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For exceptions, permission may be sought for such use through Elsevier's permissions site at: http://www.elsevier.com/locate/permissionusematerial From: ESTELA JACINTO, AGC Kinases in mTOR Signaling, In Mike Hall and Fuyuhiko Tamanoi: The Enzymes, Vol. 27, Burlington: Academic Press, 2010, pp.101-128. ISBN: 978-0-12-381539-2, © Copyright 2010 Elsevier Inc, Academic Press. Author's personal copy 7 AGC Kinases in mTOR Signaling ESTELA JACINTO Department of Physiology and Biophysics UMDNJ-Robert Wood Johnson Medical School, Piscataway New Jersey, USA I. Abstract The mammalian target of rapamycin (mTOR), a protein kinase with homology to lipid kinases, orchestrates cellular responses to growth and stress signals. Various extracellular and intracellular inputs to mTOR are known. mTOR processes these inputs as part of two mTOR protein com- plexes, mTORC1 or mTORC2. Surprisingly, despite the many cellular functions that are linked to mTOR, there are very few direct mTOR substrates identified to date. With the recent discovery of mTORC2, mounting evidence point to mTOR as a central regulator of members of a family of protein kinases, the AGC (protein kinases A/PKG/PKC) family. The AGC kinases are one of the most well-characterized among the eukaryotic protein kinase family. A multitude of cellular functions and substrates has been ascribed to these kinases and their deregulation under- lies numerous pathological conditions. mTOR phosphorylates common motifs in a number of these AGC kinases that could lead to their allosteric activation. AGC kinase activation triggers the phosphorylation of diverse targets that ultimately control cellular response to stimuli. This review will focus on the recent findings on how mTOR regulates AGC kinases. I will discuss how these kinases are wired to the mTOR signaling circuit, includ- ing examples of mTOR-dependent outputs consequent to AGC kinase activation. THE ENZYMES, Vol. XXVII 101 ISSN NO: 1874-6047 # 2010 Elsevier Inc. All rights reserved. DOI: 10.1016/S1874-6047(10)27007-5 Author's personal copy 102 ESTELA JACINTO II. Introduction Mammalian target of rapamycin (mTOR) is linked to diverse cellular and physiological functions that ultimately control cell and body growth [1, 2].In the whole organism, mTOR plays a role in development, metabolism, mem- ory, and aging. At the cellular level, mTOR responds to the presence of nutrients and other growth cues. It functions to regulate translation initiation, ribosome biogenesis, autophagy, actin cytoskeleton reorganization, and tran- scription. Deregulation of the mTOR signaling pathway leads to pathological conditions including cancer, immune-related diseases, diabetes, cardiovascu- lar, and neurological disorders. mTOR is part of a cellular signaling network and it integrates multiple intracellular signals. But what are its direct targets and what exactly does it do to its targets? Understanding TOR/mTOR signaling emerged with the use of rapamy- cin, a macrolide that binds and allosterically inhibits mTOR in the presence of the immunophilin FKBP12 [3]. Although more recent studies revealed that not all functions of mTOR are inhibited by rapamycin, this drug has been instrumental in defining the downstream targets and functions of mTOR. The new generation of mTOR active site inhibitors are also unra- veling important clues on both the rapamycin sensitive and insensitive func- tions of mTOR [4]. But perhaps genetic studies that have disrupted TOR/ mTOR functions are providing key pieces in assembling the mTOR signal- ing puzzle [2, 5]. Recently, the availability of tissue-specific knockouts and cell lines that genetically disrupt mTOR complex components have con- firmed the involvement of mTORCs in regulating AGC kinases and have also led to the identification of new mTOR downstream targets. Whether most of the cellular functions of mTOR are due to its direct regulation of AGC kinases remains to be elucidated. This review will discuss the accumu- lating evidence that reveal how mTOR and AGC kinases coregulate each other. Although there are other kinase families, such as the CMGC and CAMK families [6], that feed signals to the mTOR pathway, so far only the AGC kinases have been shown to be direct mTOR targets as well. mTOR promotes the optimal activation of several AGC kinases and in turn the AGC kinases not only mediate mTOR signals via phosphorylation of numerous intracellular targets that lead to a cellular response but also fine tune the response via feedback regulation of upstream mTOR signals. III. mTOR, an Atypical Protein Kinase mTOR is a member of a family of protein kinases termed the PIKKs (phosphatidylinositol-3-kinase-related kinases), a subgroup of the atypical protein kinases [6]. Members of this family share homology with lipid Author's personal copy 7. AGC KINASES IN mTOR SIGNALING 103 kinases but possess protein kinase activity. The kinase domain is located near the C-terminus and is flanked by two regions that are common to PIKKs called the FAT (FRAP, ATM, TRRAP) and FATC (FAT C-terminus) domains. Since these domains flank the kinase region, it is believed that they participate in kinase regulation [7]. The N-terminal region consists of HEAT (Huntingtin, Elongation factor 3, A subunit of protein phosphatase 2A, TOR1) repeats, most likely a region that mediates protein–protein interactions. PIKKs form protein complexes and seem to recognize their targets via their binding partners [8]. From yeast to man, TOR forms two distinct protein complexes. Mammalian TOR complex 1 (mTORC1) is composed of mTOR and its conserved partners, raptor, and mLST8. mTORC2 consists of mTOR, rictor, SIN1, and mLST8 [1]. While there is likely extensive signal cross talk between the two mTOR complexes, they perform distinct functions. Despite the myriad cellular roles of mTOR, little is known on its direct substrates. S6K (S6 kinase), a protein kinase belonging to the AGC kinase family was first to be identified as a rapamycin-sensitive target of mTOR [9, 10]. Another in vitro and in vivo substrate of mTOR is 4EBP, which is not an enzyme but a small regulatory protein that is highly phosphorylated. S6K and 4EBP phosphorylation is inhibited by rapamycin and both pro- teins function to regulate translation initiation. Three other mTOR sub- strates were recently identified, namely Akt/PKB, PKC, and SGK1. These substrates are also members of the AGC kinase family. Most PIKKs show preference for the phosphorylation of Ser/Thr-Gln residues [11] but mTOR does not appear to phosphorylate such motif. Instead, it phosphorylates Ser/Thr- residues followed by -Tyr/Phe (bulky hydrophobic residue) or -Pro (Table 7.1). The 4EBP sites phosphorylated by mTOR in vitro are Ser/Thr-Pro sites. Because these recognition motifs are common for a number of kinases, particularly in vitro, other factors such TABLE 7.1 SUBSTRATES AND SEQUENCE MOTIFS PHOSPHORYLATED BY MTOR COMPLEXES mTORC1 substrates mTORC2 substrates S6K1 S371PDD Akt/PKB T450PPD FLGFT389YVAPS FPQFS473YSASG EKFS401FEP IRS411PRRFIGS418PRT421PVS424PVK cPKCa T638PPD FEGFS657YVNPQ 4E-BP1 DYSTT37PGG SGK1 S397IGKS401PD (nd) (not determined) FSTT46PGG FLGFS422YAPP CRNS65PVTKT70PPR (nd) ¼ not determined Author's personal copy 104 ESTELA JACINTO as docking interactions, cellular localization, and/or scaffolding proteins could contribute to the substrate specificity of mTOR. IV. AGC Kinase, the‘‘Prototype’’of Protein Kinases The AGC kinase family belongs to the conventional ePKs (eukaryotic protein kinases). The 63 members of this family in the human genome share high homology, roughly 40% sequence identity, in their kinase domain [12]. They phosphorylate their targets on Serine or Threonine residues and include some of the most familiar regulatory enzymes that have key roles on a number of important intracellular signaling pathways. The prototypi- cal kinase structure was first resolved for an AGC kinase, the cAMP- dependent protein kinase (PKA) [13]. The X-ray structure of PKA revealed a bilobal architecture that turned out to be common to all protein kinases. Activation of AGC kinases, like other protein kinases, involves conformational changes in the key regulatory C-helix in the N-lobe [14]. Phosphorylation of the activation loop (T-loop) either by itself or by another AGC kinase, PDK1, stabilizes the kinase in a conformation that is optimal for substrate binding. In AGC kinases, repositioning of the C-helix is mediated by its carboxyl-terminal tail (C-tail) [15]. The C-tail plays a critical regulatory role and distinguishes the AGC kinases from other ePKs. It is defined by three segments: N-lobe tether (NLT) that includes the hydropho- bic motif (HM), C-lobe tether (CLT) that interacts with the C-lobe and interlobe linker spanning the N and C lobes, and the active-site tether (AST) that interacts with the ATP binding pocket [15]. The C-tail serves as a docking site for trans-acting cellular components, in addition to its cis- acting function, and is thereby essential for activity and allostery. The HM plays a pivotal role in regulating AGC kinase activity. Based on the PKA structure, the HM folds back into a hydrophobic pocket in the N-lobe. The HM is present in most AGC kinases (with the notable exception of PDK1) and contains a Ser/Thr residue that may or may not be phosphory- lated. The phosphorylation of this conserved residue could also be regulated in some of the family members.