Phosphorylation of PRAS40 on Thr246 by PKB/AKT facilitates efficient phosphorylation of Ser183 by mTORC1

Type 2 diabetes is associated with alterations in protein kinase B (PKB/Akt) and mammalian target of rapamycin complex 1 (mTORC1) signalling. The proline-rich Akt substrate of 40-kDa (PRAS40) is a component of mTORC1, which has a regulatory function at the intersection of the PKB/Akt and mTORC1 signalling pathway. Phosphorylation of PRAS40-Thr246 by PKB/Akt, and PRAS40-Ser183 and PRAS40-Ser221 by mTORC1 results in dissociation from mTORC1, and its binding to 14-3-3 proteins. Although all phosphorylation sites within PRAS40 have been implicated in 14-3-3 binding, substitution of Thr246 by Ala alone is sufficient to abolish 14-3-3 binding under conditions of intact mTORC1 signalling. This suggests that phosphorylation of PRAS40-Thr246 may facilitate efficient phosphorylation of PRAS40 on its mTORC1-dependent sites. In the present study, we investigated the mechanism of PRAS40-Ser183 phosphorylation in response to insulin. Insulin promoted PRAS40-Ser183 phosphorylation after a euglycaemic–hyperinsulinaemic clamp in human skeletal muscle. The insulin-induced PRAS40-Ser183 phosphorylation was further evidenced in vivo in rat skeletal and cardiac muscle, and in vitro in A14 fibroblasts, 3T3L1 adipocytes and L6 myotubes. Inhibition of mTORC1 by rapamycin or amino acid deprivation partially abrogated insulin-mediated PRAS40-Ser183 phosphorylation in cultured cell lines. However, lowering insulin-induced PRAS40-Thr246 phosphorylation using wortmannin or palmitate in cell lines, or by feeding rats a high-fat diet, completely abolished insulin-mediated PRAS40-Ser183 phosphorylation. In addition, replacement of Thr246 by Ala reduced insulin-mediated PRAS40-Ser183 phosphorylation. We conclude that PRAS40-Ser183 is a component of insulin action, and that efficient phosphorylation of PRAS40-Ser183 by mTORC1 requires the phosphorylation of PRAS40-Thr246 by PKB/Akt.     

PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) is positively regulated by amino acids and insulin. PRAS40 associates with mTORC1 (which contains raptor) but not mTORC2. PRAS40 interacts with raptor, and this requires an intact TOR-signaling (TOS) motif in PRAS40. Like TOS motif-containing proteins such as eIF4E-binding protein 1 (4E-BP1), PRAS40 is a substrate for phosphorylation by mTORC1. Consistent with this, starvation of cells of amino acids or treatment with rapamycin alters the phosphorylation of PRAS40. PRAS40 binds 14-3-3 proteins, and this requires both amino acids and insulin. Binding of PRAS40 to 14-3-3 proteins is inhibited by TSC1/2 (negative regulators of mTORC1) and stimulated by Rheb in a rapamycin-sensitive manner. This confirms that PRAS40 is a target for regulation by mTORC1. Small interfering RNA-mediated knockdown of PRAS40 impairs both the amino acid- and insulin-stimulated phosphorylation of 4E-BP1 and the phosphorylation of S6. However, this has no effect on the phosphorylation of Akt or TSC2 (an Akt substrate). These data place PRAS40 downstream of mTORC1 but upstream of its effectors, such as S6K1 and 4E-BP1.     

The mechanism of insulin-stimulated 4E-BP protein binding to mammalian target of rapamycin (mTOR) complex 1 and its contribution to mTOR complex 1 signaling

Insulin activation of mTOR complex 1 is accompanied by enhanced binding of substrates. We examined the mechanism and contribution of this enhancement to insulin activation of mTORC1 signaling in 293E and HeLa cells. In 293E, insulin increased the amount of mTORC1 retrieved by the transiently expressed nonphosphorylatable 4E-BP[5A] to an extent that varied inversely with the amount of PRAS40 bound to mTORC1. RNAi depletion of PRAS40 enhanced 4E-BP[5A] binding to ～70% the extent of maximal insulin, and PRAS40 RNAi and insulin together did not increase 4E-BP[5A] binding beyond insulin alone, suggesting that removal of PRAS40 from mTORC1 is the predominant mechanism of an insulin-induced increase in substrate access. As regards the role of increased substrate access in mTORC1 signaling, RNAi depletion of PRAS40, although increasing 4E-BP[5A] binding, did not stimulate phosphorylation of endogenous mTORC1 substrates S6K1(Thr(389)) or 4E-BP (Thr(37)/Thr(46)), the latter already ～70% of maximal in amino acid replete, serum-deprived 293E cells. In HeLa cells, insulin and PRAS40 RNAi also both enhanced the binding of 4E-BP[5A] to raptor but only insulin stimulated S6K1 and 4E-BP phosphorylation. Furthermore, Rheb overexpression in 293E activated mTORC1 signaling completely without causing PRAS40 release. In the presence of Rheb and insulin, PRAS40 release is abolished by Akt inhibition without diminishing mTORC1 signaling. In conclusion, dissociation of PRAS40 from mTORC1 and enhanced mTORC1 substrate binding results from Akt and mTORC1 activation and makes little or no contribution to mTORC1 signaling, which rather is determined by Rheb activation of mTOR catalytic activity, through mechanisms that remain to be fully elucidated.     

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.     

Identification of a proline-rich Akt substrate as a 14-3-3 binding partner

Akt (also called protein kinase B) is one of the major downstream targets of the phosphatidylinositol 3-kinase pathway. This protein kinase has been implicated in insulin signaling, stimulation of cellular growth, and inhibition of apoptosis as well as transformation of cells. Although a number of cellular proteins have been identified as putative targets of the enzyme, additional substrates may play a role in the varied responses elicited by this enzyme. We have used a combination of 14-3-3 binding and recognition by an antibody to the phosphorylation consensus of the enzyme to identify and isolate one of the major substrates of Akt, which is also a 14-3-3 binding protein. This 40-kDa protein, designated PRAS40, is a proline-rich Akt substrate. Demonstration that it is a substrate of Akt was accomplished by showing that 1) PRAS40 was phosphorylated in vitro by purified Akt on the same site that was phosphorylated in insulin-treated cells; 2) activation of an inducible Akt was alone sufficient to stimulate the phosphorylation of PRAS40; and 3) cells lacking Akt1 and Akt2 exhibit a diminished ability to phosphorylate this protein. Thus, PRAS40 is a novel substrate of Akt, the phosphorylation of which leads to the binding of this protein to 14-3-3.     

Proline-rich Akt substrate of 40kDa (PRAS40): a novel downstream target of PI3k/Akt signaling pathway

Modifications in signaling of the proline-rich Akt substrate of 40-kDa (PRAS40) pathway is implicated in type 2 diabetes and melanoma. PRAS40 is known for its ability to regulate the mammalian target of rapamycin complex 1 (mTORC1) kinase activity, possessing a key regulatory role at the cross point of signal transduction pathways activated by growth factor receptors. Recently it has been found that PRAS40 is regulated by its upstream phosphatidylinositol 3-kinase/Akt (PI3K/Akt) which is activated by many tyrosine kinase receptors growth factors including insulin-like growth factor 1. Also, PRAS40 functions downstream of mTORC1 and upstream from its effectors ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Phosphorylation of PRAS40 by Akt and mTORC1 disrupts the binding between mTORC1 and PRAS40, and relieves the inhibitory constraint of PRAS40 on mTORC1 activity. This review summarizes the signaling regulating PRAS40 phosphorylation, as well as the dual function of PRAS40 as substrate and inhibitor of mTORC1 upon growth factor stimulation and under pathophysiological conditions.     

Rapamycin does not improve insulin sensitivity despite elevated mammalian target of rapamycin complex 1 activity in muscles of ob/ob mice

Studies of cultured cells have indicated that the mammalian target of rapamycin complex 1 (mTORC1) mediates the development of insulin resistance. Because a role for mTORC1 in the development of skeletal muscle insulin resistance has not been established, we studied mTORC1 activity in skeletal muscles of ob/ob (OB) mice and wild-type (WT) mice. In vivo insulin action was assessed in muscles of mice 15 min following an intraperitoneal injection of insulin or an equivalent volume of saline. In the basal state, the phosphorylation of S6K on Thr(389), mTOR on Ser(2448), and PRAS40 on Thr(246) were increased significantly in muscles from OB mice compared with WT mice. The increase in basal mTORC1 signaling was associated with an increase in basal PKB phosphorylation on Thr(308) and Ser(473). In the insulin-stimulated state, no differences existed in the phosphorylation of S6K on Thr(389), but PKB phosphorylation on Thr(308) and Ser(473) was significantly reduced in muscles of OB compared with WT mice. Despite elevated mTORC1 activity in OB mice, rapamycin treatment did not improve either glucose tolerance or insulin tolerance. These results indicate that the insulin resistance of OB mice is mediated, in part, by factors other than mTORC1.     

Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40

Insulin stimulates protein synthesis and cell growth by activation of the protein kinases Akt (also known as protein kinase B, PKB) and mammalian target of rapamycin (mTOR). It was reported that Akt activates mTOR by phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2). However, in recent studies the physiological requirement of Akt phosphorylation of TSC2 for mTOR activation has been questioned. Here, we identify PRAS40 (proline-rich Akt/PKB substrate 40 kDa) as a novel mTOR binding partner that mediates Akt signals to mTOR. PRAS40 binds the mTOR kinase domain and its interaction with mTOR is induced under conditions that inhibit mTOR signalling, such as nutrient or serum deprivation or mitochondrial metabolic inhibition. Binding of PRAS40 inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. PRAS40 silencing inactivates insulin-receptor substrate-1 (IRS-1) and Akt, and uncouples the response of mTOR to Akt signals. Furthermore, PRAS40 phosphorylation by Akt and association with 14-3-3, a cytosolic anchor protein, are crucial for insulin to stimulate mTOR. These findings identify PRAS40 as an important regulator of insulin sensitivity of the Akt-mTOR pathway and a potential target for the treatment of cancers, insulin resistance and hamartoma syndromes.     

PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase

The heterotrimeric mTORC1 protein kinase nucleates a signaling network that promotes cell growth in response to insulin and becomes constitutively active in cells missing the TSC1 or TSC2 tumor suppressors. Insulin stimulates the phosphorylation of S6K1, an mTORC1 substrate, but it is not known how mTORC1 kinase activity is regulated. We identify PRAS40 as a raptor-interacting protein that binds to mTORC1 in insulin-deprived cells and whose in vitro interaction with mTORC1 is disrupted by high salt concentrations. PRAS40 inhibits cell growth, S6K1 phosphorylation, and rheb-induced activation of the mTORC1 pathway, and in vitro it prevents the great increase in mTORC1 kinase activity induced by rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which prevents its inhibition of mTORC1 in cells and in vitro. We propose that the relative strengths of the rheb- and PRAS40-mediated inputs to mTORC1 set overall pathway activity and that insulin activates mTORC1 through the coordinated regulation of both.     

Alcohol and PRAS40 knockdown decrease mTOR activity and protein synthesis via AMPK signaling and changes in mTORC1 interaction

The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/GβL and PRAS40. Previously, we reported that mTOR plays an important role in regulating protein synthesis in response to alcohol (EtOH). However, the mechanisms by which EtOH regulates mTORC1 activity have not been established. Here, we investigated the effect of EtOH on the phosphorylation and interaction of components of mTORC1 in C2C12 myocytes. We also examined the specific role that PRAS40 plays in this process. Incubation of myocytes with EtOH (100 mM, 24 h) increased raptor and PRAS40 phosphorylation. Likewise, there were increased levels of the PRAS40 upstream regulators Akt and IRS‐1. EtOH also caused changes in mTORC1 protein–protein interactions. EtOH enhanced the binding of raptor and PRAS40 with mTOR. These alterations occurred in concert with increased binding of 14‐3‐3 to raptor, while the PRAS40 and 14‐3‐3 interaction was not affected. The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to EtOH. PRAS40 KD increased raptor phosphorylation and its association with 14‐3‐3, whereas decreased GβL–mTOR binding. The effects of EtOH and PRAS40 KD were mediated by AMPK. Both factors increased in vitro AMPK activity towards the substrate raptor. In addition, KD enhanced the activity of AMPK towards TSC2. Collectively, our results indicate that EtOH stabilizes the association of raptor, PRAS40, and GβL with mTOR, while likewise increasing the interaction of raptor with 14‐3‐3. These data suggest a possible mechanism for the inhibitory effects of EtOH on mTOR kinase activity and protein synthesis in myocytes. J. Cell. Biochem. 109: 1172–1184, 2010. © 2010 Wiley‐Liss, Inc.     

Pharmacological and genetic evaluation of proposed roles of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and p90(RSK) in the control of mTORC1 protein signaling by phorbol esters

The mammalian target of rapamycin complex 1 (mTORC1) links the control of mRNA translation, cell growth, and metabolism to diverse stimuli. Inappropriate activation of mTORC1 can lead to cancer. Phorbol esters are naturally occurring products that act as potent tumor promoters. They activate isoforms of protein kinase C (PKCs) and stimulate the oncogenic MEK/ERK signaling cascade. They also activate mTORC1 signaling. Previous work indicated that mTORC1 activation by the phorbol ester PMA (phorbol 12-myristate 13-acetate) depends upon PKCs and may involve MEK. However, the precise mechanism(s) through which they activate mTORC1 remains unclear. Recent studies have implicated both the ERKs and the ERK-activated 90-kDa ribosomal S6 kinases (p90(RSK)) in activating mTORC1 signaling via phosphorylation of TSC2 (a regulator of mTORC1) and/or the mTORC1 component raptor. However, the relative importance of each of these kinases and phosphorylation events for the activation of mTORC1 signaling is unknown. The recent availability of MEK (PD184352) and p90(RSK) (BI-D1870) inhibitors of improved specificity allowed us to address the roles of these protein kinases in controlling mTORC1 in a variety of human and rodent cell types. In parallel, we used specific shRNAs against p90(RSK1) and p90(RSK2) to further test their roles in regulating mTORC1 signaling. Our data indicate that p90(RSKs) are dispensable for the activation of mTORC1 signaling by phorbol esters in all cell types tested. Our data also reveal striking diversity in the requirements for MEK/ERK in the control of mTORC1 between different cell types, pointing to additional signaling connections between phorbol esters and mTORC1, which do not involve MEK/ERK. This study provides important information for the design of efficient strategies to combat the hyperactivation of mTORC1 signaling by oncogenic pathways.     

PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding

Mammalian target of rapamycin (mTOR) functions in two distinct signaling complexes, mTORC1 and mTORC2. In response to insulin and nutrients, mTORC1, consisting of mTOR, raptor (regulatory-associated protein of mTOR), and mLST8, is activated and phosphorylates eukaryotic initiation factor 4E-binding protein (4EBP) and p70 S6 kinase to promote protein synthesis and cell size. Previously we found that activation of mTOR kinase in response to insulin was associated with increased 4EBP1 binding to raptor. Here we identify prolinerich Akt substrate 40 (PRAS40) as a binding partner for mTORC1. A putative TOR signaling motif, FVMDE, is identified in PRAS40 and shown to be required for interaction with raptor. Insulin stimulation markedly decreases the level of PRAS40 bound by mTORC1. Recombinant PRAS40 inhibits mTORC1 kinase activity in vivo and in vitro, and this inhibition depends on PRAS40 association with raptor. Furthermore, decreasing PRAS40 expression by short hairpin RNA enhances 4E-BP1 binding to raptor, and recombinant PRAS40 competes with 4E-BP1 binding to raptor. We, therefore, propose that PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.     

PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis

TOR (Target of Rapamycin) is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) based proteomic strategy to identify new mammalian TOR (mTOR) binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40) and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP) and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFalpha and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5) and was therefore named PRR5-Like (PRR5L). PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1) and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.     

The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1

The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor-binding protein that is phosphorylated directly by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) but not mTORC2 in vitro, predominantly at PRAS40 (Ser(183)). The binding of S6K1 and 4E-BP1 to raptor requires a TOR signaling (TOS) motif, which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids. PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe(129) to Ala). Immediately carboxyl-terminal to Phe(129) are two small hydrophobic amino acid followed by two acidic residues. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site, Ser(183), to Asp. PRAS40 (Ser(183)) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser(183)) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser(183)) and its binding to raptor. RNA interference-induced depletion of PRAS40 enhanced the amino acid-stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo and is therefore a potential locus of regulation.     

ER stress induces anabolic resistance in muscle cells through PKB-induced blockade of mTORC1

Background

Anabolic resistance is the inability to increase protein synthesis in response to an increase in amino acids following a meal. One potential mediator of anabolic resistance is endoplasmic reticulum (ER) stress. The purpose of the present study was to test whether ER stress impairs the response to growth factors and leucine in muscle cells.

Methods

Muscle cells were incubated overnight with tunicamycin or thapsigargin to induce ER stress and the activation of the unfolded protein response, mTORC1 activity at baseline and following insulin and amino acids, as well as amino acid transport were determined.

Results

ER stress decreased basal phosphorylation of PKB and S6K1 in a dose-dependent manner. In spite of the decrease in basal PKB phosphorylation, insulin (10–50 nM) could still activate both PKB and S6K1. The leucine (2.5–5 mM)-induced phosphorylation of S6K1 on the other hand was repressed by low concentrations of both tunicamycin and thapsigargin. To determine the mechanism underlying this anabolic resistance, several inhibitors of mTORC1 activation were measured. Tunicamycin and thapsigargin did not change the phosphorylation or content of either AMPK or JNK, both increased TRB3 mRNA expression and thapsigargin increased REDD1 mRNA. Tunicamycin and thapsigargin both decreased the basal phosphorylation state of PRAS40. Neither tunicamycin nor thapsigargin prevented phosphorylation of PRAS40 by insulin. However, since PKB is not activated by amino acids, PRAS40 phosphorylation remained low following the addition of leucine. Blocking PKB using a specific inhibitor had the same effect on both PRAS40 and leucine-induced phosphorylation of S6K1.

Conclusion

ER stress induces anabolic resistance in muscle cells through a PKB/PRAS40-induced blockade of mTORC1.     

Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1

Oxidants are well recognized for their capacity to reduce the phosphorylation of the mammalian target of rapamycin (mTOR) substrates, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1), thereby hindering mRNA translation at the level of initiation. mTOR functions to regulate mRNA translation by forming the signaling complex mTORC1 (mTOR, raptor, GβL). Insulin signaling to mTORC1 is dependent upon phosphorylation of Akt/PKB and the inhibition of the tuberous sclerosis complex (TSC1/2), thereby enhancing the phosphorylation of 4E-BP1 and S6K1. In this study we report the effect of H₂O₂ on insulin-stimulated mTORC1 activity and assembly using A549 and bovine aortic smooth muscle cells. We show that insulin stimulated the phosphorylation of TSC2 leading to a reduction in raptor–mTOR binding and in the quantity of proline-rich Akt substrate 40 (PRAS40) precipitating with mTOR. Insulin also increased 4E-BP1 coprecipitating with mTOR and the phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1. H₂O₂, on the other hand, opposed the effects of insulin by increasing raptor–mTOR binding and the ratio of PRAS40/raptor derived from the mTOR immunoprecipitates in both cell types. These effects occurred in conjunction with a reduction in 4E-BP1 phosphorylation and the 4E-BP1/raptor ratio. siRNA-mediated knockdown of PRAS40 in A549 cells partially reversed the effect of H₂O₂ on 4E-BP1 phosphorylation but not on S6K1. These findings are consistent with PRAS40 functioning as a negative regulator of insulin-stimulated mTORC1 activity during oxidant stress.     

Curcumin represses mTORC1 signaling in Caco-2 cells by a two-sided mechanism involving the loss of IRS-1 and activation of AMPK

The mechanistic target of rapamycin complex 1 (mTORC1) is a central modulator of inflammation and tumorigenesis in the gastrointestinal tract. Growth factors upregulate mTORC1 via the PI3K/AKT and/or Ras/MAPK signal pathways. Curcumin (CUR), a polyphenol found in turmeric roots (Curcuma longa) can repress mTORC1 kinase activity in colon cancer cell lines; however, key aspects of CUR mechanism of action remain to be elucidated including its primary cellular target. We investigated the molecular effects of physiologically attainable concentration of CUR (20 μM) in the intestinal lumen on mTORC1 signaling in Caco-2 cells. CUR markedly inhibited mTORC1 kinase activity as determined by the decreased phosphorylation of p70S6K (Thr389, −99%, P < 0.0001) and S6 (Ser235/236, −92%, P < 0.0001). Mechanistically, CUR decreased IRS-1 protein abundance (−80%, P < 0.0001) thereby downregulating AKT phosphorylation (Ser473, −94%, P < 0.0001) and in turn PRAS40 phosphorylation (Thr246, −99%, P < 0.0001) while total PRAS40 abundance was unchanged. The use of proteasome inhibitor MG132 showed that CUR-mediated loss of IRS-1 involved proteasomal degradation. CUR lowered Raptor protein abundance, which combined with PRAS40 hypophosphorylation, suggests CUR repressed mTORC1 activity by inducing compositional changes that hinder the complex assembly. In addition, CUR activated AMPK (Thr172 phosphorylation, P < 0.0001), a recognized repressor of mTORC1, and AMPK upstream regulator LKB1. Although cargo adapter protein p62 was decreased by CUR (−49%, P < 0.004), CUR did not significantly induce autophagy. Inhibition of AKT/mTORC1 signaling by CUR may have lifted the cross-inhibition onto MAPK signaling, which became induced; p-ERK1/2 (+670%, P < 0.0001), p-p38 (+1433%, P < 0.0001). By concomitantly targeting IRS-1 and AMPK, CUR's mechanism of mTORC1 inhibition is distinct from that of rapamycin.     

RhoA modulates signaling through the mechanistic target of rapamycin complex 1 (mTORC1) in mammalian cells

The mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1) pathway integrates signals generated by hormones and nutrients to control cell growth and metabolism. The activation state of mTORC1 is regulated by a variety of GTPases including Rheb and Rags. Recently, Rho1, the yeast ortholog of RhoA, was shown to interact directly with TORC1 and repress its activation state in yeast. Thus, the purpose of the present study was to test the hypothesis that the RhoA GTPase modulates signaling through mTORC1 in mammalian cells. In support of this hypothesis, exogenous overexpression of either wild type or constitutively active (ca)RhoA repressed mTORC1 signaling as assessed by phosphorylation of p70S6K1 (Thr389), 4E-BP1 (Ser65) and ULK1 (Ser757). Additionally, RhoA·GTP repressed phosphorylation of mTORC1-associated mTOR (Ser2481). The RhoA·GTP mediated repression of mTORC1 signaling occurred independent of insulin or leucine induced stimulation. In contrast to the action of Rho1 in yeast, no evidence was found to support a direct interaction of RhoA·GTP with mTORC1. Instead, expression of caRheb, but not caRags, was able to rescue the RhoA·GTP mediated repression of mTORC1 suggesting RhoA functions upstream of Rheb to repress mTORC1 activity. Consistent with this suggestion, RhoA·GTP repressed phosphorylation of TSC2 (Ser939), PRAS40 (Thr246), Akt (Ser473), and mTORC2-associated mTOR (Ser2481). Overall, the results support a model in which RhoA·GTP represses mTORC1 signaling upstream of Akt and mTORC2.     

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.