Structure of substrate-bound SMG1-8-9 kinase complex reveals molecular basis for phosphorylation specificity

Abstract

<jats:p>PI3K-related kinases (PIKKs) are large Serine/Threonine (Ser/Thr)-protein kinases central to the regulation of many fundamental cellular processes. PIKK family member SMG1 orchestrates progression of an RNA quality control pathway, termed nonsense-mediated mRNA decay (NMD), by phosphorylating the NMD factor UPF1. Phosphorylation of UPF1 occurs in its unstructured N- and C-terminal regions at Serine/Threonine-Glutamine (SQ) motifs. How SMG1 and other PIKKs specifically recognize SQ motifs has remained unclear. Here, we present a cryo-electron microscopy (cryo-EM) reconstruction of a human SMG1-8-9 kinase complex bound to a UPF1 phosphorylation site at an overall resolution of 2.9 Å. This structure provides the first snapshot of a human PIKK with a substrate-bound active site. Together with biochemical assays, it rationalizes how SMG1 and perhaps other PIKKs specifically phosphorylate Ser/Thr-containing motifs with a glutamine residue at position +1 and a hydrophobic residue at position -1, thus elucidating the molecular basis for phosphorylation site recognition.</jats:p>

The activation mechanisms of master kinases in the DNA damage response

“…LBE, FATC (ATM, ATR) or FRB (DNA-PK), and PRD together assemble a gate through which substrates enter, and extend into the active cleft. The same configuration has been observed within the mTOR complex (Yang et al, 2017) and the SMG1-8-9 complex (Langer et al, 2020;Zhu et al, 2019). For this reason, these enzymes exhibit auto-inhibition forms in the absence of activators.…”

Structural Insights into TOR Signaling

“…Therefore, it appears that the PRD regulates the activity and/or activation of PIKKs by modulating access to the active site. Interestingly, it has been recently proposed that the PRD could act as a pseudosubstrate, at least in some PIKKs such as SMG1 and ATM [115]. Accordingly, known phosphorylation sites of mTOR that are coupled to mitogen signaling are located in this region (Thr2446 and Ser2448) [112].…”

Stretch and Twist of HEAT Repeats Leads to Activation of DNA-PK Kinase

“…1c). Moreover, the PIKK regulatory domain (PRD, aa 4009-4039), which is partially disordered in mTOR and SMG1 (Langer et al, 2020;Yang et al, 2017), and closed in ATM, ATR (Jansma et al, 2020;Williams et al, 2020), and in all other DNA-PKcs and DNA-PK complexes, rotated 115° to expose the substrate binding groove. Complex VI also revealed that the activated DNA-PKcs has a potential binding site for inositol hexaphosphate (IP6), which has been shown to activate DNA-PK and NHEJ (Hanakahi et al, 2000).…”

“…DNA-PKcs is a Ser/Thr kinase of over 4000 residues and a member of the PI3K-related kinase (PIKK) family, which also includes mTOR, ATM, ATR and SMG1 (Baretic and Williams, 2014;Hartley et al, 1995). PIKK kinases play key roles in regulation of responses to nutrient stress (mTOR), misfolded and non-functional RNAs (SMG1), DNA double-strand breaks (DNA-PKcs and ATM) or single-stranded DNA (ATR) (Blackford and Jackson, 2017;Langer et al, 2020;Yang et al, 2017). The kinase activity of DNA-PKcs is modestly stimulated by DNA but becomes fully activated only in the presence of both DNA and Ku70/80, which is known to bind DNA ends (Chan and Lees-Miller, 1996;Hammarsten and Chu, 1998;Mimori et al, 1986;West et al, 1998).…”

“…Multiple structures of other PIKK kinases complexed with accessory subunits, activation cofactors, ATP analogs, and even a substrate peptide have been reported, representing either active mTOR and SMG1 (Langer et al, 2020;Yang et al, 2017) or inhibited ATM and ATR (Jansma et al, 2020;Williams et al, 2020). In all cases the role of the HEAT-repeat structures, which is 5 to 9-fold larger than the kinase domain, remains unclear.…”