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 k inase (PIK)-related kinase family. This gene, which we term human SMG-1 (hS MG- 1), is orthologous to Caenorhabditis elegans SMG-1, a protein that func tions 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 kin ase mTOR. Immunopurified FLAG-tagged hSMG-1 exhibits protein kinase activit y as measured by autophosphorylation and phosphorylation of the generic PIK -related kinase substrate PHAS-1. hSMG-1 kinase activity is inhibited by hi gh nanomolar concentrations of wortmannin (IC50 = 105 nM) but is not inhibi ted by a FISBP12-rapamycin complex. Mutation of conserved residues within t he kinase domain of hSMG-1 abolishes both autophosphorylation and substrate phosphorylation, demonstrating that hSMG-1 exhibits intrinsic protein kina se activity. hSMG-1 phosphorylates purified hUpf1 protein, a phosphoprotein that plays a critical role in NMD, at sites that are also phosphorylated i n whole cells. Eased on these data, we conclude that hSMG-1 is the human or thologue to C. elegans SMG-1. Our data indicate that hSMG-1 may function in NMD by directly phosphorylating hUpf1 protein at physiologically relevant sites.