Distant N- and C-terminal Domains Are Required for Intrinsic Kinase Activity of SMG-1, a Critical Component of Nonsense-mediated mRNA Decay

Tomoko Morita,Akio Yamashita,Isao Kashima,Kazuhiro Ogata,Shoichi Ishiura,Shigeo Ohno

doi:10.1074/jbc.m610159200

Abstract

Phosphatidylinositol 3-kinase-related kinases (PIKKs) consisting of SMG-1, ATM, ATR, DNA-PKcs, and mTOR are a family of proteins involved in the surveillance of gene expression in eukaryotic cells. They are involved in mechanisms responsible for genome stability, mRNA quality, and translation. They share a large N-terminal domain and a C-terminal FATC domain in addition to the unique serine/threonine protein kinase (PIKK) domain that is different from classical protein kinases. However, structure-function relationships of PIKKs remain unclear. Here we have focused on one of the PIKK members, SMG-1, which is involved in RNA surveillance, termed nonsense-mediated mRNA decay (NMD), to analyze the roles of conserved and SMG-1-specific sequences on the intrinsic kinase activity. Analyses of sets of point and deletion mutants of SMG-1 in a purified system and intact cells revealed that the long N-terminal region and the conserved leucine in the FATC domain were essential for SMG-1 kinase activity. However, the conserved tryptophan in the TOR SMG-1 (TS) homology domain and the FATC domain was not. In addition, the long insertion region between PIKK and FATC domains was not essential for SMG-1 kinase activity. These results indicated an unexpected feature of SMG-1, i.e. that distantly located N- and C-terminal sequences were essential for the intrinsic kinase activity.

Highlights

SMG-1 is the newest member of the family of phosphatidylinositol 3-kinase (PI3K)-related protein kinases (PIKKs), which includes mammalian target of rapamycin, ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase catalytic subunit (DNAPKcs) in mammalian cells
Previous reports showed that SMG-1 associated with other nonsense-mediated mRNA decay (NMD)-related proteins such as Upf1 and SMG-7 (3, 7), but immunopurified recombinant SMG-1 did not show any signs of contaminations in silver staining (Fig. 1B, lanes 6 –10) and Western blotting, indicating the high purity of the enzyme
FLAG-SMG-1-(⌬1– 617), -(⌬631– 649), -(⌬746 –791), -(⌬912– 1014), and -(⌬1375–1477), which have little or no kinase activity in vitro, did not restore sufficient Upf1 phosphorylation in vivo (Fig. 5, lanes 5– 8 and 10). These results showed that SMG-1 mutants that retained kinase activity in vitro corresponded to SMG-1 mutants that kept kinase activity in vivo

Summary

Introduction

SMG-1 is the newest member of the family of phosphatidylinositol 3-kinase (PI3K)-related protein kinases (PIKKs), which includes mammalian target of rapamycin (mTOR), ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase catalytic subunit (DNAPKcs) in mammalian cells. The fact that FLAG-SMG-1 D2331A, a kinase inactive point mutant in the PIKK catalytic domain (3), did not have any detectable kinase activity supported the validity of the assay.

Results

Conclusion