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1.
Cell ; 136(3): 521-34, 2009 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-19203585

RESUMO

Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.


Assuntos
Autofagia , Glutamina/metabolismo , Proteínas Quinases/metabolismo , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Drosophila melanogaster , Humanos , Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Proteínas , Serina-Treonina Quinases TOR , Fatores de Transcrição/metabolismo
2.
Mol Cell ; 47(4): 535-46, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22795129

RESUMO

The tuberous sclerosis complex (TSC) tumor suppressors form the TSC1-TSC2 complex, which limits cell growth in response to poor growth conditions. Through its GTPase-activating protein (GAP) activity toward Rheb, this complex inhibits the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a key promoter of cell growth. Here, we identify and biochemically characterize TBC1D7 as a stably associated and ubiquitous third core subunit of the TSC1-TSC2 complex. We demonstrate that the TSC1-TSC2-TBC1D7 (TSC-TBC) complex is the functional complex that senses specific cellular growth conditions and possesses Rheb-GAP activity. Sequencing analyses of samples from TSC patients suggest that TBC1D7 is unlikely to represent TSC3. TBC1D7 knockdown decreases the association of TSC1 and TSC2 leading to decreased Rheb-GAP activity, without effects on the localization of TSC2 to the lysosome. Like the other TSC-TBC components, TBC1D7 knockdown results in increased mTORC1 signaling, delayed induction of autophagy, and enhanced cell growth under poor growth conditions.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/genética , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica , Proteínas/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais , Serina-Treonina Quinases TOR , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética
3.
Nature ; 485(7397): 195-200, 2012 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-22575959

RESUMO

R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. Despite the biological and therapeutic significance, the molecular mechanism of R-spondin action remains unclear. Here we show that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homologue ring finger 43 (RNF43) are negative feedback regulators of Wnt signalling. ZNRF3 is associated with the Wnt receptor complex, and inhibits Wnt signalling by promoting the turnover of frizzled and LRP6. Inhibition of ZNRF3 enhances Wnt/ß-catenin signalling and disrupts Wnt/planar cell polarity signalling in vivo. Notably, R-spondin mimics ZNRF3 inhibition by increasing the membrane level of Wnt receptors. Mechanistically, R-spondin interacts with the extracellular domain of ZNRF3 and induces the association between ZNRF3 and LGR4, which results in membrane clearance of ZNRF3. These data suggest that R-spondin enhances Wnt signalling by inhibiting ZNRF3. Our study provides new mechanistic insights into the regulation of Wnt receptor turnover, and reveals ZNRF3 as a tractable target for therapeutic exploration.


Assuntos
Receptores Wnt/metabolismo , Trombospondinas/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/metabolismo , Animais , Polaridade Celular/fisiologia , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica , Feminino , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Via de Sinalização Wnt , Xenopus , Peixe-Zebra , beta Catenina/metabolismo
4.
Mol Cell ; 39(2): 171-83, 2010 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-20670887

RESUMO

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1alpha) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease.


Assuntos
Regulação da Expressão Gênica/fisiologia , Glicólise/fisiologia , Lipídeos/biossíntese , Via de Pentose Fosfato/fisiologia , Biossíntese de Proteínas/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Animais , Linhagem Celular Transformada , Proliferação de Células , Genômica/métodos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipídeos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Metabolômica/métodos , Camundongos , Complexos Multiproteicos , Neoplasias/genética , Neoplasias/metabolismo , Obesidade/genética , Obesidade/metabolismo , Proteínas , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética
5.
Nat Chem Biol ; 10(5): 343-9, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24633354

RESUMO

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.


Assuntos
Proteínas Hedgehog/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Cromatografia de Afinidade , Proteômica , Transdução de Sinais , Espectrometria de Massas em Tandem
6.
Nature ; 468(7326): 968-72, 2010 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-21107320

RESUMO

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.


Assuntos
Resistencia a Medicamentos Antineoplásicos , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Regulação Alostérica , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , MAP Quinase Quinase Quinases/genética , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Melanoma/genética , Melanoma/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fases de Leitura Aberta/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Vemurafenib
7.
Nature ; 461(7264): 614-20, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19759537

RESUMO

The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.


Assuntos
Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tanquirases/antagonistas & inibidores , Proteínas Wnt/antagonistas & inibidores , Proteína Axina , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteômica , Proteínas Repressoras/química , Tanquirases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismo
8.
Eur J Immunol ; 42(12): 3394-404, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22930133

RESUMO

The signalling molecule PI3Kγ has been reported to play a key role in the immune system and the inflammatory response. In particular, it facilitates the migration of haemato-poietic cells to the site of inflammation. In this study, we reveal a novel role for PI3Kγ in the regulation of the pro-inflammatory cytokine IL-17. Loss of PI3Kγ or expression of a catalytically inactive mutant of PI3Kγ in mice led to increased IL-17 production both in vitro and in vivo in response to various stimuli. The kinetic profile was unaltered from WT cells, with no effect on proliferation or other cytokines. Elevated levels of IL-17 were not due to an aberrant expansion of IL-17-producing cells. Furthermore, we also identified an increase in IL-17RA expression on PI3Kγ(-/-) CD4(+) T cells, yet these cells exhibited impaired PI3K-dependent signalling in response to IL-17A, and subsequent NF-κB phosphorylation. In vivo, instillation of recombinant IL-17 into the airways of mice lacking PI3Kγ signalling also resulted in reduced phosphorylation of Akt. Cell influx in response to IL-17 was also reduced in PI3Kγ(-/-) lungs. These data demonstrate PI3Kγ-dependent signalling downstream of IL-17RA, which plays a pivotal role in regulating IL-17 production in T cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-17/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores de Interleucina-17/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Interleucina-17/genética , Interleucina-17/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Fosforilação/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Transdução de Sinais/genética
9.
Proc Natl Acad Sci U S A ; 107(35): 15473-8, 2010 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-20713706

RESUMO

Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.


Assuntos
Anticorpos/farmacologia , Proteínas Relacionadas a Receptor de LDL/imunologia , Ligantes , Proteínas Wnt/metabolismo , Animais , Anticorpos/imunologia , Linhagem Celular , Transformação Celular Viral , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Nus , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Proteínas Wnt/genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Proteína Wnt3 , Proteína Wnt3A , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética , beta Catenina/metabolismo
10.
Mol Cancer Res ; 6(4): 614-23, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18403640

RESUMO

Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3'-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2alpha, beta, and gamma). In this study, we used quantitative reverse transcription-PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2alpha, but not PI3K-C2beta, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2alpha mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2alpha to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2alpha in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class Ialpha isoform but also class IIalpha may contribute to an effective anticancer strategy.


Assuntos
Apoptose , Regulação para Baixo , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Classe II de Fosfatidilinositol 3-Quinases , Perfilação da Expressão Gênica , Células HeLa , Humanos , Isoenzimas/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Tempo
11.
ACS Med Chem Lett ; 7(1): 72-6, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26819669

RESUMO

Autophagy is a dynamic process that regulates lysosomal-dependent degradation of cellular components. Until recently the study of autophagy has been hampered by the lack of reliable pharmacological tools, but selective inhibitors are now available to modulate the PI 3-kinase VPS34, which is required for autophagy. Here we describe the discovery of potent and selective VPS34 inhibitors, their pharmacokinetic (PK) properties, and ability to inhibit autophagy in cellular and mouse models.

12.
Nat Cell Biol ; 16(11): 1069-79, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25327288

RESUMO

Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.


Assuntos
Autofagia/fisiologia , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Ferritinas/metabolismo , Homeostase/fisiologia , Ferro/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Humanos , Lisossomos/metabolismo , Camundongos , Fagossomos/metabolismo , Ligação Proteica
13.
Mol Cell Biol ; 33(1): 98-110, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23109424

RESUMO

Autophagy is a vesicular trafficking pathway that regulates the degradation of aggregated proteins and damaged organelles. Initiation of autophagy requires several multiprotein signaling complexes, such as the ULK1 kinase complex and the Vps34 lipid kinase complex, which generates phosphatidylinositol 3-phosphate [PtdIns(3)P] on the forming autophagosomal membrane. Alterations in autophagy have been reported for various diseases, including myopathies. Here we show that skeletal muscle autophagy is compromised in mice deficient in the X-linked myotubular myopathy (XLMTM)-associated PtdIns(3)P phosphatase myotubularin (MTM1). Mtm1-deficient muscle displays several cellular abnormalities, including a profound increase in ubiquitin aggregates and abnormal mitochondria. Further, we show that Mtm1 deficiency is accompanied by activation of mTORC1 signaling, which persists even following starvation. In vivo pharmacological inhibition of mTOR is sufficient to normalize aberrant autophagy and improve muscle phenotypes in Mtm1 null mice. These results suggest that aberrant mTORC1 signaling and impaired autophagy are consequences of the loss of Mtm1 and may play a primary role in disease pathogenesis.


Assuntos
Autofagia/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas/metabolismo , Animais , Autofagia/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Complexos Multiproteicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas/antagonistas & inibidores , Transdução de Sinais/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Ubiquitina/metabolismo
14.
Chem Biol ; 20(7): 912-21, 2013 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-23890009

RESUMO

Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/IL-23 with an unknown target and has been evaluated in clinical trials for patients with Crohn's disease or rheumatoid arthritis. Using a chemical genetic approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/IL-23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/IL-23p40 expression. Thus, we have uncovered a phosphoinositide-mediated regulatory mechanism that controls TLR signaling.


Assuntos
Interleucina-12/antagonistas & inibidores , Interleucina-23/antagonistas & inibidores , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Triazinas/farmacologia , Animais , Linhagem Celular , Citocinas/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazonas , Camundongos , Morfolinas/metabolismo , Ligação Proteica , Pirimidinas , Especificidade por Substrato , Triazinas/metabolismo
15.
PLoS One ; 7(11): e48548, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23155392

RESUMO

The mammalian target of rapamycin (mTOR) is regulated by oncogenic growth factor signals and plays a pivotal role in controlling cellular metabolism, growth and survival. Everolimus (RAD001) is an allosteric mTOR inhibitor that has shown marked efficacy in certain cancers but is unable to completely inhibit mTOR activity. ATP-competitive mTOR inhibitors such as NVP-BEZ235 can block rapamycin-insensitive mTOR readouts and have entered clinical development as anti-cancer agents. Here, we show the degree to which RAD001 and BEZ235 can be synergistically combined to inhibit mTOR pathway activation, cell proliferation and tumor growth, both in vitro and in vivo. RAD001 and BEZ235 synergized in cancer lines representing different lineages and genetic backgrounds. Strong synergy is seen in neuronal, renal, breast, lung, and haematopoietic cancer cells harboring abnormalities in PTEN, VHL, LKB1, Her2, or KRAS. Critically, in the presence of RAD001, the mTOR-4EBP1 pathway and tumorigenesis can be fully inhibited using lower doses of BEZ235. This is relevant since RAD001 is relatively well tolerated in patients while the toxicity profiles of ATP-competitive mTOR inhibitors are currently unknown.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Imidazóis/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Everolimo , Humanos , Sirolimo/farmacologia
16.
Cancer Discov ; 2(10): 948-59, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22874768

RESUMO

The overall power of kinase inhibitors is substantially overshadowed by the acquisition of drug resistance. To address this issue, we systematically assessed the potential of secreted proteins to induce resistance to kinase inhibitors. To this end, we developed a high-throughput platform for screening a cDNA library encoding 3,432 secreted proteins in cellular assays. Using cancer cells originally dependent on either MET, FGFR2, or FGFR3, we observed a bypass of dependence through ligand-mediated activation of alternative receptor tyrosine kinases (RTK). Our findings indicate a broad and versatile potential for RTKs from the HER and FGFR families as well as MET to compensate for loss of each other. We further provide evidence that combined inhibition of simultaneously active RTKs can lead to an added anticancer effect.


Assuntos
Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor ErbB-2/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/genética , Transdução de Sinais/efeitos dos fármacos , Transplante Heterólogo
17.
Mol Cell Biol ; 31(14): 2867-76, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21576371

RESUMO

The mammalian target of rapamycin complex 1 (mTORC1) is a multiprotein signaling complex regulated by oncogenes and tumor suppressors. Outputs downstream of mTORC1 include ribosomal protein S6 kinase 1 (S6K1), eukaryotic translation initiation factor 4E (eIF4E), and autophagy, and their modulation leads to changes in cell growth, proliferation, and metabolism. Rapamycin, an allosteric mTORC1 inhibitor, does not antagonize equally these outputs, but the reason for this is unknown. Here, we show that the ability of rapamycin to activate autophagy in different cell lines correlates with mTORC1 stability. Rapamycin exposure destabilizes mTORC1, but in cell lines where autophagy is drug insensitive, higher levels of mTOR-bound raptor are detected than in cells where rapamycin stimulates autophagy. Using small interfering RNA (siRNA), we find that knockdown of raptor relieves autophagy and the eIF4E effector pathway from rapamycin resistance. Importantly, nonefficacious concentrations of an ATP-competitive mTOR inhibitor can be combined with rapamycin to synergistically inhibit mTORC1 and activate autophagy but leave mTORC2 signaling intact. These data suggest that partial inhibition of mTORC1 by rapamycin can be overcome using combination strategies and offer a therapeutic avenue to achieve complete and selective inhibition of mTORC1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antibióticos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Resistência a Medicamentos/fisiologia , Fosfoproteínas/metabolismo , Sirolimo/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular , Linhagem Celular , Corantes Fluorescentes/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Proteínas/genética , Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Regulatória Associada a mTOR , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
18.
Nat Cell Biol ; 13(5): 623-9, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21478859

RESUMO

The Wnt/ß-catenin signalling pathway plays essential roles in embryonic development and adult tissue homeostasis, and deregulation of this pathway has been linked to cancer. Axin is a concentration-limiting component of the ß-catenin destruction complex, and its stability is regulated by tankyrase. However, the molecular mechanism by which tankyrase-dependent poly(ADP-ribosyl)ation (PARsylation) is coupled to ubiquitylation and degradation of axin remains undefined. Here, we identify RNF146, a RING-domain E3 ubiquitin ligase, as a positive regulator of Wnt signalling. RNF146 promotes Wnt signalling by mediating tankyrase-dependent degradation of axin. Mechanistically, RNF146 directly interacts with poly(ADP-ribose) through its WWE domain, and promotes degradation of PARsylated proteins. Using proteomics approaches, we have identified BLZF1 and CASC3 as further substrates targeted by tankyrase and RNF146 for degradation. Thus, identification of RNF146 as a PARsylation-directed E3 ligase establishes a molecular paradigm that links tankyrase-dependent PARsylation to ubiquitylation. RNF146-dependent protein degradation may emerge as a major mechanism by which tankyrase exerts its function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Wnt/metabolismo , Sequência de Aminoácidos , Animais , Proteína Axina , Humanos , Hidrólise , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/química
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