Cloning of a Novel Phosphatidylinositol Kinase-related Kinase

Abstract

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 PIKrelated 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.The PIK 1 -related kinases are a subfamily of the phosphatidylinositol (PI) kinases based on homology to the core catalytic domain of phosphatidylinositol 3-kinase (PI3K) (1, 2). Regions of homology between PI kinases and the protein kinase superfamily include the ATP-binding site and the catalytic/substrate-binding site (3). PIK-related kinases are distinct from PI kinases in that they are high molecular weight proteins that function as serine/threonine protein kinases, rather than lipid kinases (3). PIK-related kinases can be divided into three subgroups based on structural and functional similarities shared by certain family members. The ATM/ATR/RAD3 subgroup functions in DNA damage response pathways, and members contain regions called RAD3 homology domains (4, 5). The targets of rapamycin or TORs (Saccharomyces cerevisiae TOR1 and TOR2 and human mTOR/FRAP/RAFT1/RAPT1) were originally identified as intracellular targets of the immunophilinimmunosuppressant complex FKBP12-rapamycin (6, 7). TORs share sequence similarity within the FRB domain, which binds this complex (7). The TORs function in response to mitogenic signaling to regulate cap-dependent translation (8). Finally, the catalytic subunit of DNA-dependent protein kinase (DNA-PK cs ) shares no sequence similarity to other PIK-related kinases, aside from the kinase domain. DNA-PK functions in the repair of programmed DNA breaks generated by meiotic and V(D

Cell cycle checkpoint signaling through the ATM and ATR kinases

“…On the other hand, mTOR plays no identifiable role in genome surveillance; rather, this PIKK coordinates G 1 phase progression with the supply of nutrients and growth factors (Gingras et al 2001). As discussed previously, a very recent addition to this list is hSMG-1, which has no yeast counterparts, but, based on sequence homology to the Caenorhabditis elegans SMG-1 protein, is predicted to function in NMD (Denning 2001). Our group has cloned a partially overlapping open reading frame, and we have provisionally termed the encoded polypeptide ATX (K.M.…”

“…Otterness, and R.T. Abraham, in prep.). Although the human cDNA has been designated hSMG-1 by one group (Denning et al 2001), we suggest that this name remain provisional until a role for this PIKK family member in NMD is unequivocally shown. Our own results indicate that hSMG1 is found in both the nucleus and cytoplasm, and, like ATM and ATR, exhibits phosphotransferase activity toward a variety of proteins containing Ser-Gln motifs under in vitro assay conditions.…”

“…The C. elegans gene, SMG-1, encodes a putative protein kinase that plays an essential role in the elimination of RNA species containing premature termination codons, a process termed nonsense-mediated mRNA decay (NMD; Maquat 2000). Subsequently, a human PIKK family member bearing regional sequence homology to SMG-1 was cloned independently by at least two research teams (Denning et al 2001; K.M. Brumbaugh, D.M.…”

Ser-64 and Ser-111 in PHAS-I Are Dispensable for Insulin-stimulated Dissociation from eIF4E

“…Ser-111 was first identified as the major site for in vitro phosphorylation by protein kinase CK2 (15). More recently, this site has been shown to be phosphorylated in vitro by the phosphatidylinositol 3-OH kinase-related protein kinases, ATM, ATR, and Smg1 (15)(16)(17)(18). Phosphorylation of Ser-111 has been suggested to serve as a priming event, which is necessary for both the phosphorylation of other sites in PHAS-I and the dissociation of the PHAS-I⅐eIF4E complex (8,18).…”

Mammalian Target of Rapamycin Positively Regulates Collagen Type I Production via a Phosphatidylinositol 3-Kinase-independent Pathway

“…For the most part, these proteins act as checkpoints for DNA synthesis, protein translation, and mRNA quality and mediate cellular responses to stresses such as DNA damage and nutrient deprivation. For example, family members DNA-protein kinase (14), ataxia telangiectasia-mutated (ATM), ataxia telangiectasia-related genes are regulators of DNA repair, whereas the recently identified hSMG-1 (15,16) regulates nonsense-mediated decay. Consistent with other members of this family, the mammalian target of rapamycin (mTOR) (17,18) also appears to act as a checkpoint.…”