Emerging common themes in regulation of PIKKs and PI3Ks

Phosphatidylinositol‐3 kinase‐related kinases (PIKKs) comprise a family of protein kinases that respond to various stresses, including DNA damage, blocks in DNA replication, availability of nutrients and errors in mRNA splicing. PIKKs are characterized by the presence of a conserved kinase domain (KD), whose activity is regulated by two C‐terminal regions, referred to as PIKK‐regulatory domain (PRD) and FRAP‐ATM‐TRRAP‐C‐terminal (FATC), respectively. Here, we review functional and structural data that implicate the PRD and FATC domains in regulation of PIKK activity, drawing parallels to phosphatidylinositol‐3 kinases (PI3K), lipid kinases that have sequence similarity to PIKKs. The PI3K C‐terminus, which we propose to be equivalent to the PRD and FATC domains of PIKKs, is in close proximity to the activation loop of the KD, suggesting that in PIKKs, the PRD and FATC domains may regulate kinase activity by targeting the activation loop.     

Tti1 and Tel2 Are Critical Factors in Mammalian Target of Rapamycin Complex Assembly

Mammalian target of rapamycin (mTOR) is a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family and is a major regulator of translation, cell growth, and autophagy. mTOR exists in two distinct complexes, mTORC1 and mTORC2, that differ in their subunit composition. In this study, we identified KIAA0406 as a novel mTOR-interacting protein. Because it has sequence homology with Schizosaccharomyces pombe Tti1, we named it mammalian Tti1. Tti1 constitutively interacts with mTOR in both mTORC1 and mTORC2. Knockdown of Tti1 suppresses phosphorylation of both mTORC1 substrates (S6K1 and 4E-BP1) and an mTORC2 substrate (Akt) and also induces autophagy. S. pombe Tti1 binds to Tel2, a protein whose mammalian homolog was recently reported to regulate the stability of PIKKs. We confirmed that Tti1 binds to Tel2 also in mammalian cells, and Tti1 interacts with and stabilizes all six members of the PIKK family of proteins (mTOR, ATM, ATR, DNA-PKcs, SMG-1, and TRRAP). Furthermore, using immunoprecipitation and size-exclusion chromatography analyses, we found that knockdown of either Tti1 or Tel2 causes disassembly of mTORC1 and mTORC2. These results indicate that Tti1 and Tel2 are important not only for mTOR stability but also for assembly of the mTOR complexes to maintain their activities.     

Requirement of the FATC domain of protein kinase Tel1 for localization to DNA ends and target protein recognition

Two large phosphatidylinositol 3-kinase–related protein kinases (PIKKs), ATM and ATR, play a central role in the DNA damage response pathway. PIKKs contain a highly conserved extreme C-terminus called the FRAP-ATM-TRRAP-C-terminal (FATC) domain. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. In this study, we characterized functions of the FATC domain of Tel1 by introducing substitution or truncation mutations. One substitution mutation, termed tel1-21, and a truncation mutation, called tel1-ΔC, did not significantly affect the expression level. The tel1-21 mutation impaired the cellular response to DNA damage and conferred moderate telomere maintenance defect. In contrast, the tel1-ΔC mutation behaved like a null mutation, conferring defects in both DNA damage response and telomere maintenance. Tel1-21 protein localized to DNA ends as effectively as wild-type Tel1 protein, whereas Tel1-ΔC protein failed. Introduction of a hyperactive TEL1-hy mutation suppressed the tel1-21 mutation but not the tel1-ΔC mutation. In vitro analyses revealed that both Tel1-21 and Tel1-ΔC proteins undergo efficient autophosphorylation but exhibit decreased kinase activities toward the exogenous substrate protein, Rad53. Our results show that the FATC domain of Tel1 mediates localization to DNA ends and contributes to phosphorylation of target proteins.