Cloning of a novel phosphatidylinositol kinase-related kinase: characterization of the human SMG-1 RNA surveillance protein.
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 (IC(50) = 105 nm) but is not inhibited by a FKBP12-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. Based 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.
Denning
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Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Jamieson
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Maquat
L E
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Thompson
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EA
Fields
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AP
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011494:Protein Kinases; D008666:Metalloendopeptidases; C070738:O-sialoglycoprotein endopeptidase