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ABSTRACT: Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and autophagy. Its activity is regulated by the availability of amino acids and growth factors. The activation of mTORC1 by growth factors, such as insulin and insulin-like growth factor-1 (IGF-1), is mediated by tuberous sclerosis complex (TSC) 1 and 2 and Rheb GTPase. Relative to the growth factor-regulated mTORC1 pathway, the evolutionarily ancient amino acid-mTORC1 pathway remains not yet clearly defined. The amino acid-mTORC1 pathway is mediated by Rag GTPase heterodimers. Several binding proteins of Rag GTPases were discovered in recent studies. Here, we discuss the functions and mechanisms of the newly-identified binders of Rag GTPases. In particular, this review focuses on SH3 binding protein 4 (SH3BP4), the protein recently identifed as a negative regulator of Rag GTPases.
Molecules and Cells 12/2012; · 2.18 Impact Factor
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ABSTRACT: Amino acids stimulate cell growth and suppress autophagy through activation of mTORC1. The activation of mTORC1 by amino acids is mediated by Rag guanosine triphosphatase (GTPase) heterodimers on the lysosome. The molecular mechanism by which amino acids regulate the Rag GTPase heterodimers remains to be elucidated. Here, we identify SH3 domain-binding protein 4 (SH3BP4) as a binding protein and a negative regulator of Rag GTPase complex. SH3BP4 binds to the inactive Rag GTPase complex through its Src homology 3 (SH3) domain under conditions of amino acid starvation and inhibits the formation of active Rag GTPase complex. As a consequence, the binding abrogates the interaction of mTORC1 with Rag GTPase complex and the recruitment of mTORC1 to the lysosome, thus inhibiting amino acid-induced mTORC1 activation and cell growth and promoting autophagy. These results demonstrate that SH3BP4 is a negative regulator of the Rag GTPase complex and amino acid-dependent mTORC1 signaling.
Molecular cell 05/2012; 46(6):833-46. · 14.61 Impact Factor
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ABSTRACT: ULK1 (Unc51-like kinase, hATG1) is a Ser/Thr kinase that plays a key role in inducing autophagy in response to starvation. ULK1 is phosphorylated and negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that ULK1 is not only a downstream effector of mTORC1 but also a negative regulator of mTORC1 signaling. ( 1-3) Here, we investigated how ULK1 regulates mTORC1 signaling, and found that ULK1 inhibits the kinase activity of mTORC1 and cell proliferation. Deficiency or knockdown of ULK1 or its homolog ULK2 enhanced mTORC1 signaling, cell proliferation rates and accumulation of cell mass, whereas overexpression of ULK1 had the opposite effect. Knockdown of Atg13, the binding partner of ULK1 and ULK2, mimicked the effects of ULK1 or ULK2 deficiency or knockdown. Both insulin and leucine stimulated mTORC1 signaling to a greater extent when ULK1 or ULK2 was deficient or knocked down. In contrast, Atg5 deficiency did not have a significant effect on mTORC1 signaling and cell proliferation. The stimulatory effect of ULK1 knockdown on mTORC1 signaling occurred even in the absence of tuberous sclerosis complex 2 (TSC2), the negative regulator of mTORC1 signaling. In addition, ULK1 was found to bind raptor, induce its phosphorylation, and inhibit the kinase activity of mTORC1. These results demonstrate that ULK1 negatively regulates the kinase activity of mTORC1 and cell proliferation in a manner independent of Atg5 and TSC2. The inhibition of mTORC1 by ULK1 may be important to coordinately regulate cell growth and autophagy with optimized utilization of cellular energy.
Autophagy 10/2011; 7(10):1212-21. · 7.45 Impact Factor
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Joung Hyuck Joo,
Frank C Dorsey,
Aashish Joshi,
Kristin M Hennessy-Walters,
Kristie L Rose,
Kelly McCastlain,
Ji Zhang,
Rekha Iyengar,
Chang Hwa Jung,
Der-Fen Suen,
Meredith A Steeves,
Chia-Ying Yang,
Stephanie M Prater, Do-Hyung Kim,
Craig B Thompson,
Richard J Youle,
Paul A Ney,
John L Cleveland,
Mondira Kundu
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ABSTRACT: Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologs of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1, and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13-directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.
Molecular cell 08/2011; 43(4):572-85. · 14.61 Impact Factor
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ABSTRACT: The mammalian target of rapamycin complex 1 (mTORC1) is a molecular hub that regulates protein synthesis in response to a number of extracellular stimuli. Cyclic AMP (cAMP) is considered to be an important second messenger that controls mTOR; however, the signaling components of this pathway have not yet been elucidated. Here, we identify cAMP phosphodiesterase 4D (PDE4D) as a binding partner of Rheb that acts as a cAMP-specific negative regulator of mTORC1. Under basal conditions, PDE4D binds Rheb in a noncatalytic manner that does not require its cAMP-hydrolyzing activity and thereby inhibits the ability of Rheb to activate mTORC1. However, elevated cAMP levels disrupt the interaction of PDE4D with Rheb and increase the interaction between Rheb and mTOR. This enhanced Rheb-mTOR interaction induces the activation of mTORC1 and cap-dependent translation, a cellular function of mTORC1. Taken together, our results suggest a novel regulatory mechanism for mTORC1 in which the cAMP-determined dynamic interaction between Rheb and PDE4D provides a key, unique regulatory event. We also propose a new role for PDE4 as a molecular transducer for cAMP signaling.
Molecular and cellular biology 11/2010; 30(22):5406-20. · 6.06 Impact Factor
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ABSTRACT: Nutrient starvation induces autophagy in eukaryotic cells through inhibition of TOR (target of rapamycin), an evolutionarily-conserved protein kinase. TOR, as a central regulator of cell growth, plays a key role at the interface of the pathways that coordinately regulate the balance between cell growth and autophagy in response to nutritional status, growth factor and stress signals. Although TOR has been known as a key regulator of autophagy for more than a decade, the underlying regulatory mechanisms have not been clearly understood. This review discusses the recent advances in understanding of the mechanism by which TOR regulates autophagy with focus on mammalian TOR (mTOR) and its regulation of the autophagy machinery.
FEBS letters 04/2010; 584(7):1287-95. · 3.54 Impact Factor
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ABSTRACT: Cellular nutritional and energy status regulates a wide range of nuclear processes important for cell growth, survival, and metabolic homeostasis. Mammalian target of rapamycin (mTOR) plays a key role in the cellular responses to nutrients. However, the nuclear processes governed by mTOR have not been clearly defined. Using isobaric peptide tagging coupled with linear ion trap mass spectrometry, we performed quantitative proteomics analysis to identify nuclear processes in human cells under control of mTOR. Within 3 h of inhibiting mTOR with rapamycin in HeLa cells, we observed down-regulation of nuclear abundance of many proteins involved in translation and RNA modification. Unexpectedly, mTOR inhibition also down-regulated several proteins functioning in chromosomal integrity and up-regulated those involved in DNA damage responses (DDRs) such as 53BP1. Consistent with these proteomic changes and DDR activation, mTOR inhibition enhanced interaction between 53BP1 and p53 and increased phosphorylation of ataxia telangiectasia mutated (ATM) kinase substrates. ATM substrate phosphorylation was also induced by inhibiting protein synthesis and suppressed by inhibiting proteasomal activity, suggesting that mTOR inhibition reduces steady-state (abundance) levels of proteins that function in cellular pathways of DDR activation. Finally, rapamycin-induced changes led to increased survival after radiation exposure in HeLa cells. These findings reveal a novel functional link between mTOR and DDR pathways in the nucleus potentially operating as a survival mechanism against unfavorable growth conditions.
Molecular & Cellular Proteomics 11/2009; 9(2):403-14. · 7.40 Impact Factor
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ABSTRACT: The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTOR's access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.
Molecular and cellular biology 06/2009; 29(14):3991-4001. · 6.06 Impact Factor
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ABSTRACT: Autophagy, the starvation-induced degradation of bulky cytosolic components, is up-regulated in mammalian cells when nutrient supplies are limited. Although mammalian target of rapamycin (mTOR) is known as the key regulator of autophagy induction, the mechanism by which mTOR regulates autophagy has remained elusive. Here, we identify that mTOR phosphorylates a mammalian homologue of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2. The mammalian Atg13 binds both ULK1 and ULK2 and mediates the interaction of the ULK proteins with FIP200. The binding of Atg13 stabilizes and activates ULK and facilitates the phosphorylation of FIP200 by ULK, whereas knockdown of Atg13 inhibits autophagosome formation. Inhibition of mTOR by rapamycin or leucine deprivation, the conditions that induce autophagy, leads to dephosphorylation of ULK1, ULK2, and Atg13 and activates ULK to phosphorylate FIP200. These findings demonstrate that the ULK-Atg13-FIP200 complexes are direct targets of mTOR and important regulators of autophagy in response to mTOR signaling.
Molecular biology of the cell 03/2009; 20(7):1992-2003. · 5.98 Impact Factor
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ABSTRACT: TOR kinases integrate environmental and nutritional signals to regulate cell growth in eukaryotic organisms. Here, we describe results from a study combining quantitative proteomics and comparative expression analysis in the budding yeast, S. cerevisiae, to gain insights into TOR function and regulation. We profiled protein abundance changes under conditions of TOR inhibition by rapamycin treatment, and compared this data to existing expression information for corresponding gene products measured under a variety of conditions in yeast. Among proteins showing abundance changes upon rapamycin treatment, almost 90% of them demonstrated homodirectional (i.e., in similar direction) transcriptomic changes under conditions of heat/oxidative stress. Because the known downstream responses regulated by Tor1/2 did not fully explain the extent of overlap between these two conditions, we tested for novel connections between the major regulators of heat/oxidative stress response and the TOR pathway. Specifically, we hypothesized that activation of regulator(s) of heat/oxidative stress responses phenocopied TOR inhibition and sought to identify these putative TOR inhibitor(s). Among the stress regulators tested, we found that cells (hsf1-R206S, F256S and ssa1-3 ssa2-2) constitutively activated for heat shock transcription factor 1, Hsf1, inhibited rapamycin resistance. Further analysis of the hsf1-R206S, F256S allele revealed that these cells also displayed multiple phenotypes consistent with reduced TOR signaling. Among the multiple Hsf1 targets elevated in hsf1-R206S, F256S cells, deletion of PIR3 and YRO2 suppressed the TOR-regulated phenotypes. In contrast to our observations in cells activated for Hsf1, constitutive activation of other regulators of heat/oxidative stress responses, such as Msn2/4 and Hyr1, did not inhibit TOR signaling. Thus, we propose that activated Hsf1 inhibits rapamycin resistance and TOR signaling via elevated expression of specific target genes in S. cerevisiae. Additionally, these results highlight the value of comparative expression analyses between large-scale proteomic and transcriptomic datasets to reveal new regulatory connections.
PLoS ONE 02/2008; 3(2):e1598. · 4.09 Impact Factor
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ABSTRACT: The protein kinase mammalian target of rapamycin (mTOR) plays an important role in the coordinate regulation of cellular responses to nutritional and growth factor conditions. mTOR achieves these roles through interacting with raptor and rictor to form two distinct protein complexes, mTORC1 and mTORC2. Previous studies have been focused on mTORC1 to elucidate the central roles of the complex in mediating nutritional and growth factor signals to the protein synthesis machinery. Functions of mTORC2, relative to mTORC1, have remained little understood. Here we report identification of a novel component of mTORC2 named PRR5 (PRoline-Rich protein 5), a protein encoded by a gene located on a chromosomal region frequently deleted during breast and colorectal carcinogenesis (Johnstone, C. N., Castellvi-Bel, S., Chang, L. M., Sung, R. K., Bowser, M. J., Pique, J. M., Castells, A., and Rustgi, A. K. (2005) Genomics 85, 338-351). PRR5 interacts with rictor, but not raptor, and the interaction is independent of mTOR and not disturbed under conditions that disrupt the mTOR-rictor interaction. PRR5, unlike Sin1, another component of mTORC2, is not important for the mTOR-rictor interaction and mTOR activity toward Akt phosphorylation. Despite no significant effect of PRR5 on mTORC2-mediated Akt phosphorylation, PRR5 silencing inhibits Akt and S6K1 phosphorylation and reduces cell proliferation rates, a result consistent with PRR5 roles in cell growth and tumorigenesis. The inhibition of Akt and S6K1 phosphorylation by PRR5 knock down correlates with reduction in the expression level of platelet-derived growth factor receptor beta (PDGFRbeta). PRR5 silencing impairs PDGF-stimulated phosphorylation of S6K1 and Akt but moderately reduces epidermal growth factor- and insulin-stimulated phosphorylation. These findings propose a potential role of mTORC2 in the cross-talk with the cellular machinery that regulates PDGFRbeta expression and signaling.
Journal of Biological Chemistry 09/2007; 282(35):25604-12. · 4.77 Impact Factor
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ABSTRACT: The protein kinase mammalian target of rapamycin (mTOR) plays an important role in the coordinate regulation of cellular responses
to nutritional and growth factor conditions. mTOR achieves these roles through interacting with raptor and rictor to form
two distinct protein complexes, mTORC1 and mTORC2. Previous studies have been focused on mTORC1 to elucidate the central roles
of the complex in mediating nutritional and growth factor signals to the protein synthesis machinery. Functions of mTORC2,
relative to mTORC1, have remained little understood. Here we report identification of a novel component of mTORC2 named PRR5
(PRoline-Rich protein 5), a protein encoded by a gene located on a chromosomal region frequently deleted during breast and
colorectal carcinogenesis (Johnstone, C. N., Castellvi-Bel, S., Chang, L. M., Sung, R. K., Bowser, M. J., Pique, J. M., Castells,
A., and Rustgi, A. K. (2005) Genomics 85, 338–351). PRR5 interacts with rictor, but not raptor, and the interaction is independent of mTOR and not disturbed under
conditions that disrupt the mTOR-rictor interaction. PRR5, unlike Sin1, another component of mTORC2, is not important for
the mTOR-rictor interaction and mTOR activity toward Akt phosphorylation. Despite no significant effect of PRR5 on mTORC2-mediated
Akt phosphorylation, PRR5 silencing inhibits Akt and S6K1 phosphorylation and reduces cell proliferation rates, a result consistent
with PRR5 roles in cell growth and tumorigenesis. The inhibition of Akt and S6K1 phosphorylation by PRR5 knock down correlates
with reduction in the expression level of platelet-derived growth factor receptor β (PDGFRβ). PRR5 silencing impairs PDGF-stimulated
phosphorylation of S6K1 and Akt but moderately reduces epidermal growth factor- and insulin-stimulated phosphorylation. These
findings propose a potential role of mTORC2 in the cross-talk with the cellular machinery that regulates PDGFRβ expression
and signaling.
Journal of Biological Chemistry 08/2007; 282(35):25604-25612. · 4.77 Impact Factor
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ABSTRACT: 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.
Nature Cell Biology 04/2007; 9(3):316-23. · 19.49 Impact Factor
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ABSTRACT: Mammalian target-of-rapamycin (mTOR), which is a master controller of cell growth, senses a mitogenic signal in part through the lipid second messenger phosphatidic acid (PA), generated by phospholipase D (PLD). To understand further which isozymes of PLD are involved in this process, we compared the effect of PLD isozymes on mTOR activation. We found that PLD2 has an essential role in mitogen-induced mTOR activation as the siRNA-mediated knockdown of PLD2, not of PLD1, profoundly reduced the phosphorylations of S6K1 and 4EBP1, well-known mTOR effectors. Furthermore, exogenous PA-induced mTOR activation was abrogated by PLD2 knockdown, but not by PLD1 knockdown. This abrogation was found to be the result of complex formation between PLD2 and mTOR/raptor. PLD2 possesses a TOS-like motif (Phe-Glu-Val-Gln-Val, a.a. 265-269), through which it interacts with raptor independently of the other TOS motif-containing proteins, S6K1 and 4EBP1. PLD2-dependent mTOR activation appears to require PLD2 binding to mTOR/raptor with lipase activity, since lipase-inactive PLD2 cannot trigger mTOR activation despite its ability to interact with mTOR/raptor. Abrogation of mitogen-dependent mTOR activation by PLD2 knockdown was rescued only by wild type PLD2, but not by raptor binding-deficient and lipase-inactive PLD2. Our results demonstrate the importance of localized PA generation for the mitogen-induced activation of mTOR, which is achieved by a specific interaction between PLD2 and mTOR/raptor.
Cellular Signalling 01/2007; 18(12):2283-91. · 4.06 Impact Factor
