Cloning of a novel phosphatidylinositol kinase-related kinase - Characterization of the human SMG-1 RNA surveillance protein
Cloning of a novel phosphatidylinositol kinase-related kinase - Characterization of the human SMG-1 RNA surveillance protein
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DOI：
10.1074/jbc.c100144200
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发表时间：
2001-06-22
影响因子：
4.8
通讯作者：
Fields, AP
中科院分区：
文献类型：
--
作者：
Denning, G;Jamieson, L;Fields, AP
我们已经克隆并鉴定了磷脂酰肌醇激酶（PIK）相关激酶家族的一个新成员。这个基因，我们称之为人SMG-1（hSMG- 1），是直链的秀丽隐杆线虫SMG-1，蛋白质的无义介导的mRNA衰变（NMD）的功能。cDNA测序显示hSMG-1编码一个3031个氨基酸的蛋白质，包含一个保守的激酶结构域，一个PIK相关激酶特有的C-末端结构域和一个类似于PIK相关激酶mTOR的FKBP 12-雷帕霉素结合样结构域。免疫纯化的FLAG标记的hSMG-1表现出蛋白激酶活性，如通过通用PIK相关激酶底物PHAS-1的自磷酸化和磷酸化所测量的。hSMG-1激酶活性被高纳摩尔浓度的渥曼青霉素（IC 50 = 105 nM）抑制，但不被FISBP 12-雷帕霉素复合物抑制。hSMG-1激酶结构域中保守残基的突变消除了自磷酸化和底物磷酸化，表明hSMG-1具有内在的蛋白激酶活性。hSMG-1磷酸化纯化的hUpf 1蛋白，一种在NMD中起关键作用的磷蛋白，在整个细胞中也被磷酸化的位点。根据这些数据，我们得出结论，hSMG-1是C. elegans SMG-1.我们的数据表明，hSMG-1可能通过直接磷酸化hUpf 1蛋白在生理相关位点在NMD中发挥作用。
We have cloned and characterized a new member of the phosphatidylinositol kinase (PIK)-related kinase family. This gene, which we term human SMG-1 (hSMG- 1), is orthologous to Caenorhabditis elegans SMG-1, a protein that functions in nonsense-mediated mRNA decay (NMD). cDNA sequencing revealed that hSMG-1 encodes a protein of 3031 amino acids containing a conserved kinase domain, a C-terminal domain unique to the PIK-related kinases and an FKBP12-rapamycin binding-like domain similar to that found in the PIK-related kinase mTOR. Immunopurified FLAG-tagged hSMG-1 exhibits protein kinase activity as measured by autophosphorylation and phosphorylation of the generic PIK-related kinase substrate PHAS-1. hSMG-1 kinase activity is inhibited by high nanomolar concentrations of wortmannin (IC50 = 105 nM) but is not inhibited by a FISBP12-rapamycin complex. Mutation of conserved residues within the kinase domain of hSMG-1 abolishes both autophosphorylation and substrate phosphorylation, demonstrating that hSMG-1 exhibits intrinsic protein kinase activity. hSMG-1 phosphorylates purified hUpf1 protein, a phosphoprotein that plays a critical role in NMD, at sites that are also phosphorylated in whole cells. Eased on these data, we conclude that hSMG-1 is the human orthologue to C. elegans SMG-1. Our data indicate that hSMG-1 may function in NMD by directly phosphorylating hUpf1 protein at physiologically relevant sites.