Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1)

p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1.     

TRAF2 phosphorylation promotes NF-κB-dependent gene expression and inhibits oxidative stress-induced cell death

Tumor necrosis factor α (TNF-α) receptor-associated factor 2 (TRAF2) regulates activation of the c-Jun N-terminal kinase (JNK)/c-Jun and the inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling cascades in response to TNF-α stimulation. Gene knockout studies have revealed that TRAF2 inhibits TNF-α-induced cell death but promotes oxidative stress-induced apoptosis. Here we report that TNF-α and oxidative stress both induce TRAF2 phosphorylation at serines 11 and 55 and that this dual phosphorylation promotes the prolonged phase of IKK activation while inhibiting the prolonged phase of JNK activation. Prolonged IKK activation trigged by TNF-α plays an essential role in efficient expression of a subset of NF-κB target genes but has no substantial role in TNF-α-induced cell death. On the other hand, TRAF2 phosphorylation in response to oxidative stress significantly promotes cell survival by inducing prolonged IKK activation and by inhibiting the prolonged phase of JNK activation. Notably, stable expression of phospho-null mutant TRAF2 in cancer cells leads to an increase in the basal and inducible JNK activation and B-cell lymphoma 2 (Bcl-2) phosphorylation. In addition, exposure of cells expressing phospho-null mutant TRAF2 to sublethal oxidative stress results in a rapid degradation of Bcl-2 and cellular inhibitor of apoptosis 1 as well as significantly increased cell death. These results suggest that TRAF2 phosphorylation is essential for cell survival under conditions of oxidative stress.     

S6K directly phosphorylates IRS-1 on Ser-270 to promote insulin resistance in response to TNF-(alpha) signaling through IKK2

S6K1 (p70S6K) is a serine kinase downstream from Akt in the insulin signaling pathway that is involved in negative feedback regulation of insulin action. S6K1 is also activated by TNF-alpha, a pro-inflammatory cytokine. However, its role remains to be characterized. In the current study, we elucidated a mechanism for S6K1 to mediate TNF-alpha-induced insulin resistance in adipocytes and hepatocytes. S6K1 was phosphorylated at Thr-389 in response to TNF-alpha. This led to phosphorylation of IRS-1 by S6K1 at multiple serine residues including Ser-270, Ser-307, Ser-636, and Ser-1101 in human IRS-1 (Ser-265, Ser-302, Ser-632, and Ser-1097, in rodent IRS-1). Direct phosphorylation of these sites by S6K1 was observed in an in vitro kinase assay using purified IRS-1 and S6K1. Phosphorylation of all these serines was increased in the adipose tissue of obese mice. RNAi knockdown demonstrated an important role for S6K1 in mediating TNF-alpha-induced IRS-1 inhibition that led to impaired insulin-stimulated glucose uptake in adipocytes. A point mutant of IRS-1 (S270A) impaired association of IRS-1 with S6K1 resulting in diminished phosphorylation of IRS-1 at three other S6K1 phosphorylation sites (Ser-307, Ser-636, and Ser-1101). Expression of a dominant negative S6K1 mutant prevented TNF-induced Ser-270 phosphorylation and IRS-1 protein degradation. Moreover, in IKK2 (but not IKK1)-null cells, TNF-alpha treatment did not result in Thr-389 phosphorylation of S6K1. We present a new mechanism for TNF-alpha to induce insulin resistance that involves activation of S6K by an IKK2-dependent pathway. S6K directly phosphorylates IRS-1 on multiple serine residues to inhibit insulin signaling.     

Enterovirus 71 2C protein inhibits TNF-α-mediated activation of NF-κB by suppressing IκB kinase β phosphorylation

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α-mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α-mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α-mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.     

Signal transduction pathways involved in phosphorylation and activation of p70S6K following exposure to UVA irradiation

Ultraviolet light A (UVA) plays an important role in the etiology of human skin cancer, and UVA-induced signal transduction has a critical role in UVA-induced skin carcinogenesis. The upstream signaling pathways leading to p70(S6K) phosphorylation and activation are not well understood. Here, we observed that UVA induces phosphorylation and activation of p70(S6K). Further, UVA-stimulated p70(S6K) activity and phosphorylation at Thr(389) were blocked by wortmannin, rapamycin, PD98059, SB202190, and dominant negative mutants of phosphatidylinositol (PI) 3-kinase p85 subunit (DNM-Deltap85), ERK2 (DNM-ERK2), p38 kinase (DNM-p38), and JNK1 (DNM-JNK1) and were absent in Jnk1-/- or Jnk2-/- knockout cells. The p70(S6K) phosphorylation at Ser(411) and Thr(421)/Ser(424) was inhibited by rapamycin, PD98059, or DNM-ERK2 but not by wortmannin, SB202190, DNM-Deltap85, or DNM-p38. However, Ser(411), but not Thr(421)/Ser(424) phosphorylation, was suppressed in DNM-JNK1 and abrogated in Jnk1-/- or Jnk2-/- cells. In vitro assays indicated that Ser(411) on immunoprecipitated p70(S6K) proteins is phosphorylated by active JNKs and ERKs, but not p38 kinase, and Thr(421)/Ser(424) is phosphorylated by ERK1, but not ERK2, JNKs, or p38 kinase. Moreover, p70(S6K) co-immunoprecipitated with PI 3-kinase and possibly PDK1. The complex possibly possessed a partial basal level of phosphorylation, but not at MAPK sites, which was available for its activation by MAPKs in vitro. Thus, these results suggest that activation of MAPKs, like PI 3-kinase/mTOR, may be involved in UVA-induced phosphorylation and activation of p70(S6K).     

Effects of PI3K catalytic subunit and Akt isoform deficiency on mTOR and p70S6K activation in myoblasts

The PI3K/Akt/mTOR signaling pathway is critical for cellular growth and survival in skeletal muscle, and is activated in response to growth factors such as insulin-like growth factor-I (IGF-I). We found that in C2C12 myoblasts, deficiency of PI3K p110 catalytic subunits or Akt isoforms had distinct effects on phosphorylation of mTOR and p70S6K. siRNA-mediated knockdown of PI3K p110α, p110β, and simultaneous knockdown of p110α and p110β resulted in increased basal and IGF-I-stimulated phosphorylation of mTOR S2448 and p70S6K T389; however, phosphorylation of S6 was reduced in p110β-deficient cells, possibly due to reductions in total S6 protein. We found that IGF-I-stimulated Akt1 activity was enhanced in Akt2- or Akt3-deficient cells, and that knockdown of individual Akt isoforms increased mTOR/p70S6K activation in an isoform-specific fashion. Conversely, levels of IGF-I-stimulated p70S6K phosphorylation in cells simultaneously deficient in both Akt1 and Akt3 were increased beyond those seen with loss of any single Akt isoform, suggesting an alternate, Akt-independent mechanism that activates mTOR/p70S6K. Our results collectively suggest that mTOR/p70S6K is activated in a PI3K/Akt-dependent manner, but that in the absence of p110α or Akt, alternate pathway(s) may mediate activation of mTOR/p70S6K in C2C12 myoblasts.     

Intracellular parasitism with Toxoplasma gondii stimulates mammalian‐target‐of‐rapamycin‐dependent host cell growth despite impaired signalling to S6K1 and 4E‐BP1

The Ser/Thr kinase mammalian‐target‐of‐rapamycin (mTOR) is a central regulator of anabolism, growth and proliferation. We investigated the effects of Toxoplasma gondii on host mTOR signalling. Toxoplasma invasion of multiple cell types rapidly induced sustained mTOR activation that was restricted to infected cells, as determined by rapamycin‐sensitive phosphorylation of ribosomal protein S6; however, phosphorylation of the growth‐associated mTOR substrates 4E‐BP1 and S6K1 was not detected. Infected cells still phosphorylated S6K1 and 4E‐BP1 in response to insulin, although the S6K1 response was blunted. Parasite‐induced S6 phosphorylation was independent of S6K1 and did not require activation of canonical mTOR‐inducing pathways mediated by phosphatidylinositol 3‐kinase–Akt and ERK. Host mTOR was localized in a vesicular pattern surrounding the parasitophorous vacuole, suggesting potential activation by phosphatidic acid in the vacuolar membrane. In spite of a failure to phosphorylate 4E‐BP1 and S6K1, intracellular T. gondii triggered host cell cycle progression in an mTOR‐dependent manner and progression of infected cells displayed increased sensitivity to rapamycin. Moreover, normal cell growth was maintained during parasite‐induced cell cycle progression, as indicated by total cellular S6 levels. The Toxoplasma‐infected cell provides a unique example of non‐canonical mTOR activation supporting growth that is independent of signalling through either S6K1 or 4E‐BP1.     

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.     

AMPK activation suppresses mTOR/S6K1 phosphorylation and induces leucine resistance in rats with sepsis

Although it has been known that protein synthesis is suppressed in sepsis, which cannot be corrected by leucine supplement (also known as leucine resistance), the molecular signaling mechanism remains unclear. This study aimed to investigate the AMP‐activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway in sepsis‐induced leucine resistance and its upstream signals, and to seek a way to correct leucine resistance in sepsis. Sepsis was produced by cecal ligation and puncture (CLP) model in rat. Both septic rats and sham operation rat received total parenteral nutrition (TPN) with or without leucine for 24 h, and then protein synthesis and AMPK/mTOR and protein kinase B (PKB) were tested. In vitro C2C12 cells were treated with or without leucine, and we tested the AMPK/mTOR pathway and protein synthesis. We blocked AMPK by compound C and stimulated it by 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) individually. The results showed that AMPK was highly phosphorylated and suppressed mTOR/S6K1 activation in CLP rats. In vitro when AMPK was activated by AICAR, protein synthesis was suppressed and leucine resistance was observed. High phosphorylation of AMPK was accompanied by PKB inactivation in CLP rats. When PKB was blocked, both AMPK activation and leucine resistance were observed. In CLP rats, nutrition support with intensive insulin therapy reversed leucine resistance by activating PKB and suppressing AMPK phosphorylation. These findings suggest that high phosphorylation of AMPK induced by PKB inactivation in sepsis suppresses mTOR, S6K1 phosphorylation, and protein synthesis and leads to leucine resistance. Intensive insulin treatment can reverse leucine resistance by suppressing AMPK activation through activation of PKB.     

Lyn kinase controls TLR4-dependent IKK and MAPK activation modulating the activity of TRAF-6/TAK-1 protein complex in mast cells

Mast cells (MCs) control allergic reactions and contribute to protective innate immune responses through TLR4 activation. The tyrosine kinase Lyn is important to the high affinity IgE receptor (FcεRI) signal transduction system in MCs, but its role on the TLR4 signalling cascade is still elusive. Here, we characterized several TLR4-triggered responses in bone marrow-derived mast cells (BMMCs) from wild-type (WT) and Lyn(-/-) mice. We found that Lyn(-/-) MCs secreted lower amounts of TNF-α after LPS challenge when compared with WT cells. Lyn(-/-) BMMCs showed less MAPK, IκB phosphorylation and NF-κB nuclear translocation after TLR-4 triggering than WT cells. LPS-induced MAPK and inhibitor of IκB kinase (IKK) phosphorylation were importantly reduced in the absence of Lyn. A constitutive interaction between TNF receptor associated factor 6 (TRAF-6) and phosphorylated TGF-β-activated kinase (TAK-1) was observed in Lyn(-/-) BMMCs and this complex was insensitive to LPS addition. Lyn kinase was activated and associated to TRAF-6 shortly after LPS addition in WT MCs. Analyzing two local MC-dependent innate immune responses in?vivo, we found that Lyn positively controls early TNF-α production and immune cell recruitment after an intraperitoneal injection of LPS. Our results indicate that Lyn plays a positive role in TLR4-induced production of TNF-α in MCs controlling the activity of the TRAF-6/TAK-1 protein complex.     

Mammalian target of rapamycin inhibition in macrophages of asymptomatic HIV+ persons reverses the decrease in TLR-4-mediated TNF-α release through prolongation of MAPK pathway activation

TLR-4-mediated signaling is significantly impaired in macrophages from HIV(+) persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4-mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4-mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R-associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4-mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV(+) macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4-mediated TNF-α release in HIV(+) macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV(+) persons.     

Improved insulin sensitivity by rapamycin is associated with reduction of mTOR and S6K1 activities in L6 myotubes

This study was designed to evaluate the role of mammalian target of rapamycin (mTOR)/p70S61 kinase (S6K1) pathways in ER stress-induced insulin resistance in L6 myotubes. Pretreatment with 5μg/ml of tunicamycin or 600nM thapsigargin for 3h decreased insulin-mediated tyrosine phosphorylation of IRS-1 and glucose uptake, and increased the level of mTOR/S6K1 phosphorylation in L6 myotubes. However, the inhibition of mTOR activity by rapamycin (inhibitor of several intracellular pathways including S6K1 pathways) reversed the ER stress-reduced tyrosine phosphorylation of IRS-1 and glucose uptake. Furthermore, pretreatment of cells with rapamycin decreased ER stress-induced phosphorylation of mTOR and S6K1. Interestingly, inhibition of mTOR by rapamycin did not affect ER stress markers such as PERK and JNK activity under the ER stress condition. Similar results were obtained with or without pretreatment with tunicamycin in the absence or presence of S6K1 RNAi. Moreover, S6K1 RNAi-mediated knockdown preserved insulin-stimulated Akt phosphorylation and glucose uptake in ER-stressed L6 myotubes, which was blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. Taken together, these results suggest that rapamycin improved ER stress-induced insulin resistance via inhibition of mTOR/S6K1 hyperphosphorylation in L6 myotubes.     

Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1

Nutrient overload leads to obesity, insulin resistance, and often type 2 diabetes. Whereas increased fat intake is commonly cited as the major factor in diet-induced dysmetabolic states, increased protein consumption also contributes, through elevated circulating amino acids. Recent studies have revealed that ribosomal protein S6 kinase 1, S6K1, an effector of mTOR, is sensitive to both insulin and nutrients, including amino acids. Although S6K1 is an effector of growth, recent reports show that amino acids also negatively affect insulin signaling through mTOR/S6K1 phosphorylation of IRS1. Moreover, rather than signaling through the class 1 PI3K pathway, amino acids appear to mediate mTOR activation through class 3 PI3K, or hVps34. Consistent with this, infusion of amino acids into humans leads to S6K1 activation, inhibition of insulin-induced class 1 PI3K activation, and insulin resistance. Thus, S6K1 may mediate deleterious effects, like insulin resistance, and potentially type 2 diabetes in the face of nutrient excess.     

Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts

The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.     

Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector.     

Alpha-synuclein overexpression negatively regulates insulin receptor substrate 1 by activating mTORC1/S6K1 signaling

Alpha-synuclein (α-Syn) is a major component of Lewy bodies, a pathological feature of Parkinson's and other neurodegenerative diseases collectively known as synucleinopathies. Among the possible mechanisms of α-Syn-mediated neurotoxicity is interference with cytoprotective pathways such as insulin signaling. Insulin receptor substrate (IRS)-1 is a docking protein linking IRs to downstream signaling pathways such as phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K)1; the latter exerts negative feedback control on insulin signaling, which is impaired in Alzheimer's disease. Our previous study found that α-Syn overexpression can inhibit protein phosphatase (PP)2A activity, which is involved in the protective mechanism of insulin signaling. In this study, we found an increase in IRS-1 phosphorylation at Ser636 and decrease in tyrosine phosphorylation, which accelerated IRS-1 turnover and reduced insulin-Akt signaling in α-Syn-overexpressing SK-N-SH cells and transgenic mice. The mTOR complex (C)1/S6K1 blocker rapamycin inhibited the phosphorylation of IRS-1 at Ser636 in cells overexpressing α-Syn, suggesting that mTORC1/S6K1 activation by α-Syn causes feedback inhibition of insulin signaling via suppression of IRS-1 function. α-Syn overexpression also inhibited PP2A activity, while the PP2A agonist C2 ceramide suppressed both S6K1 activation and IRS-1 Ser636 phosphorylation upon α-Syn overexpression. Thus, α-Syn overexpression negatively regulated IRS-1 via mTORC1/S6K1 signaling while activation of PP2A reverses this process. These results provide evidence for a link between α-Syn and IRS-1 that may represent a novel mechanism for α-Syn-associated pathogenesis.