药。MAK-1在体外可磷酸化抽动蛋白NL-Kin-CRD。遗传数据表明另外两个mak-1旁系同源物和两个p38丝裂原活化蛋白激酶直系同源物也参与其中。这些结果表明MAK-1是抽动蛋白激酶的激活剂，并且p38丝裂原活化蛋白激酶途径可能参与抽动蛋白的调节。
