Tyrosine phosphorylation of DEPTOR functions as a molecular switch to activate mTOR signaling
2021; Elsevier BV; Volume: 297; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2021.101291
ISSN1083-351X
AutoresLaurence M. Gagné, Nadine Morin, Noémie Lavoie, Nicolas Bisson, Jean‐Philippe Lambert, Frédérick A. Mallette, Marc‐Étienne Huot,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoMetabolic dysfunction is a major driver of tumorigenesis. The serine/threonine kinase mechanistic target of rapamycin (mTOR) constitutes a key central regulator of metabolic pathways promoting cancer cell proliferation and survival. mTOR activity is regulated by metabolic sensors as well as by numerous factors comprising the phosphatase and tensin homolog/PI3K/AKT canonical pathway, which are often mutated in cancer. However, some cancers displaying constitutively active mTOR do not carry alterations within this canonical pathway, suggesting alternative modes of mTOR regulation. Since DEPTOR, an endogenous inhibitor of mTOR, was previously found to modulate both mTOR complexes 1 and 2, we investigated the different post-translational modification that could affect its inhibitory function. We found that tyrosine (Tyr) 289 phosphorylation of DEPTOR impairs its interaction with mTOR, leading to increased mTOR activation. Using proximity biotinylation assays, we identified SYK (spleen tyrosine kinase) as a kinase involved in DEPTOR Tyr 289 phosphorylation in an ephrin (erythropoietin-producing hepatocellular carcinoma) receptor–dependent manner. Altogether, our work reveals that phosphorylation of Tyr 289 of DEPTOR represents a novel molecular switch involved in the regulation of both mTOR complex 1 and mTOR complex 2. Metabolic dysfunction is a major driver of tumorigenesis. The serine/threonine kinase mechanistic target of rapamycin (mTOR) constitutes a key central regulator of metabolic pathways promoting cancer cell proliferation and survival. mTOR activity is regulated by metabolic sensors as well as by numerous factors comprising the phosphatase and tensin homolog/PI3K/AKT canonical pathway, which are often mutated in cancer. However, some cancers displaying constitutively active mTOR do not carry alterations within this canonical pathway, suggesting alternative modes of mTOR regulation. Since DEPTOR, an endogenous inhibitor of mTOR, was previously found to modulate both mTOR complexes 1 and 2, we investigated the different post-translational modification that could affect its inhibitory function. We found that tyrosine (Tyr) 289 phosphorylation of DEPTOR impairs its interaction with mTOR, leading to increased mTOR activation. Using proximity biotinylation assays, we identified SYK (spleen tyrosine kinase) as a kinase involved in DEPTOR Tyr 289 phosphorylation in an ephrin (erythropoietin-producing hepatocellular carcinoma) receptor–dependent manner. Altogether, our work reveals that phosphorylation of Tyr 289 of DEPTOR represents a novel molecular switch involved in the regulation of both mTOR complex 1 and mTOR complex 2. The serine/threonine kinase mechanistic target of rapamycin (mTOR) is a central regulator of cellular metabolism, and its dysfunction is frequently observed in cancer. Indeed, constitutive activation of mTOR-regulated pathways is now considered as a potent inducer of tumor growth and cancer cell survival (1Machado L.E. Alvarenga A.W. da Silva F.F. Roffe M. Begnami M.D. Torres L.F.B. da Cunha I.W. Martins V.R. Hajj G.N.M. Overexpression of mTOR and p(240-244)S6 in IDH1 wild-type human glioblastomas is predictive of low survival.J. Histochem. Cytochem. 2018; 66: 403-414Crossref PubMed Scopus (11) Google Scholar, 2Hutt-Cabezas M. Karajannis M.A. Zagzag D. Shah S. Horkayne-Szakaly I. Rushing E.J. Cameron J.D. Jain D. Eberhart C.G. Raabe E.H. Rodriguez F.J. Activation of mTORC1/mTORC2 signaling in pediatric low-grade glioma and pilocytic astrocytoma reveals mTOR as a therapeutic target.Neuro Oncol. 2013; 15: 1604-1614Crossref PubMed Scopus (56) Google Scholar, 3Korkolopoulou P. Levidou G. El-Habr E.A. Piperi C. Adamopoulos C. Samaras V. Boviatsis E. Thymara I. Trigka E.A. Sakellariou S. Kavantzas N. Patsouris E. Saetta A.A. 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Cell. 2011; 44: 317-324Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 25Zhao Y. Xiong X. Sun Y. DEPTOR, an mTOR inhibitor, is a physiological substrate of SCF(betaTrCP) E3 ubiquitin ligase and regulates survival and autophagy.Mol. Cell. 2011; 44: 304-316Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 26Gao D. Inuzuka H. Tan M.K. Fukushima H. Locasale J.W. Liu P. Wan L. Zhai B. Chin Y.R. Shaik S. Lyssiotis C.A. Gygi S.P. Toker A. Cantley L.C. Asara J.M. et al.mTOR drives its own activation via SCF(betaTrCP)-dependent degradation of the mTOR inhibitor DEPTOR.Mol. Cell. 2011; 44: 290-303Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). While it is clear that DEPTOR degradation can affect mTOR-associated biological processes and pathology, the precise mechanisms underlying DEPTOR endogenous modulation allowing the fine-tuning of mTOR activity remained unexplored. Erythropoietin-producing hepatocellular carcinoma (EPH) receptors are the largest family of receptor protein tyrosine kinase. They are involved in a vast variety of cellular processes, such as proliferation, differentiation, cell shape, and mobility (27Lemmon M.A. Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2010; 141: 1117-1134Abstract Full Text Full Text PDF PubMed Scopus (2944) Google Scholar). Their dysregulation is often associated with cancers and developmental disorders (28Pasquale E.B. Eph receptors and ephrins in cancer: Bidirectional signalling and beyond.Nat. Rev. Cancer. 2010; 10: 165-180Crossref PubMed Scopus (877) Google Scholar). The EPH receptors are divided in either type A subgroup that contains receptors EPHA1–A8 and EPHA10, or the type B subgroup, comprising receptors EPHB1–B4 and EPHB6. Aside from the pseudokinases EPHA10 and EPHB6, all EPH receptors display catalytic activity (29Dai D. Huang Q. Nussinov R. Ma B. 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Neurosci. 2004; 7: 417-418Crossref PubMed Scopus (125) Google Scholar). EPH receptor activation is mainly triggered by cell–cell contact or by an extracellular signaling from neighboring cells. The complexity of EPH receptor–ephrin signaling results in a vast variety of functions and regulation. For this reason, dysregulation of the expression of EPH receptors is often associated with cancer (33Murai K.K. Pasquale E.B. 'Eph'ective signaling: Forward, reverse and crosstalk.J. Cell Sci. 2003; 116: 2823-2832Crossref PubMed Scopus (290) Google Scholar, 34Bruckner K. Pasquale E.B. Klein R. Tyrosine phosphorylation of transmembrane ligands for Eph receptors.Science. 1997; 275: 1640-1643Crossref PubMed Scopus (346) Google Scholar). We identified a novel phosphorylation event targeting DEPTOR tyrosine (Tyr) 289, which rapidly affects its inhibitory function on both mTORC1 and mTORC2. Phosphorylation of DEPTOR on Tyr 289 weakens its association with mTOR, thus allowing a rapid and sustained activation of both mTORC1 and mTORC2. Proximity biotinylation experiments identified SYK (spleen tyrosine kinase) as a kinase implicated on DEPTOR Tyr phosphorylation. Using combinatory methods, we found that the EPHB2 receptor signaling pathway also promotes DEPTOR Tyr 289 phosphorylation through SYK activation. Taken together, our findings identified a novel molecular mechanism involved in modulating mTOR activity and a previously unidentified potential pathway contributing to PTEN/PI3K/AKT-independent increase of mTOR activity observed in a subset of cancers. Increasing evidence is pointing toward multiple PTMs playing a disruptive role on DEPTOR function as an endogenous inhibitor of mTOR. While serine phosphorylation was shown to trigger DEPTOR proteasomal degradation (12Peterson T.R. Laplante M. Thoreen C.C. Sancak Y. Kang S.A. Kuehl W.M. Gray N.S. Sabatini D.M. DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival.Cell. 2009; 137: 873-886Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar, 24Duan S. Skaar J.R. Kuchay S. Toschi A. Kanarek N. Ben-Neriah Y. Pagano M. mTOR generates an auto-amplification loop by triggering the betaTrCP- and CK1alpha-dependent degradation of DEPTOR.Mol. Cell. 2011; 44: 317-324Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 25Zhao Y. Xiong X. Sun Y. DEPTOR, an mTOR inhibitor, is a physiological substrate of SCF(betaTrCP) E3 ubiquitin ligase and regulates survival and autophagy.Mol. Cell. 2011; 44: 304-316Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 26Gao D. Inuzuka H. Tan M.K. Fukushima H. Locasale J.W. Liu P. Wan L. Zhai B. Chin Y.R. Shaik S. Lyssiotis C.A. Gygi S.P. Toker A. Cantley L.C. Asara J.M. et al.mTOR drives its own activation via SCF(betaTrCP)-dependent degradation of the mTOR inhibitor DEPTOR.Mol. Cell. 2011; 44: 290-303Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), no PTM has been identified as a reversible molecular switch to modulate DEPTOR functions and in turn rapidly activate/inhibit mTORC1 and mTORC2. We hypothesized that DEPTOR is Tyr phosphorylated following growth factor stimulation and that this regulates DEPTOR stability and/or activity. We first used a phospho-Tyr-specific antibody (4G10) on denatured DEPTOR immunoprecipitate extract and were able to detect a Tyr phosphorylation event on DEPTOR (Fig. 1A). Since Tyr phosphorylation is known to modulate protein interactions and activity in a rapid and reversible manner (35Ardito F. Giuliani M. Perrone D. Troiano G. Lo Muzio L. The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (review).Int. J. Mol. Med. 2017; 40: 271-280Crossref PubMed Scopus (437) Google Scholar), we sought to map DEPTOR residue(s) undergoing Tyr phosphorylation. To do this, we assessed if Tyr phosphorylation was restricted to a specific portion of DEPTOR. Using different fragments of DEPTOR (Fig. 1B), we found Tyr phosphorylation on the C-terminal half of the protein (amino acids 214–409) (Fig. 1C). We further divided the C-terminal fragment in two smaller fragments (D3 and D4 fragments) and found that Tyr phosphorylation was restricted to the D4 fragment (amino acids 276–409) (Fig. 1D). We observed multiple bands above the major band for DEPTOR C-terminal and DEPTOR D4 fragment (Fig. 1, C and D). For both of them, the upper band seems to be the one harboring most of the Tyr phosphorylation. We then investigated the nature of this bands pattern by using a DEPTOR D4 fragment tagged on both its N terminus (FLAG) and C terminus (hemagglutinin [HA]). Detection of both bands of DEPTOR fragment D4 using FLAG and HA antibodies suggested that these forms of DEPTOR are not resulting from proteolytic cleavage but rather resulting of PTMs (Fig. S1A). Only the higher molecular weight band of DEPTOR fragment D4 was Tyr phosphorylated and recognized using 4G10 antibody (Fig. S1B). In addition, we validated that the ∼18 kDa band was not solely the result of DEPTOR Tyr phosphorylation, since it was still observable using anti-FLAG in Tyr phosphatase-treated immunoprecipitates (Fig. S1B). Sequence alignment revealed that three tyrosines are present within D4 fragment and that only two are evolutionarily conserved, namely Tyr 289 and Tyr 326 (Fig. S1C). By generating point mutation of each conserved tyrosine within the D4 fragment into nonphosphorylatable phenylalanine (Y to F), we determined that only Tyr 289 can be phosphorylated (Fig. 1E). DEPTOR association to mTOR is mediated by their respective PDZ (DEPTOR) and FAT (mTOR) domain (12Peterson T.R. Laplante M. Thoreen C.C. Sancak Y. Kang S.A. Kuehl W.M. Gray N.S. Sabatini D.M. DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival.Cell. 2009; 137: 873-886Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar). We investigated whether phosphorylation on Tyr 289, which is within the PDZ domain, could be involved in modulating DEPTOR ability to bind mTOR. The dynamics of the DEPTOR–mTOR association are regulated through energy levels within the cell, as a low energy level favors tight binding between DEPTOR and mTOR, while high nutrient availability promotes their dissociation (24Duan S. Skaar J.R. Kuchay S. Toschi A. Kanarek N. Ben-Neriah Y. Pagano M. mTOR generates an auto-amplification loop by triggering the betaTrCP- and CK1alpha-dependent degradation of DEPTOR.Mol. Cell. 2011; 44: 317-324Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 25Zhao Y. Xiong X. Sun Y. DEPTOR, an mTOR inhibitor, is a physiological substrate of SCF(betaTrCP) E3 ubiquitin ligase and regulates survival and autophagy.Mol. Cell. 2011; 44: 304-316Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 26Gao D. Inuzuka H. Tan M.K. Fukushima H. Locasale J.W. Liu P. Wan L. Zhai B. Chin Y.R. Shaik S. Lyssiotis C.A. Gygi S.P. Toker A. Cantley L.C. Asara J.M. et al.mTOR drives its own activation via SCF(betaTrCP)-dependent degradation of the mTOR inhibitor DEPTOR.Mol. Cell. 2011; 44: 290-303Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). To assess the impact of Tyr 289 phosphorylation on DEPTOR binding to mTOR, we starved cells prior to serum stimulation and then performed DEPTOR immunoprecipitation (IP). Endogenous DEPTOR was first depleted from HeLa cells using validated DEPTOR shRNAs targeting the 3′UTR of DEPTOR (Fig. S2A). We then infected these cells with shRNA-resistant WT DEPTOR or mutants harboring Tyr 289 phosphomimetic Y to E substitution (DEPTORY289E) or a nonphosphorylatable Y to F substitution (DEPTORY289F). In starved cells, nonphosphorylatable DEPTORY289F showed increased association to mTOR when compared with DEPTORWT, whereas the phosphomimetic version of DEPTOR (DEPTORY289E) showed a slight decreased binding when compared with DEPTORWT (Fig. 2). This difference in association of DEPTORY289E was even more noticeable following growth factor stimulation, while both DEPTORWT and DEPTORY289F showed similar association to mTOR (Fig. 2). These results suggest that Tyr 289 phosphorylation negatively modulates DEPTOR ability to bind mTOR in serum-starved and serum-stimulated conditions. Thus, by modulating DEPTOR–mTOR association, DEPTOR phosphorylation on Y289 might then promote mTOR activation. Growth factor–induced DEPTOR dissociation from mTOR was shown to positively modulate mTOR activity (24Duan S. Skaar J.R. Kuchay S. Toschi A. Kanarek N. Ben-Neriah Y. Pagano M. mTOR generates an auto-amplification loop by triggering the betaTrCP- and CK1alpha-dependent degradation of DEPTOR.Mol. Cell. 2011; 44: 317-324Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 25Zhao Y. Xiong X. Sun Y. DEPTOR, an mTOR inhibitor, is a physiological substrate of SCF(betaTrCP) E3 ubiquitin ligase and regulates survival and autophagy.Mol. Cell. 2011; 44: 304-316Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 26Gao D. Inuzuka H. Tan M.K. Fukushima H. Locasale J.W. Liu P. Wan L. Zhai B. Chin Y.R. Shaik S. Lyssiotis C.A. Gygi S.P. Toker A. Cantley L.C. Asara J.M. et al.mTOR drives its own activation via SCF(betaTrCP)-dependent degradation of the mTOR inhibitor DEPTOR.Mol. Cell. 2011; 44: 290-303Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). To determine whether DEPTOR Tyr phosphorylation impacts mTOR activity, we investigated the repressive function of DEPTOR phosphorylation mutant on mTORC1 (phospho-S6S240/244) and mTORC2 (phospho-AKTS473) activation. To monitor the regulation of mTOR activity, cells were first serum starved for 16 h, then stimulated with complete media for 30 min, and then replaced with starving media for 12 h in order to follow kinetics of the regulation of mTOR activity. We observed a faster decrease in phosphorylation of S6 in DEPTORY289F-expressing cells, compared with cells expressing DEPTORWT, whereas those expressing DEPTORY289E showed a sustained mTORC1 activation until 4 h poststimulation (Fig. 3A), suggesting that phosphorylation on Tyr 289 impairs DEPTOR ability to suppress mTORC1 activity. All DEPTOR mutants showed similar behavior regarding phospho-AKTS473, a readout for mTORC2 activation, after serum stimulation, followed by rapid decrease of phosphorylated AKT within the first hour (Fig. 3A). Because of the faster regulation of mTORC2 following stimulation, phospho-AKTS473 levels in mutant DEPTOR-expressing cells was rather determined within the first hour after serum stimulation. Like mTORC1 activity, cells expressing DEPTORY289F mutant showed a faster decrease of phospho-AKTS473 compared with cells expressing DEPTORWT, whereas DEPTORY289E-expressing cells displayed sustained mTORC2 activation following serum removal (Fig. 3B). These results confirmed that DEPTOR phosphorylation on Tyr 289 decreases its inhibitory functions on mTOR activity. To further investigate the cellular impact of DEPTOR Tyr 289 phosphorylation on mTOR-dependent mechanisms, we assessed its effect on cytoskeletal reorganization by measuring cell size area upon stimulation. We measured cell size area of HeLa cells depleted of endogenous DEPTOR expressing DEPTORWT, DEPTORY289F, or DEPTORY289E at different time points for 18 h after growth factor stimulation (Fig. 4A). Cell area rapidly increased 1 to 3 h following stimulation and reached maximum size at 12 h poststimulation. The increase in cell area was even more noticeable in cells expressing DEPTORY289E comparatively to cells expressing either DEPTORWT or nonphosphorylatable DEPTORY289F (Fig. 4A). These results are consistent with our assessment of mTORC1 and mTORC2 activation (Fig. 3). Moreover, DEPTORY289F was found to be more potent than DEPTORWT to repress mTOR activity, suggesting that the phosphorylation status of Tyr 289 could directly affect the ability of DEPTOR to bind and repress activated mTOR. To confirm the specific involvement of mTORC2 in this process, we compared the effect of rapamycin (inhibitor of mTORC1) and Torin 1 (inhibitor of both mTOR complexes) on growth factor–induced cytoskeletal modulation of cells expressing DEPTORY289E. We observed a significant decrease in cell area upon treatment with Torin 1. When only mTORC1 was inhibited, the decrease in cell area was less marked, confirming the impact of mTORC2 on cell area (Fig. 4B). These results confirm that DEPTOR phosphorylation on Tyr 289 sustained mTORC2 activity leading to increased cytoskeletal reorganization. Ubiquitin-mediated degradation of DEPTOR is an important molecular process allowing the release of mTOR from DEPTOR-mediated inhibition (24Duan S. Skaar J.R. Kuchay S. Toschi A. Kanarek N. Ben-Neriah Y. Pagano M. mTOR generates an auto-amplification loop by triggering the betaTrCP- and CK1alpha-dependent degradation of DEPTOR.Mol. Cell. 2011; 44: 317-324Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 25Zhao Y. Xiong X. Sun Y. DEPTOR, an mTOR inhibitor, is a physiological substrate of SCF(betaTrCP) E3 ubiquitin ligase and regulates survival and autophagy.Mol. Cell. 2011; 44: 304-316Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 26Gao D. Inuzuka H. Tan M.K. Fukushima H. Locasale J.W. Liu P. Wan L. Zhai B. Chin Y.R. Shaik S. Lyssiotis C.A. Gygi S.P. Toker A. Cantley L.C. Asara J.M. et al.mTOR drives its own activation via SCF(betaTrCP)-dependent degradation of the mTOR inhibitor DEPTOR.Mol. Cell. 2011; 44: 290-303Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Thus, we assessed to determine whether Tyr 289 phosphorylation affected DEPTOR protein stability by performing cycloheximide chase experiments following serum stimulation. We found that DEPTORY289E was more stable through time than DEPTORWT and the unphosphorylated version DEPTORY289F (Fig. 5A). To assess if DEPTOR Tyr 289 phosphorylation was
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