Serine/Threonine-Protein Kinase SMG1
61E3.4; ATX; hSMG-1; KIAA0421; Lambda/iota protein kinase C-interacting protein; Lambda-interacting protein; LIP; Phosphatidylinositol 3-kinase-related kinase; Phosphatidylinositol 3-kinase-related protein kinase; PI-3-kinase-related kinase SMG-1; SMG1; SMG1 homolog, phosphatidylinositol 3-kinase-related kinase; Smg-1 homolog, phosphatidylinositol 3-kinase-related kinase (C. elegans); SMG-1; Suppressor with morphological effect on Genitalia 1
In 1993, the Anderson lab reported that loss of function mutations affecting seven Caenorhabditis elegans smg genes (smg-1∼smg-7) eliminates nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism which degrades mRNA containing nonsense mutation (Pulak and Anderson 1993). Later, the Anderson lab reported cloning of C. elegans smg-2, a nematode ortholog of UPF1, and in vivo phosphorylation of SMG-2 at 1999 (Page et al. 1999). SMG-2/UPF1 is an evolutionally conserved central component of NMD. In that paper, they discussed smg-1 gene product is a strong candidate of a kinase for the phosphorylation of SMG-2 and submits a SMG-1 sequence in the public database at early September 1999 (Page et al. 1999). They report smg-1 cloning and demonstrate that SMG-1 kinase activity was essential for NMD in vivo and SMG-2 phosphorylation in vitro at 2004 (Grimson et al. 2004).
Part of the human SMG1 gene product firstly reported in 1996 as an atypical PKCλ/ι (aPKCλ/ι) interacting protein, LIP (lambda-interacting protein) (Diaz-Meco et al. 1996). However, this LIP cDNA, that encoded a 713 amino acid (aa) protein, contained a frameshift mutation and the carboxyl (C) -terminus FAT-C (FRAP, ATM, and TRRAP-C-terminus) domain which conserved among PI3-kinase related protein kinases (PIKKs), was not identified. In 1997, another fragment of the SMG1 gene product was identified that encoded a 1302-aa protein and was named KIAA0421. KIAA0421 contained most LIP sequences and contained C-terminus FAT-C domain (Ishikawa et al. 1997).
Three groups have independently cloned and reported the protein kinase activity of human SMG1. In 2001, the Fields lab reported a partial sequence of human SMG1 that encoding 3031 aa protein, as C. elegans SMG-1 related protein. This protein showed in vitro phosphorylation of 4E-BP1 and UPF1 by immunoprecipitated SMG1 (Denning et al. 2001). The same year, the Ohno lab reported the full-length sequence for human SMG1. This gene encoded a 3657 aa protein that was a novel PIKK. They showed the involvement of SMG1 in mammalian NMD, identified the SMG1 mediated-phosphorylation of UPF1 at Ser-1078 and Ser-1096 in vitro and in vivo, and showed the complex formation of SMG1 and UPF1 in cell lysate (Yamashita et al. 2001). In 2004, the Abraham lab reported that a splice variant of human SMG1, encoding a 3521 aa novel PIKK “ATX”. They showed the activation of SMG1 by DNA damage, the SMG1 mediated-phosphorylation of p53 at Ser-15 and also demonstrated the involvement of SMG1 in genotoxic stress-induced phosphorylation of p53 at Ser-15 (Brumbaugh et al. 2004).
Assembly of the human genome sequence revealed that SMG1 gene is represented eight times within the human genome with a 97∼98% of sequence identity (SMG1 and SMG1 pseudogene (SMG1P) 1∼7). The annotated SMG1 gene encodes 3661 aa protein, which has a 6 aa difference from the 3657-aa isoform identified earlier by the Ohno lab and a 108-aa difference from the 3521-aa isoform identified in the Abraham lab. Except SMG1P4, SMG1P1∼7 express long noncoding (lnc) RNA (Martin et al. 2004). Note that the 5′ of 719 nucleotides encoding 131 aa of SMG1 cDNA reported the Ohno lab comes from SMG1P5, and 5′ of 102 nucleotides encoding 4 aa of SMG1 cDNA reported the Abraham lab comes from SMG1P2. Consequently, care needs to be taken with microarray and RT-qPCR probe design to avoid detection of the lncRNA from these pseudogenes instead of SMG1 mRNA. Moreover, SMG1 expression profile data of might need to be reevaluation if non-specific probes were used.
According to immunological analysis, the human SMG1 has at least two isoforms, p430 and p400. The antibody against the amino terminal 106 amino acids of the 3657-aa protein recognizes only the SMG1 p430 isoform suggesting the SMG1 p400 isoform lacks this amino-terminal sequence (Yamashita et al. 2001). Although the two isoforms are conversed among mammals, the biological significance of the sequence variation at the amino terminus of SMG-1 remains unclear.
Structure and Biochemical Characterization of SMG1 as a PIKK Family Kinase
SMG1 belongs the member of the PIKKs that include ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3 related), mTOR (mammalian target of rapamycin), DNA-PKcs (DNA-dependent protein kinases catalytic subunit), and TRRAP (transformation/transcription domain-associated protein) (Baretic and Williams 2014). With the exception of TRRAP, PIKKs have intrinsic serine/threonine kinase activity. PIKKs can be distinguished from other protein kinases by their unique catalytic domain (PIKK domain) similar to lipid PI3K catalytic domain, FAT-C domain and large molecular weight (270–470 kDa) (Izumi et al. 2012b; Baretic and Williams 2014). The unique structural feature of SMG-1 relative to other PIKKs is a large insert between the PIKK and FAT-C domains (Fig. 1b) (Izumi et al. 2012b; Baretic and Williams 2014). The conservation of this SMG1 insert across all species examined to date suggests its importance. However, this insert is not required for the intrinsic kinase activity of SMG1 (Morita et al. 2007), and it has no reported function apart from an interaction with aPKCλ/ι (Diaz-Meco et al. 1996). It is also notable that the binding of this insert with LIP and aPKCλ/ι failed to capture the 3031-aa partial-length, or the 106-3657-aa full-length, SMG1 (Denning et al. 2001; Yamashita et al. 2001).
SMG1, ATM, ATR, and DNP-PKcs are termed S/T-Q-directed kinase, based on strong preferences for the phosphorylation of serine (S)/threonine (T) residue followed by a glutamine (Q) residue (Yamashita et al. 2001; Izumi et al. 2012b). Similar to other PIKKs, SMG1 exhibit a preference for Mn2+ ions over Mg2+ ions and is inhibited by wortmannin, IC50; 60∼105 nM in vitro/1∼2 μM in vivo, and caffeine, IC50; 0.3 mM in vitro, but is not inhibited by staurosporine and rapamycin in vitro (Denning et al. 2001; Yamashita et al. 2001; Brumbaugh et al. 2004). Although a SMG1 specific inhibitor is not commercially available, SMG1 preferential inhibitor, which have tenfold higher EC50 value, ∼0.1 μM, than mTOR, ∼1 μM, in vivo, is reported (Gopalsamy et al. 2012).
SMG1 in Nonsense-Mediated mRNA Decay (NMD)
As described in historical background section, SMG1 functions in NMD (Schweingruber et al. 2013). NMD selectively degrades premature termination codon (PTC)-containing mRNAs, which can be generated by gene mutations, splicing or transcription errors. This NMD process suppresses the production of potentially harmful or nonfunctional polypeptides and ensures the accuracy of gene expression (Schweingruber et al. 2013). NMD also plays a more general role in regulating gene expression by controlling the decay of a significant fraction of mRNAs containing uORF, 3′UTR intron, seleno-cysteine codon (UGA), and PTC-containing alternative exons (Schweingruber et al. 2013).
SMG1 plays an essential role in NMD by phosphorylating UPF1 helicase, a central regulator of NMD (Schweingruber et al. 2013). When a ribosome recognizes a PTC, SMG1, UPF1, and eukaryotic releasing factors (eRF1 and eRF3) assemble to form the SMG1C-UPF1-eRF1-eRF3 (SURF) complex on the PTC-recognizing stalled ribosome (Kashima et al. 2006; Yamashita et al. 2009; Izumi et al. 2010). If an exon junction complex (EJC), a multiprotein complex deposited on an exon-junction in a splicing dependent manner, exists downstream of the PTC, the SURF associates with the EJC through UPF2-UPF3 to form DECay InDuing (DECID) complex (Kashima et al. 2006; Yamashita et al. 2009; Izumi et al. 2010). The DECID complex formation establishes PTC recognition and induces SMG1-mediated UPF1 phosphorylation (Kashima et al. 2006; Yamashita et al. 2009; Izumi et al. 2010). Phosphorylated-UPF1 most likely marks PTC-containing mRNA (Johns et al. 2007; Yamashita et al. 2009; Kurosaki et al. 2014). Since SMG8 is a kinase repressor subunit of SMG1 and is required for the DECID formation, the presence of SMG8 ensures recruitment of kinase activity repressed SMG1 to SURF, thereby avoiding undesirable UPF1 phosphorylation (Yamashita et al. 2009; Arias-Palomo et al. 2011). It is notable that UPF2 can be recruited to SMG1C independently of UPF1, which may permit downstream exon junction independent NMD (Melero et al. 2014; Lopez-Perrote et al. 2016). This observation also implies that UPF2 binding to SMG1 is insufficient for SMG1 activation. Another protein, DHX34 may also be involved with formation of the SMG1C, UPF1 and UPF2 complex (Melero et al. 2016).
Roles of SMG1 Beyond NMD
UPF1 is a multifunctional protein that is involved not only in NMD, but also histone mRNA decay, Staufen-mediated mRNA decay, Regnase-1-associated mRNA decay, telomere leading-strand replication, and DNA polymeraseδ-mediated DNA repair in addition to NMD (Izumi et al. 2012b). Of these functions, SMG1 is thought to regulate Staufen-mediated mRNA decay (Cho et al. 2013b). Intriguingly, instead of SMG1 there are alternative PIKKs for UPF1 associated with histone mRNA decay (ATR and DNA-PK), DNA polymeraseδ-mediated DNA repair (ATR), and telomere leading-strand replication (ATR) (Izumi et al. 2012b).
Homozygous SMG1 gene trap mice have an early embryonic lethal phenotype, while haploinsufficient mice have elevated rates of cancer and inflammation-related cytokines (e.g., IL-2, IL-6, IL-10, and TNFα) (McIlwain et al. 2010).
In contrast, SMG1 null mutants in C. elegans and D. melanogaster are viable. SMG1 inactivation increases oxidative stress resistance and longevity in analogy to TOR in C. elegans (Masse et al. 2008). On the other hand, SMG1 and mTORC1 act antagonistically to regulate response to injury and growth in planarians (Schmidtea mediterranea) (Gonzalez-Estevez et al. 2012).
SMG1 is a PIKK family member kinase forms a stable heterotrimeric complex with SMG8 and SMG9, termed SMG1C. SMG1C is conserved among various eukaryotes including metazoan and plant. SMG1 plays an essential role in NMD by phosphorylating specific serine/threonine residues of UPF1. When a ribosome is stalled at a PTC, SMG1 forms the SURF (SMG1-UPF1-eRF1-eRF3) complex on the PTC recognizing ribosome. If an EJC exists downstream of the PTC, the SURF associates with the EJC through UPF2-UPF3 to form the DECID complex. The DECID complex establishes PTC recognition and induces the SMG1-mediated UPF1 phosphorylation to recruit mRNA decay factors. SMG1 also implicates various cellular functions including Staufen-mediated mRNA decay, DNA damage, oxidative stress, hypoxia, TNFα signaling, and sorafenib sensitivity. The detailed mechanisms of these SMG1 functions are yet to be resolved.
I would like to thank Ms. Kae Suzuki for reading manuscript.
This project was funded by the Japan Society for the Promotion of Science KAKENHI .
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