Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin

Phosphorylation of the ribosomal S6 subunit is tightly correlated with enhanced translation initiation of a subset of mRNAs that encodes components of the protein synthesis machinery, which is an important early event that controls mammalian cell growth and proliferation. The recently identified S6 kinase 2 (S6K2), together with its homologue S6K1, is likely responsible for the mitogen-stimulated phosphorylation of S6. Like S6K1, the activation of S6K2 requires signaling from both the phosphatidylinositol 3-kinase and the mammalian target of rapamycin (mTOR). Here we report the investigation of the mechanisms of S6K2 regulation by mTOR. We demonstrate that similar to S6K1 the serum activation of S6K2 in cells is dependent on mTOR kinase activity, amino acid sufficiency, and phosphatidic acid. Previously we have shown that mTOR is a cytoplasmic-nuclear shuttling protein. As a predominantly nuclear protein, S6K2 activation was facilitated by enhanced mTOR nuclear import with the tagging of an exogenous nuclear localization signal and diminished by enhanced mTOR nuclear export with the tagging of a nuclear export sequence. However, further increase of mTOR nuclear import by the tagging of four copies of nuclear localization signal resulted in its decreased ability to activate S6K2, suggesting that mTOR nuclear export may also be an integral part of the activation process. Consistently, the nuclear export inhibitor leptomycin B inhibited S6K2 activation. Taken together, our observations suggest a novel regulatory mechanism in which an optimal cytoplasmic-nuclear distribution or shuttling rate for mTOR is required for maximal activation of the nuclear S6K2.     

Identification of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)-phosphorylating kinase

Here we demonstrate that mammalian target of rapamycin (mTOR) is phosphorylated in a rapamycin-sensitive manner. We show that S6 kinase 1 (S6K1), but not Akt, directly phosphorylates mTOR in cell-free in vitro system and in cells. Expression of a constitutively active, rapamycin- and wortmannin-resistant S6K1 leads to constitutive phosphorylation of mTOR, whereas knock-down of S6K1 using small inhibitory RNA greatly reduces mTOR phosphorylation despite elevated Akt activity. Importantly, phosphorylation of mTOR by S6K1 occurs at threonine 2446/serine 2448. This region has been shown previously to be part of a regulatory repressor domain. These sites are also constitutively phosphorylated in the breast cancer cell line MCF7 carrying an amplification of the S6K1 gene, but not in a less tumorigenic cell line, MCF10a. Many models for Akt signaling to mTOR have been presented, suggesting direct phosphorylation by Akt. These models must be reconsidered in light of the present findings.     

Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status

The mammalian target of rapamycin (mTOR) is a key regulator of protein translation. Signaling via mTOR is increased by growth factors but decreased during nutrient deprivation. Previous studies have identified Ser2448 as a nutrient-regulated phosphorylation site located in the mTOR catalytic domain, insulin stimulates Ser2448 phosphorylation via protein kinase B (PKB), while Ser2448 phosphorylation is attenuated with amino acid starvation. Here we have identified Thr2446 as a novel nutrient-regulated phosphorylation site on mTOR. Thr2446 becomes phosphorylated when CHO-IR cells are nutrient-deprived, but phosphorylation is reduced by insulin stimulation. Nutrient deprivation activates AMP-activated protein kinase (AMPK). To test whether this could be involved in regulating phoshorylation of mTOR, we treated cultured murine myotubes with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or dinitrophenol (DNP). Both treatments activated AMPK and also caused a concomitant increase in phosphorylation of Thr2446 and a parallel decrease in insulin's ability to phosphorylate p70 S6 kinase. In vitro kinase assays using peptides based on the sequence in amino acids 2440-2551 of mTOR found that PKB and AMPK are capable of phosphorylating sites in this region. However, phosphorylation by PKB is restricted when Thr2446 is mutated to an acidic residue mimicking phosphorylation. Conversely, AMP-kinase-induced phosphorylation is reduced when Ser2448 is phosphorylated. These data suggest differential phosphorylation Thr2446 and Ser2448 could act as a switch mechanism to integrate signals from nutrient status and growth factors to control the regulation of protein translation.     

Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro

p70 S6 kinase alpha (p70alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70alpha activation in a manner that can be overcome by coexpression of p70alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70alpha phosphorylation in vitro is accompanied by a substantial restoration in p70alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70alpha activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined.     

A downstream kinase of the mammalian target of rapamycin, p70S6K1, regulates human double minute 2 protein phosphorylation and stability

Human double minute 2 (HDM2) is an oncoprotein overexpressed in many human cancers. HDM2 expression is regulated at multiple levels in cells. Phosphorylation of HDM2 plays an important role in its post-translational regulation. In this study, we have shown that the phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, have similar effects on the inhibition of HDM2 phosphorylation and protein turnover. Rapamycin inhibited p70S6K1, but not AKT activation, indicating that rapamycin affects HDM2 phosphorylation via an AKT-independent mechanism. Rapamycin also decreased HDM2 protein stability. Knockdown of p70S6K1 by a p70S6K1 siRNA resulted in the inhibition of HDM2 phosphorylation and a decrease in HDM2 protein turnover. Overexpression of p70S6K1 enhanced HDM2 phosphorylation and led to an increase in HDM2 protein turnover. Our results suggest that p70S6K1 regulates turnover of HDM2 protein for cancer development.     

A nuclear transport signal in mammalian target of rapamycin is critical for its cytoplasmic signaling to S6 kinase 1

The mammalian target of rapamycin (mTOR) regulates nutrient-dependent cell growth and proliferation through cytoplasmic targets, such as S6 kinase 1 (S6K1). Consistent with its main function in the cytoplasm, mTOR is predominantly cytoplasmic. However, previously we have found that mTOR shuttles between the nucleus and cytoplasm, and we have proposed that the nucleocytoplasmic shuttling of mTOR is required for the maximal activation of S6K1. The intrinsic signals directing mTOR nuclear transport and the underlying mechanisms are unknown. In this study we initially set out to identify nuclear export signals in mTOR. A systematic scan of the mTOR sequence revealed 16 peptides conforming to the canonical leucine-rich nuclear export signal, of which 3 were found by reporter assays to contain leptomycin B-sensitive and leucine-dependent nuclear export activity. Unexpectedly, mTOR proteins with those conserved leucines mutated to alanines were unable to enter the nucleus. Further investigation revealed that the L982A/L984A and L1287A/L1289A mutations likely induced a global structural change in mTOR, whereas the L545A/L547A mutation directly impaired the nuclear import of the protein, potentially regulated by a nucleocytoplasmic shuttling signal. The loss of nuclear import was accompanied by the significantly reduced ability of the L545A/L547A mutant to activate S6K1 in cells. Most importantly, when nuclear import was restored in the L545A/L547A mutant by the addition of an exogenous nuclear import signal, signaling to S6K1 was rescued. Taken together, our observations suggest the existence of a nuclear shuttling signal in mTOR and provide definitive evidence for the requirement of mTOR nuclear import in its cytoplasmic signaling to S6K1.     

Glycogen synthase kinase (GSK)-3 and mammalian target of rapamycin complex 1 (mTORC1) cooperate to regulate protein S6 kinase 1 (S6K1)

Comment on: Shin S, et al. Proc Natl Acad Sci USA 2011; 108:1204–13     

Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and cap-dependent initiation of translation by the c-Abl protein-tyrosine kinase

The c-Abl protein-tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The rapamycin and FKBP-target 1 (RAFT1), also known as FKBP12-rapamycin-associated protein (FRAP, mTOR), regulates the p70S6 kinase (p70(S6k)) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The present results demonstrate that c-Abl binds directly to RAFT1 and phosphorylates RAFT1 in vitro and in vivo. c-Abl inhibits autophosphorylation of RAFT1 and RAFT1-mediated phosphorylation p70(S6k). The functional significance of the c-Abl-RAFT1 interaction is further supported by the finding that eIF4E-dependent translation in mouse embryo fibroblasts from Abl(-/-) mice is significantly higher than that compared in wild-type cells. The results also demonstrate that exposure of cells to ionizing radiation is associated with c-Abl-mediated binding of 4E-BP1 to eIF4E and inhibition of translation. These findings with the c-Abl tyrosine kinase represent the first demonstration of a negative physiologic regulator of RAFT1-mediated 5' cap-dependent translation.     

p21-activated kinase 1 is activated through the mammalian target of rapamycin/p70 S6 kinase pathway and regulates the replication of hepatitis C virus in human hepatoma cells

Cellular mechanisms that regulate the replication of hepatitis C virus (HCV) RNA are poorly understood. p21-activated kinase 1 (PAK1) is a serine/threonine kinase that has been suggested to participate in antiviral signaling. We studied its role in the cellular control of HCV replication. Transfection of PAK1-specific small interfering RNA enhanced viral RNA and protein abundance in established replicon cell lines as well as cells infected with chimeric genotype 1a/2a HCV, despite reducing cellular proliferation, suggesting specific regulation of HCV replication. PAK1 knockdown did not reduce interferon regulatory factor 3-dependent gene expression, indicating that this regulation is independent of the retinoic acid-inducible gene I/interferon regulatory factor 3 pathway. On the other hand, LY294002 and rapamycin abolished PAK1 phosphorylation and enhanced HCV abundance, suggesting that the mammalian target of rapamycin (mTOR) is involved in PAK1 regulation of HCV. Small interfering RNA knockdown of the mTOR substrate p70 S6 kinase abrogated PAK1 phosphorylation and enhanced HCV RNA abundance, whereas overexpression of a constitutively active alternate substrate, eukaryotic translation initiation factor 4E-binding protein 1, increased cap-independent viral translation and viral RNA abundance without influencing PAK1 phosphorylation. Similar data indicated that mTOR is regulated by both phosphatidylinositol 3-kinase/Akt and ERK. Taken together, the data indicate that p70 S6 kinase activates PAK1 and contributes to phosphatidylinositol 3-kinase- and ERK-mediated regulation of HCV RNA replication.     

Regulation of ribosomal protein S6 phosphorylation by casein kinase 1 and protein phosphatase 1

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5'-m(7)GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.     

Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase

The mammalian target of rapamycin (mTOR) coordinates cell growth with the growth factor and nutrient/energy status of the cell. The phosphatidylinositol 3-kinase-AKT pathway is centrally involved in the transmission of mitogenic signals to mTOR. Previous studies have shown that mTOR is a direct substrate for the AKT kinase and identified Ser-2448 as the AKT target site in mTOR. In this study, we demonstrate that rapamycin, a specific inhibitor of mTOR function, blocks serum-stimulated Ser-2448 phosphorylation and that this drug effect is not explained by the inhibition of AKT. Furthermore, the phosphorylation of Ser-2448 was dependent on mTOR kinase activity, suggesting that mTOR itself or a protein kinase downstream from mTOR was responsible for the modification of Ser-2448. Here we show that p70S6 kinase phosphorylates mTOR at Ser-2448 in vitro and that ectopic expression of rapamycin-resistant p70S6 kinase restores Ser-2448 phosphorylation in rapamycin-treated cells. In addition, we show that cellular amino acid status, which modulates p70S6 kinase (S6K1) activity via the TSC/Rheb pathway, regulates Ser-2448 phosphorylation. Finally, small interfering RNA-mediated depletion of p70S6 kinase reduces Ser-2448 phosphorylation in cells. Taken together, these results suggest that p70S6 kinase is a major effector of mTOR phosphorylation at Ser-2448 in response to both mitogen- and nutrient-derived stimuli.     

PAP-1, a novel target protein of phosphorylation by pim-1 kinase.

Protooncogene, pim-1, has been reported to be a predisposition for lymphomagenesis along with myc, and its protein product, Pim-1, has been shown to be a serine/threonine protein kinase, whose activity is involved in proliferation and differentiation of blood cells. The signal transduction pathways neither to nor from Pim-1, however, have been clarified. We have cloned a cDNA encoding a novel Pim-1 binding protein, PAP-1, comprising 213 amino acids with a basic amino-acid cluster near the C-terminus. PAP-1 was colocalized with Pim-1 in human HeLa cell nuclei. The in vitro binding assays using GST fusion proteins of the wild-type and various deletion mutants revealed that the whole molecule of Pim-1 is required for the binding activity to PAP-1 and that Pim-1 binds to the region from amino-acid numbers 1-147 of PAP-1, or to two segments in the region. The association of PAP-1 with Pim-1 was also shown in vivo in transfected cells. Furthermore, PAP-1 was phosphorylated in vitro by Pim-1, but not a kinase-negative Pim-1 mutant. The two serine residues of PAP-1 at amino acids 204 and 206 near the C-terminus were phosphorylated by Pim-1. PAP-1 is thus thought to be a target protein for Pim-1 kinase.     

Regulation of B-Raf kinase activity by tuberin and Rheb is mammalian target of rapamycin (mTOR)-independent

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome with manifestations that can include seizures, mental retardation, autism, and tumors in the brain, retina, kidney, heart, and skin. The products of the TSC1 and TSC2 genes, hamartin and tuberin, respectively, heterodimerize and inhibit the mammalian target of rapamycin (mTOR). We found that tuberin expression increases p42/44 MAPK phosphorylation and B-Raf kinase activity. Short interfering RNA down-regulation of tuberin decreased the p42/44 MAPK phosphorylation and B-Raf activity. Expression of Rheb, the target of the GTPase-activating domain of tuberin, inhibited wild-type B-Raf kinase but not activated forms of B-Raf. The interaction of endogenous Rheb with B-Raf was enhanced by serum and by Ras overexpression. A farnesylation-defective mutant of Rheb co-immunoprecipitated with and inhibited B-Raf but did not activate ribosomal protein S6 kinase, indicating that farnesylation is not required for B-Raf inhibition by Rheb and that B-Raf inhibition and S6 kinase activation are separable activities of Rheb. Consistent with this, inhibition of B-Raf and p42/44 MAPK by Rheb was resistant to rapamycin in contrast to Rheb activation of S6 kinase, which is rapamycin-sensitive. Taken together these data demonstrate that inhibition of B-Raf kinase via Rheb is an mTOR-independent function of tuberin.     

Regulation of placental amino acid transporter activity by mammalian target of rapamycin

The activity of placental amino acid transporters is decreased in intrauterine growth restriction (IUGR), but the underlying regulatory mechanisms have not been established. Inhibition of the mammalian target of rapamycin (mTOR) signaling pathway has been shown to decrease the activity of the system L amino acid transporter in human placental villous fragments, and placental mTOR activity is decreased in IUGR. In the present study, we used cultured primary trophoblast cells to study mTOR regulation of placental amino acid transporters in more detail and to test the hypothesis that mTOR alters amino acid transport activity by changes in transporter expression. Inhibition of mTOR by rapamycin significantly reduced the activity of system A (-17%), system L (-28%), and taurine (-40%) amino acid transporters. mRNA expression of isoforms of the three amino acid transporter systems in response to mTOR inhibition was measured using quantitative real-time PCR. mRNA expression of l-type amino acid transporter 1 (LAT1; a system L isoform) and taurine transporter was reduced by 13% and 50%, respectively; however, mTOR inhibition did not alter the mRNA expression of system A isoforms (sodium-coupled neutral amino acid transporter-1, -2, and -4), LAT2, or 4F2hc. Rapamycin treatment did not significantly affect the protein expression of any of the transporter isoforms. We conclude that mTOR signaling regulates the activity of key placental amino acid transporters and that this effect is not due to a decrease in total protein expression. These data suggest that mTOR regulates placental amino acid transporters by posttranslational modifications or by affecting transporter translocation to the plasma membrane.     

The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain

Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.     

Ribosomal S6 kinase (RSK) regulates phosphorylation of filamin A on an important regulatory site

The Ras-mitogen-activated protein (Ras-MAP) kinase pathway regulates various cellular processes, including gene expression, cell proliferation, and survival. Ribosomal S6 kinase (RSK), a key player in this pathway, modulates the activities of several cytoplasmic and nuclear proteins via phosphorylation. Here we report the characterization of the cytoskeletal protein filamin A (FLNa) as a membrane-associated RSK target. We show that the N-terminal kinase domain of RSK phosphorylates FLNa on Ser(2152) in response to mitogens. Inhibition of MAP kinase signaling with UO126 or mutation of Ser(2152) to Ala on FLNa prevents epidermal growth factor (EGF)-stimulated phosphorylation of FLNa in vivo. Furthermore, phosphorylation of FLNa on Ser(2152) is significantly enhanced by the expression of wild-type RSK and antagonized by kinase-inactive RSK or specific reduction of endogenous RSK. Strikingly, EGF-induced, FLNa-dependent migration of human melanoma cells is significantly reduced by UO126 treatment. Together, these data provide substantial evidence that RSK phosphorylates FLNa on Ser(2152) in vivo. Given that phosphorylation of FLNa on Ser(2152) is required for Pak1-mediated membrane ruffling, our results suggest a novel role for RSK in the regulation of the actin cytoskeleton.     

Mammalian target of rapamycin regulates IRS-1 serine 307 phosphorylation

Insulin signaling can be negatively regulated by phosphorylation of serine 307 of the insulin receptor substrate (IRS)-1. Rapamycin, an inhibitor of the kinase mTOR, can prevent serine 307 phosphorylation and the development of insulin resistance. We further investigated the role of mTOR in regulating serine 307 phosphorylation, demonstrating that serine 307 phosphorylation in response to insulin, anisomycin, or tumor necrosis factor was quantitatively and temporally associated with activation of mTOR and could be inhibited by rapamycin. Amino acid stimulation activated mTOR and resulted in IRS-1 serine 307 phosphorylation without activating PKB or JNK. Okadaic acid, an inhibitor of the phosphatase PP2A, activated mTOR and stimulated the phosphorylation of serine 307 in a rapamycin-sensitive manner, indicating serine 307 phosphorylation requires mTOR activity but not PP2A, suggesting that mTOR itself may be responsible for phosphorylating serine 307. Finally, we demonstrated that serine 307 phosphorylated IRS-1 is detected primarily in the cytosolic fraction.     

Regulation of proline-rich Akt substrate of 40 kDa (PRAS40) function by mammalian target of rapamycin complex 1 (mTORC1)-mediated phosphorylation

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [(32)P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition.     

Threonine-120 phosphorylation regulated by phosphoinositide-3-kinase/Akt and mammalian target of rapamycin pathway signaling limits the antitumor activity of mammalian sterile 20-like kinase 1

Mst1/Stk4, a hippo-like serine-threonine kinase, is implicated in many cancers, including prostate cancer. However, the mechanisms regulating Mst1 remain obscure. Here, we characterized the effects of phospho-Thr-120 on Mst1 in prostate cancer cells. We demonstrated that phospho-Thr-120 did not alter the nuclear localization or cleavage of Mst1 in a LNCaP or castration-resistant C4-2 prostate tumor cell model, as revealed by a mutagenesis approach. Phospho-Thr-120 appeared to be specific to cancer cells and predominantly localized in the nucleus. In contrast, phospho-Thr-183, a critical regulator of Mst1 cell death, was exclusively found in the cytoplasm. As assessed by immunohistochemistry, a similar distribution of phospho-Mst1-Thr-120/Thr-183 was also observed in a prostate cancer specimen. In addition, the blockade of PI3K signaling by a small molecule inhibitor, LY294002, increased cytoplasmic phospho-Mst1-Thr-183 without having a significant effect on nuclear phospho-Mst1-Thr-120. However, the attenuation of mammalian target of rapamycin (mTOR) activity by a selective pharmacologic inhibitor, Ku0063794 or CCI-779, caused the up-regulation of nuclear phospho-Mst1-Thr-120 without affecting cytoplasmic phospho-Mst1-Thr-183. This suggests that PI3K and mTOR pathway signaling differentially regulate phospho-Mst1-Thr-120/Thr-183. Moreover, mutagenesis and RNAi data revealed that phospho-Thr-120 resulted in C4-2 cell resistance to mTOR inhibition and reduced the Mst1 suppression of cell growth and androgen receptor-driven gene expression. Collectively, these findings indicate that phospho-Thr-120 leads to the loss of Mst1 functions, supporting cancer cell growth and survival.