Distant N- and C-terminal domains are required for intrinsic kinase activity of SMG-1, a critical component of nonsense-mediated mRNA decay
Distant N- and C-terminal domains are required for intrinsic kinase activity of SMG-1, a critical component of nonsense-mediated mRNA decay
复制标题
DOI：
10.1074/jbc.m610159200
复制
发表时间：
2007-03-16
影响因子：
4.8
通讯作者：
Ohno, Shigeo
中科院分区：
文献类型：
--
作者：
Morita, Tomoko;Yamashita, Akio;Ohno, Shigeo
磷脂酰肌醇3-激酶相关激酶（PIKKs）由SMG-1、ATM、ATR、DNA-PKcs和mTOR组成，是参与真核细胞中基因表达监视的蛋白质家族。它们参与基因组稳定性、mRNA质量和翻译的机制。除了与经典蛋白激酶不同的独特丝氨酸/苏氨酸蛋白激酶（PIKK）结构域之外，它们还共享大的N-末端结构域和C-末端FATC结构域。然而，PIKK的结构-功能关系仍不清楚。在这里，我们集中在PIKK成员之一，SMG-1，这是参与RNA监视，称为无义介导的mRNA衰变（NMD），分析保守和SMG-1特异性序列的内在激酶活性的作用。在一个纯化的系统和完整的细胞中的SMG-1的点和缺失突变体集的分析表明，长的N-末端区域和保守的亮氨酸在FATC结构域中的SMG-1激酶活性是必不可少的。然而，TOR SMG-1（TS）同源结构域和FATC结构域中的保守色氨酸不是。此外，PIKK和FATC结构域之间的长插入区域对于SMG-1激酶活性不是必需的。这些结果表明SMG-1的一个意想不到的特征，即位于远处的N-和C-末端序列对于内在激酶活性是必需的。
Phosphatidylinositol 3-kinase-related kinases (PIKKs) consisting of SMG-1, ATM, ATR, DNA-PKcs, and mTOR are a family of proteins involved in the surveillance of gene expression in eukaryotic cells. They are involved in mechanisms responsible for genome stability, mRNA quality, and translation. They share a large N-terminal domain and a C-terminal FATC domain in addition to the unique serine/threonine protein kinase (PIKK) domain that is different from classical protein kinases. However, structure-function relationships of PIKKs remain unclear. Here we have focused on one of the PIKK members, SMG-1, which is involved in RNA surveillance, termed nonsense-mediated mRNA decay (NMD), to analyze the roles of conserved and SMG-1-specific sequences on the intrinsic kinase activity. Analyses of sets of point and deletion mutants of SMG-1 in a purified system and intact cells revealed that the long N-terminal region and the conserved leucine in the FATC domain were essential for SMG-1 kinase activity. However, the conserved tryptophan in the TOR SMG-1 (TS) homology domain and the FATC domain was not. In addition, the long insertion region between PIKK and FATC domains was not essential for SMG-1 kinase activity. These results indicated an unexpected feature of SMG-1, i.e. that distantly located N- and C-terminal sequences were essential for the intrinsic kinase activity.