Distant N- and C-terminal domains are required for intrinsic kinase activity of SMG-1, a critical component of nonsense-mediated mRNA decay.
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.
Morita
Tomoko
T
Department of Life Sciences, Graduate School of Arts and Science, The University of Tokyo 153-8902, Japan.
Yamashita
Akio
A
Kashima
Isao
I
Ogata
Kazuhiro
K
Ishiura
Shoichi
S
Ohno
Shigeo
S
D002460:Cell Line; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism*; D014162:Transfection
D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; C514704:SMG1 protein, human