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Crit. Rev. in Biochem. and Molec. Biol. 2011 Critical Reviews in Biochemistry and Molecular Biology Critical Reviews in Biochemistry and Molecular Biology, 2011, 1–21, Early Online 2011 © 2011 Informa Healthcare USA, Inc. ISSN 1040-9238 print/ISSN 1549-7798 online 00 DOI: 10.3109/10409238.2011.618113 00 000 REVIEW 000 18 June 2011 Mammalian TOR signaling to the AGC kinases 15 August 2011 Bing Su1 and Estela Jacinto2 24 August 2011 1Department of Immunobiology and The Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT, USA and 2Department of Physiology and Biophysics, UMDNJ-RWJMS, Piscataway, NJ, USA 1040-9238 1549-7798 Abstract The mechanistic (or mammalian) target of rapamycin (mTOR), an evolutionarily conserved protein kinase, orchestrates © 2011 Informa Healthcare USA, Inc. cellular responses to growth, metabolic and stress signals. mTOR processes various extracellular and intracellular inputs as part of two mTOR protein complexes, mTORC1 or mTORC2. The mTORCs have numerous cellular targets but 10.3109/10409238.2011.618113 members of a family of protein kinases, the protein kinase (PK)A/PKG/PKC (AGC) family are the best characterized direct mTOR substrates. The AGC kinases control multiple cellular functions and deregulation of many members of BBMG this family underlies numerous pathological conditions. mTOR phosphorylates conserved motifs in these kinases to allosterically augment their activity, influence substrate specificity, and promote protein maturation and 618113 stability. Activation of AGC kinases in turn triggers the phosphorylation of diverse, often overlapping, targets that ultimately control cellular response to a wide spectrum of stimuli. This review will highlight recent findings on how mTOR regulates AGC kinases and how mTOR activity is feedback regulated by these kinases. We will discuss how this regulation can modulate downstream targets in the mTOR pathway that could account for the varied cellular functions of mTOR. Keywords: mTOR, AGC kinases, Sin1, rictor, Akt, mTOR complexes, mTORC1, mTORC2 For personal use only. Introduction associated with a conserved cellular protein, FKBP12, mTOR regulates diverse cellular and physiological it binds and allosterically inhibits mTOR (Jacinto and functions that ultimately control cell and body growth Hall, 2003). Rapamycin was originally identified from the (Wullschleger et al., 2006, Polak and Hall, 2009, Zoncu bacteria, Streptomyces hygroscopicus, isolated from soil et al., 2011). In whole organisms, mTOR plays a role in samples collected from Easter Island, known to locals as metabolism, growth, differentiation and aging. At the cel- Rapa nui. Although it has later been revealed that not all lular level, mTOR responds to the changes in nutrient sta- functions of mTOR are inhibited by rapamycin, this drug tus and many other cellular or stress cues that ultimately has remained instrumental in defining the downstream impact cell growth and division. At the molecular level, it targets and functions of mTOR. The new generation of functions to regulate translation initiation, ribosome bio- mTOR active site inhibitors is now unraveling important genesis, protein maturation, autophagy, actin cytoskel- clues on both the rapamycin sensitive and insensitive eton reorganization, and transcription. Abnormal mTOR functions of mTOR (Guertin and Sabatini, 2009). signaling has been associated with numerous pathologi- More recent studies using gene deficient mice that Critical Reviews in Biochemistry and Molecular Biology Downloaded from informahealthcare.com by Yale University on 10/21/11 cal conditions including cancer, immune disorders, dia- impair functions of mTOR and its regulators are expand- betes, cardiovascular and neurological diseases. mTOR ing our knowledge about how mTORCs function in the integrates multiple intracellular signaling pathways but development and homeostasis of different tissues (Polak the direct cellular targets of mTOR and exactly how it and Hall, 2009, Alessi et al., 2009). Tissue-specific knock- regulates its targets at the molecular level remain to be outs and mTORC2-disrupted cell lines have further fully elucidated. advanced our understanding of mTOR in regulation of Our knowledge about mTOR functions and signaling AGC kinases and have also led to the identification of emerged with the use of rapamycin. When rapamycin is new mTOR downstream targets. This review will focus (Received 18 June 2011; revised 15 August 2011; accepted 24 August 2011) 1 2 B. Su and E. Jacinto on the recent advances in our understanding of how aromatic residues (Phe, Trp, Tyr) at the +1 position. Some of mTOR and its best characterized substrates, the AGC the canonical mTOR target phosphorylation sites are shown kinases, coregulate each other. mTOR is critical for the in Table 1. Recently, a phosphoproteomic screen identified optimal activation of several well known AGC kinases. hundreds of possible direct targets of mTOR and a posi- Reciprocally, the AGC kinases not only mediate mTOR tional scanning peptide library assay validated the motif signals via phosphorylation of downstream intracellular preferences of mTOR (Hsu et al., 2011, Yu et al., 2011). targets but also fine tune the mTOR response through Mammalian TOR can be directly regulated via feedback regulation of mTOR activity, which is accom- phosphorylation. Some of these phosphosites, such plished by phosphorylating upstream mTOR activators. as Thr2446, Ser2448, and Ser2481, have been shown or suggested to be modulated by growth signals (Figure 1) mTOR, an atypical protein kinase (Cheng et al., 2004, Copp et al., 2009). Thr2446 and Ser2448 lie in a repressor domain region of mTOR Mammalian TOR is a 290 kDa protein and a member of (Sekulic et al., 2000) and whereas the former is linked to a subgroup of the atypical protein kinases termed phos- nutrient stimulation, the latter site responds to growth phoinositide 3-kinase-related kinases (PIKKs) (Manning factor stimulation (Cheng et al., 2004). More recent stud- et al., 2002b). Members of this family share homology with ies demonstrated that these sites can be phosphorylated lipid kinases but possess Ser/Thr protein kinase activity. by S6K (Holz and Blenis, 2005, Chiang and Abraham, The kinase (catalytic) domain of mTOR is located near the 2005). mTOR becomes autophosphorylated at Ser2481 C-terminus and is flanked by the FAT (FRAP, ATM, TRRAP) and this phosphorylation was earlier shown to be and FATC (FAT C-terminus) domains that are common insensitive to amino acid or serum-withdrawal but was to PIKKs (Bosotti et al., 2000) (Figure 1). The N-terminal abolished upon wortmannin treatment (Peterson et al., region consists of HEAT (Huntingtin, Elongation factor 3, 2000). More recently, Ser2481 phosphorylation was dem- A subunit of protein phosphatase 2A, TOR1) repeats, most onstrated to be induced by insulin in a PI3K-dependent likely to mediate protein-protein interactions (Figure 1). manner within both mTORC1 and mTORC2 (Copp et al., The N-terminal region also contains the helix turn helix 2009, Soliman et al., 2010). In addition, the mTORC1- (HTH) motif that was shown to bind to the promoter of associated mTOR S2481 autophosphorylation, similar 36S rDNA, and an ER and Golgi localization sequence to mTORC1 signaling, was regulated not only by serum (LS) (Liu and Zheng, 2007). An alternatively spliced iso- but also by amino acids and energy stress (Soliman et al., form, mTOR-b, that is mostly devoid of the HEAT and 2010). Phosphorylation of this site, along with Ser2448 FAT domains but retains the capacity to phosphorylate its is conserved among vertebrate species but absent in substrates has been reported (Panasyuk et al., 2009). invertebrates (Copp et al., 2009), hence regulation of Most PIKKs phosphorylate Ser/Thr followed by Gln this site may have evolved in vertebrates in response to For personal use only. residues (Abraham, 2004) but mTOR displays preference growth factors. Consistent with the earlier finding that toward substrates with Pro, hydrophobic (Leu, Val), and Ser2481 phosphorylation is insensitive to conditions Figure 1. Schematic structure of human mTOR protein. Human mTOR is a 2549 amino acid protein. It contains multiple HEAT repeats, a helix turn helix (HTH) motif, and a Golgi/ER localization signal sequence (LS) at its N-terminus. Its catalytic domain is flanked by a FAT and FATC sequences. Immediate upstream to the catalytic domain is the FKBP12-rapamycin binding (FRB) motif. A phosphomimetic residue Critical Reviews in Biochemistry and Molecular Biology Downloaded from informahealthcare.com by Yale University on 10/21/11 Asp (E2363) at its putative activation loop, three known phosphorylation sites, T2446, S2448, and S2481, and three other less characterized phosphorylation sites, S1261, S2159, T2164, are shown. (See colour version of this figure online at www.informahealthcare.com|bmg) Table 1. Best characterized mTOR substrates and sequence motifs mTORC1 substrates mTORC2 substrates S6K1 S371PDD (TM) Akt/PKB T450PPD (TM) FLGFT389YVAPS (HM) FPQFS473YSASG (HM) EKFS401FEP cPKCα T638PPD (TM) 4E-BP1 IRS411PRRFIGS418PRT421PVS424PVK DYSTT37PGG FEGFS657YVNPQ (HM) FSTT46PGG SGK1 S397IGKS401PD (TM) CRNS65PVTKT70PPR FLGFS422YAPP(HM) BBMG 618113 Critical Reviews in Biochemistry and Molecular Biology mTOR signaling to the AGC kinases 3 that inhibit mTORC1 activity (Peterson et al., 2000), this In addition to phosphorylation regulation, mutations of phosphosite was not sensitive to acute rapamycin treat- specific amino acid residues in the mTOR kinase
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