Mammalian TOR signaling to the AGC kinases

The mechanistic (or mammalian) target of rapamycin (mTOR), an evolutionarily conserved protein kinase, orchestrates 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 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 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 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.     

mTOR complex 2 signaling and functions

The mechanistic target of rapamycin (mTOR) plays a central role in cellular growth and metabolism. mTOR forms two distinct protein complexes, mTORC1 and mTORC2. Much is known about the regulation and functions of mTORC1 due to availability of a natural compound, rapamycin, that inhibits this complex. Studies that define mTORC2 cellular functions and signaling have lagged behind. The development of pharmacological inhibitors that block mTOR kinase activity, and thereby inhibit both mTOR complexes, along with availability of mice with genetic knockouts in mTOR complex components have now provided new insights on mTORC2 function and regulation. Since prolonged effects of rapamycin can also disrupt mTORC2, it is worth re-evaluating the contribution of this less-studied mTOR complex in cancer, metabolic disorders and aging. In this review, we focus on recent developments on mammalian mTORC2 signaling mechanisms and its cellular and tissue-specific functions.Key words: mTOR, mTORC2, rictor, cancer, metabolism, ribosomes, protein synthesis, protein maturation, AGC kinases, growth factor signaling     

mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1)

SGK1 (serum- and glucocorticoid-induced protein kinase 1) is a member of the AGC (protein kinase A/protein kinase G/protein kinase C) family of protein kinases and is activated by agonists including growth factors. SGK1 regulates diverse effects of extracellular agonists by phosphorylating regulatory proteins that control cellular processes such as ion transport and growth. Like other AGC family kinases, activation of SGK1 is triggered by phosphorylation of a threonine residue within the T-loop of the kinase domain and a serine residue lying within the C-terminal hydrophobic motif (Ser(422) in SGK1). PDK1 (phosphoinositide-dependent kinase 1) phosphorylates the T-loop of SGK1. The identity of the hydrophobic motif kinase is unclear. Recent work has established that mTORC1 [mTOR (mammalian target of rapamycin) complex 1] phosphorylates the hydrophobic motif of S6K (S6 kinase), whereas mTORC2 (mTOR complex 2) phosphorylates the hydrophobic motif of Akt (also known as protein kinase B). In the present study we demonstrate that SGK1 hydrophobic motif phosphorylation and activity is ablated in knockout fibroblasts possessing mTORC1 activity, but lacking the mTORC2 subunits rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated-protein-kinase-interacting protein 1) or mLST8 (mammalian lethal with SEC13 protein 8). Furthermore, phosphorylation of NDRG1 (N-myc downstream regulated gene 1), a physiological substrate of SGK1, was also abolished in rictor-, Sin1- or mLST8-deficient fibroblasts. mTORC2 immunoprecipitated from wild-type, but not from mLST8- or rictor-knockout cells, phosphorylated SGK1 at Ser(422). Consistent with mTORC1 not regulating SGK1, immunoprecipitated mTORC1 failed to phosphorylate SGK1 at Ser(422), under conditions which it phosphorylated the hydrophobic motif of S6K. Moreover, rapamycin treatment of HEK (human embryonic kidney)-293, MCF-7 or HeLa cells suppressed phosphorylation of S6K, without affecting SGK1 phosphorylation or activation. The findings of the present study indicate that mTORC2, but not mTORC1, plays a vital role in controlling the hydrophobic motif phosphorylation and activity of SGK1. Our findings may explain why in previous studies phosphorylation of substrates, such as FOXO (forkhead box O), that could be regulated by SGK, are reduced in mTORC2-deficient cells. The results of the present study indicate that NDRG1 phosphorylation represents an excellent biomarker for mTORC2 activity.     

Autoregulation of the Mechanistic Target of Rapamycin (mTOR) Complex 2 Integrity Is Controlled by an ATP-dependent Mechanism

Nutrients are essential for living organisms because they fuel biological processes in cells. Cells monitor nutrient abundance and coordinate a ratio of anabolic and catabolic reactions. Mechanistic target of rapamycin (mTOR) signaling is the essential nutrient-sensing pathway that controls anabolic processes in cells. The central component of this pathway is mTOR, a highly conserved and essential protein kinase that exists in two distinct functional complexes. The nutrient-sensitive mTOR complex 1 (mTORC1) controls cell growth and cell size by phosphorylation of the regulators of protein synthesis S6K1 and 4EBP1, whereas its second complex, mTORC2, regulates cell proliferation by functioning as the regulatory kinase of Akt and other members of the AGC kinase family. The regulation of mTORC2 remains poorly characterized. Our study shows that the cellular ATP balance controls a basal kinase activity of mTORC2 that maintains the integrity of mTORC2 and phosphorylation of Akt on the turn motif Thr-450 site. We found that mTOR stabilizes SIN1 by phosphorylation of its hydrophobic and conserved Ser-260 site to maintain the integrity of mTORC2. The optimal kinase activity of mTORC2 requires a concentration of ATP above 1.2 mm and makes this kinase complex highly sensitive to ATP depletion. We found that not amino acid but glucose deprivation of cells or acute ATP depletion prevented the mTOR-dependent phosphorylation of SIN1 on Ser-260 and Akt on Thr-450. In a low glucose medium, the cells carrying a substitution of SIN1 with its phosphomimetic mutant show an increased rate of cell proliferation related to a higher abundance of mTORC2 and phosphorylation of Akt. Thus, the homeostatic ATP sensor mTOR controls the integrity of mTORC2 and phosphorylation of Akt on the turn motif site.     

PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle

Activation of members of the protein kinase AGC (cAMP dependent, cGMP dependent, and protein kinase C) family is regulated primarily by phosphorylation at two sites: a conserved threonine residue in the activation loop and a serine/threonine residue in a hydrophobic motif (HM) near the COOH terminus. Although phosphorylation of these kinases in the activation loop has been found to be mediated by phosphoinositide-dependent protein kinase-1 (PDK1), the kinase(s) that catalyzes AGC kinase phosphorylation in the HM remains uncharacterized. So far, at least 10 kinases have been suggested to function as an HM kinase or the so-called "PDK2," including mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MK2), integrin-linked kinase (ILK), p38 MAP kinase, protein kinase Calpha (PKCalpha), PKCbeta, the NIMA-related kinase-6 (NEK6), the mammalian target of rapamycin (mTOR), the double-stranded DNA-dependent protein kinase (DNK-PK), and the ataxia telangiectasia mutated (ATM) gene product. However, whether any or all of these kinases act as a physiological HM kinase remains to be established. Nonetheless, available data suggest that multiple systems may be used in cells to regulate the activation of the AGC family kinases. It is possible that, unlike activation loop phosphorylation, phosphorylation of the HM site in the different AGC family kinases is mediated by distinct kinases. In addition, phosphorylation of the AGC family kinase at the HM site could be cell type, signaling pathway, and substrate specific. Identification and characterization of the bonafide HM kinase(s) will be essential to verify these hypotheses.     

Spatial regulation of the mTORC1 system in amino acids sensing pathway

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine protein kinase that regulates numerous cellular processes including cell growth, proliferation, cell cycle, and autophagy. mTOR forms two different multi-protein complexes referred to as mTOR complex 1 (mTORC1) and mTORC2, and each complex exerts distinct functions exclusively. mTORC1 activity is sensitive to the selective inhibitor rapamycin, whereas mTORC2 is resistant. mTORC1 is regulated by many intra- and extra-cellular cues such as growth factors, nutrients, and energy-sensing signals, while mTORC2 senses ribosome maturation and growth factor signaling. This review focuses on current understandings by which mTORC1 pathway senses cellular nutrient availability for its activation.     

Mammalian NDR protein kinases: from regulation to a role in centrosome duplication

The NDR (nuclear Dbf2-related) family of kinases is highly conserved from yeast to human, and has been classified as a subgroup of the AGC group of protein kinases based on the sequence of the catalytic domain. Like all other members of the AGC class of protein kinases, NDR kinases require the phosphorylation of conserved Ser/Thr residues for activation. Importantly, NDR family members have two unique stretches of primary sequence: an N-terminal regulatory (NTR) domain and an insert of several residues between subdomains VII and VIII of the kinase domain. The kinase domain insert functions as an auto-inhibitory sequence (AIS), while binding of the co-activator MOB (Mps-one binder) proteins to the NTR domain releases NDR kinases from inhibition of autophosphorylation. However, despite such advances in our understanding of the molecular activation mechanism(s) and physiological functions of NDR kinases in yeast and invertebrates, most biological NDR substrates still remain to be identified. Nevertheless, by showing that the centrosomal subpopulation of human NDR1/2 is required for proper centrosome duplication, the first biological role of human NDR1/2 kinases has been defined recently. How far NDR-driven centrosome overduplication could actually contribute to cellular transformation will also be discussed.     

Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth and is a key target for therapeutic intervention in cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1), consisting of mTOR, Raptor (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with SEC13 protein 8) and complex 2 (mTORC2) consisting of mTOR, Rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated protein kinase-interacting protein 1), mLST8 and Protor-1 or Protor-2. Both complexes phosphorylate the hydrophobic motifs of AGC kinase family members: mTORC1 phosphorylates S6K (S6 kinase), whereas mTORC2 regulates phosphorylation of Akt, PKCα (protein kinase Cα) and SGK1 (serum- and glucocorticoid-induced protein kinase 1). To investigate the roles of the Protor isoforms, we generated single as well as double Protor-1- and Protor-2-knockout mice and studied how activation of known mTORC2 substrates was affected. We observed that loss of Protor-1 and/or Protor-2 did not affect the expression of the other mTORC2 components, nor their ability to assemble into an active complex. Moreover, Protor knockout mice display no defects in the phosphorylation of Akt and PKCα at their hydrophobic or turn motifs. Strikingly, we observed that Protor-1 knockout mice displayed markedly reduced hydrophobic motif phosphorylation of SGK1 and its physiological substrate NDRG1 (N-Myc downregulated gene 1) in the kidney. Taken together, these results suggest that Protor-1 may play a role in enabling mTORC2 to efficiently activate SGK1, at least in the kidney.     

TOR regulation of AGC kinases in yeast and mammals

The TOR (target of rapamycin), an atypical protein kinase, is evolutionarily conserved from yeast to man. Pharmacological studies using rapamycin to inhibit TOR and yeast genetic studies have provided key insights on the function of TOR in growth regulation. One of the first bona fide cellular targets of TOR was the mammalian protein kinase p70 S6K (p70 S6 kinase), a member of a family of kinases called AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases, which include PKA (cAMP-dependent protein kinase A), PKG (cGMP-dependent kinase) and PKC (protein kinase C). AGC kinases are also highly conserved and play a myriad of roles in cellular growth, proliferation and survival. The AGC kinases are regulated by a common scheme that involves phosphorylation of the kinase activation loop by PDK1 (phosphoinositide-dependent kinase 1), and phosphorylation at one or more sites at the C-terminal tail. The identification of two distinct TOR protein complexes, TORC1 (TOR complex 1) and TORC2, with different sensitivities to rapamycin, revealed that TOR, as part of either complex, can mediate phosphorylation at the C-terminal tail for optimal activation of a number of AGC kinases. Together, these studies elucidated that a fundamental function of TOR conserved throughout evolution may be to balance growth versus survival signals by regulating AGC kinases in response to nutrients and environmental conditions. This present review highlights this emerging function of TOR that is conserved from budding and fission yeast to mammals.     

Conserved herpesvirus protein kinases

Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however, along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task.     

Isolation of hyperactive mutants of mammalian target of rapamycin

The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that plays essential roles in the regulation of a wide array of growth-related processes such as protein synthesis, cell sizing, and autophagy. mTOR forms two functionally distinct complexes, termed the mTOR complex 1 (mTORC1) and 2 (mTORC2); only the former of which is inhibited by rapamycin. Based on the similarity between the cellular responses caused by rapamycin treatment and by nutrient starvation, it has been widely accepted that modulation in the mTORC1 activity in response to nutrient status directs these cellular responses, although direct evidence has been scarce. Here we report isolation of hyperactive mutants of mTOR. The isolated mTOR mutants exhibited enhanced kinase activity in vitro and rendered cells refractory to the dephosphorylation of the mTORC1 substrates upon amino acid starvation. Cells expressing the hyperactive mTOR mutant displayed larger cell size in a normal growing condition and were resistant to cell size reduction and autophagy induction in an amino acid-starved condition. These results indicate that the activity of mTORC1 actually directs these cellular processes in response to nutrient status and confirm the biological functions of mTORC1, which had been proposed solely from loss-of-function analyses using rapamycin and (molecular)genetic techniques. Additionally, the hyperactive mTOR mutant did not induce cellular transformation of NIH/3T3 cells, suggesting that concomitant activation of additional pathways is required for tumorigenesis. This hyperactive mTOR mutant will be a valuable tool for establishing physiological consequences of mTOR activation in cells as well as in organisms.     

The discovery and optimisation of pyrido[2,3-d]pyrimidine-2,4-diamines as potent and selective inhibitors of mTOR kinase

We describe a novel series of potent inhibitors of the kinase activity of mTOR. The compounds display good selectivity relative to other PI3K-related kinase family members and, in cellular assays, inhibit both mTORC1 and mTORC2 complexes and exhibit good antiproliferative activity.     

Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase

Elongation factor 2 kinase (eEF2k) phosphorylates and inactivates eEF2. Insulin induces dephosphorylation of eEF2 and inactivation of eEF2 kinase, and these effects are blocked by rapamycin, which inhibits the mammalian target of rapamycin, mTOR. However, the signalling mechanisms underlying these effects are unknown. Regulation of eEF2 phosphorylation and eEF2k activity is lost in cells in which phosphoinositide-dependent kinase 1 (PDK1) has been genetically knocked out. This is not due to loss of mTOR function since phosphorylation of another target of mTOR, initiation factor 4E-binding protein 1, is not defective. PDK1 is required for activation of members of the AGC kinase family; we show that two such kinases, p70 S6 kinase (regulated via mTOR) and p90(RSK1) (activated by Erk), phosphorylate eEF2k at a conserved serine and inhibit its activity. In response to insulin-like growth factor 1, which activates p70 S6 kinase but not Erk, regulation of eEF2 is blocked by rapamycin. In contrast, regulation of eEF2 by stimuli that activate Erk is insensitive to rapamycin, but blocked by inhibitors of MEK/Erk signalling, consistent with the involvement of p90(RSK1).     

The TSC1-TSC2 complex is required for proper activation of mTOR complex 2

The mammalian target of rapamycin (mTOR) is a protein kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. Both complexes phosphorylate a hydrophobic motif on downstream protein kinases, which contributes to the activation of these kinases. mTOR complex 1 (mTORC1) phosphorylates S6K1, while mTORC2 phosphorylates Akt. The TSC1-TSC2 complex is a critical negative regulator of mTORC1. However, how mTORC2 is regulated and whether the TSC1-TSC2 complex is involved are unknown. We find that mTORC2 isolated from a variety of cells lacking a functional TSC1-TSC2 complex is impaired in its kinase activity toward Akt. Importantly, the defect in mTORC2 activity in these cells can be separated from effects on mTORC1 signaling and known feedback mechanisms affecting insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Our data also suggest that the TSC1-TSC2 complex positively regulates mTORC2 in a manner independent of its GTPase-activating protein activity toward Rheb. Finally, we find that the TSC1-TSC2 complex can physically associate with mTORC2 but not mTORC1. These data demonstrate that the TSC1-TSC2 complex inhibits mTORC1 and activates mTORC2, which through different mechanisms promotes Akt activation.     

mTOR complex 2 signaling and functions

The mechanistic target of rapamycin (mTOR) plays a central role in cellular growth and metabolism. mTOR forms two distinct protein complexes, mTORC1 and mTORC2. Much is known about the regulation and functions of mTORC1 due to availability of a natural compound, rapamycin, that inhibits this complex. Studies that define mTORC2 cellular functions and signaling have lagged behind. The development of pharmacological inhibitors that block mTOR kinase activity, and thereby inhibit both mTOR complexes, along with availability of mice with genetic knockouts in mTOR complex components have now provided new insights on mTORC2 function and regulation. Since prolonged effects of rapamycin can also disrupt mTORC2, it is worth re-evaluating the contribution of this less-studied mTOR complex in cancer, metabolic disorders and aging. In this review, we focus on recent developments on mammalian mTORC2 signaling mechanisms and its cellular and tissue-specific functions.     

mTOR-Dependent Cell Survival Mechanisms

The mechanistic target of rapamycin (mTOR) kinase is a conserved regulator of cell growth, proliferation, and survival. In cells, mTOR is the catalytic subunit of two complexes called mTORC1 and mTORC2, which have distinct upstream regulatory signals and downstream substrates. mTORC1 directly senses cellular nutrient availability while indirectly sensing circulating nutrients through growth factor signaling pathways. Cellular stresses that restrict growth also impinge on mTORC1 activity. mTORC2 is less well understood and appears only to sense growth factors. As an integrator of diverse growth regulatory signals, mTOR evolved to be a central signaling hub for controlling cellular metabolism and energy homoeostasis, and defects in mTOR signaling are important in the pathologies of cancer, diabetes, and aging. Here we discuss mechanisms by which each mTOR complex might regulate cell survival in response to metabolic and other stresses.The mechanistic target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase belonging to the phosphatidylinositol kinase-related kinase (PIKK) family and in mammalian cells is a central regulator of cell growth, proliferation, and survival (for review, see Sengupta et al. 2010; Zoncu et al. 2010). As its name implies, mTOR is the target of the naturally occurring compound rapamycin, which in association with the FK506-binding protein (FKBP12) is an allosteric inhibitor of mTOR. Although rapamycin is now known to only partially inhibit mTOR activity, derivatives of the drug have important clinical applications in oncology, in preventing restenosis after angioplasty, and as an immunosuppressant following organ transplants.     

Translational deregulation in PDK-1-/- embryonic stem cells

PDK-1 is a protein kinase that is critical for the activation of many downstream protein kinases in the AGC superfamily, through phosphorylation of the activation loop site on these substrates. Cells lacking PDK-1 show decreased activity of these protein kinases, including protein kinase B (PKB) and p70S6K, whereas mTOR activity remains largely unaffected. Here we show, by assessing both association of cellular RNAs with polysomes and by metabolic labeling, that PDK-1-/- embryonic stem (ES) cells exhibit defects in mRNA translation. We identify which mRNAs are most dramatically translationally regulated in cells lacking PDK-1 expression by performing microarray analysis of total and polysomal RNA in these cells. In addition to the decreased translation of many RNAs, a smaller number of RNAs show increased association with polyribosomes in PDK-1-/- ES cells relative to PDK-1+/+ ES cells. We show that PKB activity is a critical downstream component of PDK-1 in mediating translation of cystatin C, RANKL, and Rab11a, whereas mTOR activity is less important for effective translation of these targets.     

Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.     

Simultaneous inhibition of mTORC1 and mTORC2 by mTOR kinase inhibitor AZD8055 induces autophagy and cell death in cancer cells

mTOR is a major biological switch, coordinating an adequate response to changes in energy uptake (amino acids, glucose), growth signals (hormones, growth factors) and environmental stress. mTOR kinase is highly conserved through evolution from yeast to man and in both cases, controls autophagy and cellular translation in response to nutrient stress. mTOR kinase is the catalytic component of two distinct multiprotein complexes called mTORC1 and mTORC2. In addition to mTOR, mTORC1 contains Raptor, mLST8 and PRAS40. mTORC2 contains mTOR, Rictor, mSIN1 and Protor-1. mTORC1 activates p70S6K, which in turn phosphorylates the ribosomal protein S6 and 4E-BP1, both involved in protein translation. mTORC2 activates AKT directly by phosphorylating Serine 473. pAKT(S473) phosphorylates TSC2 (tuberin) and inactivates it, preventing its association with TSC1 (hamartin) and the inhibition of Rheb, an activator of mTOR. pAKT also phosphorylates PRAS40, releasing it from the mTORC1 complex, increasing its kinase activity. Finally, AKT regulates FOXO3 phosphorylation, sequestering it in the cytosol in an inactive state.     

mTOR kinase domain phosphorylation promotes mTORC1 signaling, cell growth, and cell cycle progression

The mammalian target of rapamycin complex 1 (mTORC1) functions as an environmental sensor to promote critical cellular processes such as protein synthesis, cell growth, and cell proliferation in response to growth factors and nutrients. While diverse stimuli regulate mTORC1 signaling, the direct molecular mechanisms by which mTORC1 senses and responds to these signals remain poorly defined. Here we investigated the role of mTOR phosphorylation in mTORC1 function. By employing mass spectrometry and phospho-specific antibodies, we demonstrated novel phosphorylation on S2159 and T2164 within the mTOR kinase domain. Mutational analysis of these phosphorylation sites indicates that dual S2159/T2164 phosphorylation cooperatively promotes mTORC1 signaling to S6K1 and 4EBP1. Mechanistically, S2159/T2164 phosphorylation modulates the mTOR-raptor and raptor-PRAS40 interactions and augments mTORC1-associated mTOR S2481 autophosphorylation. Moreover, mTOR S2159/T2164 phosphorylation promotes cell growth and cell cycle progression. We propose a model whereby mTOR kinase domain phosphorylation modulates the interaction of mTOR with regulatory partner proteins and augments intrinsic mTORC1 kinase activity to promote biochemical signaling, cell growth, and cell cycle progression.