Li Zhu-Nan, Hongo Seiji, Sugawara Kanetsu, Sugahara Kazuhiko, Tsuchiya Emi, Matsuzaki Yoko, Nakamura Kiyoto
Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1.
J Gen Virol. 2001 May;82(Pt 5):1085-1093. doi: 10.1099/0022-1317-82-5-1085.
The sites for fatty acylation, disulphide bond formation and phosphorylation of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoylation. Removal of one or more of three cysteine residues in the ectodomain showed that all of cysteines 1, 6 and 20 can participate in the formation of disulphide-linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found that serine 78, located within the recognition motifs for mammary gland casein kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline-dependent protein kinase. The effects of acylation and phosphorylation on the formation of disulphide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylation accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulphide bond formation were all transported to the cell surface, suggesting that none of these modifications is required for proper oligomerization. When proteins solubilized in detergent were analysed on sucrose gradients, however, the mutant lacking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulphide bond formation, although not essential for proper oligomerization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis, which showed that the triple-cysteine mutant can form multimers.
通过位点特异性诱变研究了丙型流感病毒CM2的脂肪酰化、二硫键形成和磷酸化位点。胞质尾中的半胱氨酸65被确定为棕榈酰化位点。去除胞外域中三个半胱氨酸残基中的一个或多个表明，半胱氨酸1、6和20都可以参与二硫键连接的二聚体和/或四聚体的形成，尽管半胱氨酸20可能在四聚体形成中起最重要的作用。此外，发现位于乳腺酪蛋白激酶和酪蛋白激酶I识别基序内的丝氨酸78是主要的磷酸化位点，尽管丝氨酸103在较小程度上被脯氨酸依赖性蛋白激酶磷酸化。还研究了酰化和磷酸化对二硫键连接的寡聚体形成的影响。结果表明，虽然棕榈酰化在寡聚体形成中不起作用，但磷酸化加速了四聚体的形成而不影响二聚体的形成。在酰化、磷酸化或二硫键形成方面有缺陷的CM2突变体都被转运到细胞表面，这表明这些修饰对于正确的寡聚化都不是必需的。然而，当在蔗糖梯度上分析用去污剂溶解的蛋白质时，缺乏半胱氨酸1、6和20的突变体以单体形式沉降，这增加了二硫键形成虽然对于正确的寡聚化不是必需的，但可能稳定CM2多聚体的可能性。化学交联分析结果支持了这一点，该结果表明三半胱氨酸突变体可以形成多聚体。
