Simultaneous inhibition of mTOR-containing complex 1 (mTORC1) and MNK induces apoptosis of cutaneous T-cell lymphoma (CTCL) cells

Background

mTOR kinase forms the mTORC1 complex by associating with raptor and other proteins and affects a number of key cell functions. mTORC1 activates p70S6kinase 1 (p70S6K1) and inhibits 4E-binding protein 1 (4E-BP1). In turn, p70S6K1 phosphorylates a S6 protein of the 40S ribosomal subunit (S6rp) and 4E-BP1, with the latter negatively regulating eukaryotic initiation factor 4E (eIF-4E). MNK1 and MNK2 kinases phosphorylate and augment activity of eIF4E. Rapamycin and its analogs are highly specific, potent, and relatively non-toxic inhibitors of mTORC1. Although mTORC1 activation is present in many types of malignancies, rapamycin-type inhibitors shows relatively limited clinical efficacy as single agents. Initially usually indolent, CTCL displays a tendency to progress to the aggressive forms with limited response to therapy and poor prognosis. Our previous study (M. Marzec et al. 2008) has demonstrated that CTCL cells display mTORC1 activation and short-term treatment of CTCL-derived cells with rapamycin suppressed their proliferation and had little effect on the cell survival.

Methods

Cells derived from CTCL were treated with mTORC1 inhibitor rapamycin and MNK inhibitor and evaluated for inhibition of the mTORC1 signaling pathway and cell growth and survival.

Results

Whereas the treatment with rapamycin persistently inhibited mTORC1 signaling, it suppressed only partially the cell growth. MNK kinase mediated the eIF4E phosphorylation and inhibition or depletion of MNK markedly suppressed proliferation of the CTCL cells when combined with the rapamycin-mediated inhibition of mTORC1. While MNK inhibition alone mildly suppressed the CTCL cell growth, the combined MNK and mTORC1 inhibition totally abrogated the growth. Similarly, MNK inhibitor alone displayed a minimal pro-apoptotic effect; in combination with rapamycin it triggered profound cell apoptosis.

Conclusions

These findings indicate that the combined inhibition of mTORC1 and MNK may prove beneficial in the treatment of CTCL and other malignancies.     

Distribution and association of mTOR with its cofactors,raptor and rictor,in cumulus cells and oocytes during meiotic maturation in mice

Mammalian target of rapamycin (mTOR), a Ser/Thr protein kinase, is the catalytic component of two distinct signaling complexes, mTOR‐raptor complex (mTORC1) and mTOR‐rictor complex (mTORC2). Recently, studies have demonstrated mitosis‐specific roles for mTORC1, but the functions and expression dynamics of mTOR complexes during meiotic maturation remain unclear. In the present study, to evaluate the roles of respective mTOR complexes in maternal meiosis and compare them with those in mitosis, we sought to elucidate the spatiotemporal immunolocalization of mTOR, the kinase‐active Ser2448‐ and Ser2481‐phosphorylated mTOR, and raptor and rictor during cumulus‐cell mitosis and oocyte meiotic maturation in mice. mTOR principally accumulated around the chromosomes and on the spindle. Phosphorylated mTOR (Ser2448 and Ser2481) exhibited elevated fluorescence intensities in the cytoplasm and punctate localization adjacent to the chromosomes, on the spindle poles, and on the midbody during mitotic and meiotic maturation, suggesting functional homology of mTOR between the two cell division systems, despite their mechanistically distinctive spindles. Raptor colocalized with mTOR during both types of cell division, indicating that mTORC1 is predominantly associated with these events. Mitotic rictor uniformly distributed through the cytoplasm, and meiotic rictor localized around the spindle poles of metaphase‐I oocytes, suggesting functional divergence of mTORC2 between mitosis and female meiosis. Based on the general function of mTORC2 in the organization of the actin cytoskeleton, we propose that mTORC1 controls spindle function during mitosis and meiosis, while mTORC2 contributes to actin‐dependent asymmetric division during meiotic maturation in mice. Mol. Reprod. Dev. 80: 334–348, 2013. © 2013 Wiley Periodicals, Inc.     

Metformin Protects Rat Hepatocytes against Bile Acid-Induced Apoptosis

Background

Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD). Metformin activates AMP-activated protein kinase (AMPK), the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR). Both AMPK and mTOR are able to modulate cell death.

Aim

To evaluate the effects of metformin on hepatocyte cell death.

Methods

Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA) or TNFα in combination with actinomycin D (actD). AMPK, mTOR and phosphoinositide-3 kinase (PI3K)/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively.

Results

Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation.

Conclusion

Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.     

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

Aims

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.

Methods

We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.

Results

In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC₅₀ concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.

Conclusion

Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.     

Mitotic Raptor Promotes mTORC1 Activity,G2/M Cell Cycle Progression,and Internal Ribosome Entry Site-Mediated mRNA Translation

The mTOR signaling complex integrates signals from growth factors and nutrient availability to control cell growth and proliferation, in part through effects on the protein-synthetic machinery. Protein synthesis rates fluctuate throughout the cell cycle but diminish significantly during the G₂/M transition. The fate of the mTOR complex and its role in coordinating cell growth and proliferation signals with protein synthesis during mitosis remain unknown. Here we demonstrate that the mTOR complex 1 (mTORC1) pathway, which stimulates protein synthesis, is actually hyperactive during mitosis despite decreased protein synthesis and reduced activity of mTORC1 upstream activators. We describe previously unknown G₂/M-specific phosphorylation of a component of mTORC1, the protein raptor, and demonstrate that mitotic raptor phosphorylation alters mTORC1 function during mitosis. Phosphopeptide mapping and mutational analysis demonstrate that mitotic phosphorylation of raptor facilitates cell cycle transit through G₂/M. Phosphorylation-deficient mutants of raptor cause cells to delay in G₂/M, whereas depletion of raptor causes cells to accumulate in G₁. We identify cyclin-dependent kinase 1 (cdk1 [cdc2]) and glycogen synthase kinase 3 (GSK3) pathways as two probable mitosis-regulated protein kinase pathways involved in mitosis-specific raptor phosphorylation and altered mTORC1 activity. In addition, mitotic raptor promotes translation by internal ribosome entry sites (IRES) on mRNA during mitosis and is demonstrated to be associated with rapamycin resistance. These data suggest that this pathway may play a role in increased IRES-dependent mRNA translation during mitosis and in rapamycin insensitivity.Cell growth and cell division are tightly coordinated processes required for cells to remain equal in size after division. In unicellular organisms, cell growth and proliferation are coordinated by nutrient availability, whereas their multicellular counterparts must also respond to growth factor input. Both processes lead to organismal growth as well as to increased cell number and cell mass. Cell growth and cell proliferation are also linked via the mTOR signaling pathway (16, 17). The mTOR kinase forms a distinct signaling complex (mTORC1) that participates in the coordination of nutrient and growth factor signaling. mTORC1 is composed of the kinase mTOR, the adaptor protein raptor, and the regulatory protein LST8 (25, 33, 34, 72).Accumulation of cellular proteins leads to cell growth and cell division. However, cell growth occurs only during certain phases of the cell cycle, necessitating that protein synthesis rates oscillate during cell cycling (40). In addition, in quiescent cells in G₀, protein synthesis rates are significantly reduced, whereas a select group of mRNAs maintain active translation (20, 68). During the G₁ phase, overall protein synthesis rates increase through S phase to allow cells to grow and enter another round of cell division while maintaining cell size (2, 3, 42, 45). As with G₀, entrance into mitosis (G₂/M phase) results in a global downregulation by as much as 60 to 80% of cap-dependent mRNA translation in primary, immortalized, and some transformed cells (5, 14, 29).Studies report several possible mechanisms for inhibition of protein synthesis during mitosis. Translation initiation requires the formation of an initiation factor complex known as eukaryotic translation initiation factor 4F (eIF4F), which consists of cap binding protein eIF4E, molecular scaffold protein eIF4G, and RNA helicase eIF4A. Together, they recruit ribosomes to mRNAs via bridging interactions between the 7-methyl-GTP (m⁷GTP) 5′ cap and the small 40S ribosomal subunit. Downregulation of protein synthesis during G₂/M was first ascribed to hypophosphorylation of eIF4E and the eIF4E binding proteins (4E-BPs) (5, 46). 4E-BPs are activated by hypophosphorylation, which allows them to bind and sequester eIF4E, preventing it from binding eIF4G and thereby blocking cap-dependent mRNA translation. More recently, several studies suggest that 4E-BP1, the major 4E-BP and a key target of mTORC1, is actually hyperphosphorylated (inactivated) during mitosis (26, 49). It is puzzling, then, that the phosphatidylinositol 3-kinase (PI3K)/AKT network and AKT itself (which modulate mTORC1 activity) are reportedly inactivated during late mitosis (1, 9, 22). In addition, phosphorylation of another mTORC1 target, ribosomal S6 kinase 1 (S6K1), and its activity are actually highest during G₂/M phase, consistent with elevated mTORC1 activity during mitosis (6).In this study we show that, despite repression of AKT and other activators of mTORC1 activity in mitosis, mTORC1 remains active and phosphorylates 4E-BP1 and S6K1 during G₂/M. We describe the multisite phosphorylation of raptor during mitosis, and we identify seven mitosis-specific raptor phosphorylation sites. By developing phosphomimetic and phosphorylation-deficient mutants of raptor, we show that hyperphosphorylated raptor promotes cell cycle transit through G₂/M, whereas hypophosphorylated raptor promotes transit through G₁. Raptor phosphorylation is shown to involve kinase pathways that are known to be active during mitosis, including cyclin-dependent kinase 1 (cdk1 [cdc2]) and glycogen synthase kinase 3 (GSK3) pathways that are also upregulated in certain human cancers, including breast cancers. These and other findings disclose a novel regulatory network for mTORC1 that is active during mitosis, important for G₂/M progression and increased internal ribosome entry site (IRES)-dependent translation during mitosis, and indirectly associated with rapamycin resistance.     

Rapamycin Ameliorates Inflammation and Fibrosis in the Early Phase of Cirrhotic Portal Hypertension in Rats through Inhibition of mTORC1 but Not mTORC2

Objective

Hepatic stellate cells (HSCs) transdifferentiation and subsequent inflammation are important pathological processes involved in the formation of cirrhotic portal hypertension. This study characterizes the pathogenetic mechanisms leading to cholestatic liver fibrosis and portal hypertension, and focuses on mammalian target of rapamycin (mTOR) pathway as a potential modulator in the early phase of cirrhotic portal hypertension.

Methods

Early cirrhotic portal hypertension was induced by bile duct ligation (BDL) for three weeks. One week after operation, sham-operated (SHAM) and BDL rats received rapamycin (2 mg/kg/day) by intraperitoneal injection for fourteen days. Vehicle-treated SHAM and BDL rats served as controls. Fibrosis, inflammation, and portal pressure were evaluated by histology, morphometry, and hemodynamics. Expressions of pro-fibrogenic and pro-inflammatory genes in liver were measured by RT-PCR; alpha smooth muscle actin (α-SMA) and antigen Ki67 were detected by immunohistochemistry; expressions of AKT/mTOR signaling molecules, extracellular-signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, and interleukin-1 beta (IL-1β) were assessed by western blot.

Results

The AKT/mTOR signaling pathway was markedly activated in the early phase of cirrhotic portal hypertension induced by BDL in rats. mTOR blockade by rapamycin profoundly improved liver function by limiting inflammation, fibrosis and portal pressure. Rapamycin significantly inhibited the expressions of phosphorylated 70KD ribosomal protein S6 kinase (p-P70S6K) and phosphorylated ribosomal protein S6 (p-S6) but not p-AKT Ser473 relative to their total proteins in BDL-Ra rats. Those results suggested that mTOR Complex 1 (mTORC1) rather than mTORC2 was inhibited by rapamycin. Interestingly, we also found that the level of p-ERK1/2 to ERK1/2 was significantly increased in BDL rats, which was little affected by rapamycin.

Conclusions

The AKT/mTOR signaling pathway played an important role in the early phase of cirrhotic portal hypertension in rats, which could be a potential target for therapeutic intervention in the early phase of such pathophysiological progress.     

AMPK phosphorylation of raptor mediates a metabolic checkpoint

AMPK is a highly conserved sensor of cellular energy status that is activated under conditions of low intracellular ATP. AMPK responds to energy stress by suppressing cell growth and biosynthetic processes, in part through its inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK phosphorylation of the TSC2 tumor suppressor contributes to suppression of mTORC1; however, TSC2-deficient cells remain responsive to energy stress. Using a proteomic and bioinformatics approach, we sought to identify additional substrates of AMPK that mediate its effects on growth control. We report here that AMPK directly phosphorylates the mTOR binding partner raptor on two well-conserved serine residues, and this phosphorylation induces 14-3-3 binding to raptor. The phosphorylation of raptor by AMPK is required for the inhibition of mTORC1 and cell-cycle arrest induced by energy stress. These findings uncover a conserved effector of AMPK that mediates its role as a metabolic checkpoint coordinating cell growth with energy status.     

Distinct Pools of cdc25C Are Phosphorylated on Specific TP Sites and Differentially Localized in Human Mitotic Cells

Background

The dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1.

Methodology/Principal Findings

Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD) binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis.

Conclusions/Significance

These results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C/cdk1 multi-site auto amplification loop is implausible.     

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.     

mTOR phosphorylated at S2448 binds to raptor and rictor

In mammalian cells, the mammalian target of rapamycin (mTOR) forms an enzyme complex with raptor (together with other proteins) named mTOR complex 1 (mTORC1), of which a major target is the p70 ribosomal protein S6 kinase (p70S6K). A second enzyme complex, mTOR complex 2 (mTORC2), contains mTOR and rictor and regulates the Akt kinase. Both mTORC1 and mTORC2 are regulated by phosphorylation, complex formation and localization. So far, the role of p70S6K-mediated mTOR S2448 phosphorylation has not been investigated in detail. Here, we report that endogenous mTOR phosphorylated at S2448 binds to both, raptor and rictor. Experiments with chemical inhibitors of the mTOR kinase and of the phosphatidylinositol-3-kinase revealed that downregulation of mTOR S2448 phosphorylation correlates with decreased mTORC1 activity but can occur decoupled of effects on mTORC2 activity. In addition, we found that the correlation of the mTOR S2448 phosphorylation status with mTORC1 activity is not a consequence of effects on the assembly of mTOR protein and raptor. Our data allow new insights into the role of mTOR phosphorylation for the regulation of its kinase activity.     

Mammalian Target of Rapamycin Complex 1 (mTORC1) Activity Is Associated with Phosphorylation of Raptor by mTOR

mTORC1 contains multiple proteins and plays a central role in cell growth and metabolism. Raptor (regulatory-associated protein of mammalian target of rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential for mTORC1 activity and critical for the regulation of mTORC1 activity in response to insulin signaling and nutrient and energy sufficiency. Herein we demonstrate that mTOR phosphorylates raptor in vitro and in vivo. The phosphorylated residues were identified by using phosphopeptide mapping and mutagenesis. The phosphorylation of raptor is stimulated by insulin and inhibited by rapamycin. Importantly, the site-directed mutation of raptor at one phosphorylation site, Ser⁸⁶³, reduced mTORC1 activity both in vitro and in vivo. Moreover, the Ser⁸⁶³ mutant prevented small GTP-binding protein Rheb from enhancing the phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate that mTOR-mediated raptor phosphorylation plays an important role on activation of mTORC1.Mammalian target of rapamycin (mTOR)² has been shown to function as a critical controller in cellular growth, survival, metabolism, and development (1). mTOR, a highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase, structurally forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), each of which catalyzes the phosphorylation of different substrates (1). The best characterized substrates for mTORC1 are eIF4E-binding protein (4E-BP, also known as PHAS) and p70 S6 kinase (S6K) (1), whereas mTORC2 phosphorylates the hydrophobic and turn motifs of protein kinase B (Akt/protein kinase B) (2) and protein kinase C (3, 4). mTORC1 constitutively consists of mTOR, raptor, and mLst8/GβL (1), whereas the proline-rich Akt substrate of 40 kDa (PRAS40) is a regulatory component of mTORC1 that disassociates after growth factor stimulation (5, 6). Raptor is essential for mTORC1 activity by providing a substrate binding function (7) but also plays a regulatory role on mTORC1 with stimuli of growth factors and nutrients (8). In response to insulin, raptor binding to substrates is elevated through the release of the competitive inhibitor PRAS40 from mTORC1 (9, 10) because PRAS40 and the substrates of mTORC1 (4E-BP and S6K) appear to bind raptor through a consensus sequence, the TOR signaling (TOS) motif (10–14). In response to amino acid sufficiency, raptor directly interacts with a heterodimer of Rag GTPases and promotes mTORC1 localization to the Rheb-containing vesicular compartment (15).mTORC1 integrates signaling pathways from growth factors, nutrients, energy, and stress, all of which generally converge on the tuberous sclerosis complex (TSC1-TSC2) through the phosphorylation of TSC2 (1). Growth factors inhibit the GTPase-activating protein activity of TSC2 toward the small GTPase Rheb via the PI3K/Akt pathway (16, 17), whereas energy depletion activates TSC2 GTPase-activating protein activity by stimulating AMP-activated protein kinase (AMPK) (18). Rheb binds directly to mTOR, albeit with very low affinity (19), and upon charging with GTP, Rheb functions as an mTORC1 activator (6). mTORC1 complexes isolated from growth factor-stimulated cells show increased kinase activity yet do not contain detectable levels of associated Rheb. Therefore, how Rheb-GTP binding to mTOR leads to an increase in mTORC1 activity toward substrates, and what the role of raptor is in this activation is currently unknown. More recently, the AMPK and p90 ribosomal S6 kinase (RSK) have been reported to directly phosphorylate raptor and regulate mTORC1 activity. The phosphorylation of raptor directly by AMPK reduced mTORC1 activity, suggesting an alternative regulation mechanism independent of TSC2 in response to energy supply (20). RSK-mediated raptor phosphorylation enhances mTORC1 activity and provides a mechanism whereby stress may activate mTORC1 independent of the PI3K/Akt pathway (21). Therefore, the phosphorylation status of raptor can be critical for the regulation of mTORC1 activity.In this study, we investigated phosphorylation sites in raptor catalyzed by mTOR. Using two-dimensional phosphopeptide mapping, we found that Ser⁸⁶³ and Ser⁸⁵⁹ in raptor were phosphorylated by mTOR both in vivo and in vitro. mTORC1 activity in vitro and in vivo is associated with the phosphorylation of Ser⁸⁶³ in raptor.     

Screen for Chemical Modulators of Autophagy Reveals Novel Therapeutic Inhibitors of mTORC1 Signaling

Background

Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy, a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties.

Methodology/Principal Findings

Using an automated cell-based assay, we screened a collection of >3,500 chemicals and identified three approved drugs (perhexiline, niclosamide, amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2, a negative regulator of mTORC1, was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline, niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions, by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2, an upstream regulator of mTORC1. By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation.

Conclusion/Significance

The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis, diabetes, cardiovascular disease and cancer.     

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.     

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.     

Inhibition of mTORC1 induces loss of E-cadherin through AKT/GSK-3β signaling-mediated upregulation of E-cadherin repressor complexes in non-small cell lung cancer cells

Background

mTOR, which can form mTOR Complex 1 (mTORC1) or mTOR Complex 2 (mTORC2) depending on its binding partners, is frequently deregulated in the pulmonary neoplastic conditions and interstitial lung diseases of the patients treated with rapalogs. In this study, we investigated the relationship between mTOR signaling and epithelial mesenchymal transition (EMT) by dissecting mTOR pathways.

Methods

Components of mTOR signaling pathway were silenced by shRNA in a panel of non-small cell lung cancer cell lines and protein expression of epithelial and mesenchymal markers were evaluated by immunoblotting and immunocytochemistry. mRNA level of the E-cadherin repressor complexes were evaluated by qRT-PCR.

Results

IGF-1 treatment decreased expression of the E-cadherin and rapamycin increased its expression, suggesting hyperactivation of mTOR signaling relates to the loss of E-cadherin. Genetic ablation of rapamycin-insensitive companion of mTOR (Rictor), a component of mTORC2, did not influence E-cadherin expression, whereas genetic ablation of regulatory-associated protein of mTOR (Raptor), a component of mTORC1, led to a decrease in E-cadherin expression at the mRNA level. Increased phosphorylation of AKT at Ser473 and GSK-3β at Ser9 were observed in the Raptor-silenced NSCLC cells. Of the E-cadherin repressor complexes tested, Snail, Zeb2, and Twist1 mRNAs were elevated in raptor-silenced A549 cells, and Zeb2 and Twist1 mRNAs were elevated in Raptor-silenced H2009 cells. These findings were recapitulated by treatment with the GSK-3β inhibitor, LiCl. Raptor knockdown A549 cells showed increased expression of N-cadherin and vimentin with mesenchymal phenotypic changes.

Conclusions

In conclusion, selective inhibition of mTORC1 leads to hyperactivation of the AKT/GSK-3β pathway, inducing E-cadherin repressor complexes and EMT. These findings imply the existence of a feedback inhibition loop of mTORC1 onto mTORC2 that plays a role in the homeostasis of E-cadherin expression and EMT, requiring caution in the clinical use of rapalog and selective mTORC1 inhibitors.     

Amino acid signaling to mTOR mediated by inositol polyphosphate multikinase

mTOR complex 1 (mTORC1; mammalian target of rapamycin [mTOR] in complex with raptor) is a key regulator of protein synthesis and cell growth in response to nutrient amino acids. Here we report that inositol polyphosphate multikinase (IPMK), which possesses both inositol phosphate kinase and lipid kinase activities, regulates amino acid signaling to mTORC1. This regulation is independent of IPMK's catalytic function, instead reflecting its binding with mTOR and raptor, which maintains the mTOR-raptor association. Thus, IPMK appears to be a physiologic mTOR cofactor, serving as a determinant of mTORC1 stability and amino acid-induced mTOR signaling. Substances that block IPMK-mTORC1 binding may afford therapeutic benefit in nutrient amino acid-regulated conditions such as obesity and diabetes.     

cdc2-cyclin B regulates eEF2 kinase activity in a cell cycle- and amino acid-dependent manner

The calcium/calmodulin-dependent kinase that phosphorylates and inactivates eukaryotic elongation factor 2 (eEF2 kinase; eEF2K) is subject to multisite phosphorylation, which regulates its activity. Phosphorylation at Ser359 inhibits eEF2K activity even at high calcium concentrations. To identify the kinase that phosphorylates Ser359 in eEF2K, we developed an extensive purification protocol. Tryptic mass fingerprint analysis identified it as cdc2 (cyclin-dependent kinase 1). cdc2 co-purifies with Ser359 kinase activity and cdc2-cyclin B complexes phosphorylate eEF2K at Ser359. We demonstrate that cdc2 contributes to controlling eEF2 phosphorylation in cells. cdc2 is activated early in mitosis. Kinase activity against Ser359 in eEF2K also peaks at this stage of the cell cycle and eEF2 phosphorylation is low in mitotic cells. Inactivation of eEF2K by cdc2 may serve to keep eEF2 active during mitosis (where calcium levels rise) and thereby permit protein synthesis to proceed in mitotic cells. Amino-acid starvation decreases cdc2's activity against eEF2K, whereas loss of TSC2 (a negative regulator of mammalian target of rapamycin complex 1(mTORC1)) increases it. These data closely match the control of Ser359 phosphorylation and indicate that cdc2 may be regulated by mTORC1.     

Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1

Background

Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known.

Methodology/Principal Findings

Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus.

Conclusions/Significance

hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.