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AIMS: Nitidine chloride (NC), a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb.) DC, exhibits multiple bioactivities, including antitumor, anti-inflammatory, and other therapeutic effects. However, the primary targets of NC and the mechanism of action (MOA) have not been explicitly defined. METHODS: We explored the effects of NC on mTORC1 signaling by immunoblotting and fluorescence microscopy in wild-type and gene knockout cell lines generated by the CRISPR/Cas9 gene editing technique. We identified IGF2R as a direct target of NC via the drug affinity-responsive target stability (DARTS) method. We investigated the antitumor effects of NC using a mouse melanoma B16 tumor xenograft model. KEY FINDINGS: NC inhibits mTORC1 activity by targeting amino acid-sensing signaling through activating transcription factor 4 (ATF4)-mediated Sestrin2 induction. NC directly binds to IGF2R and promotes its lysosomal degradation. Moreover, NC displayed potent cytotoxicity against various cancer cells and inhibited B16 tumor xenografts. SIGNIFICANCE: NC inhibits mTORC1 signaling through nutrient sensing and directly targets IGF2R for lysosomal degradation, providing mechanistic insights into the MOA of NC.
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Factor de Transcripción Activador 4 , Benzofenantridinas , Lisosomas , Diana Mecanicista del Complejo 1 de la Rapamicina , Transducción de Señal , Animales , Ratones , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Factor de Transcripción Activador 4/metabolismo , Factor de Transcripción Activador 4/genética , Transducción de Señal/efectos de los fármacos , Humanos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Benzofenantridinas/farmacología , Ratones Endogámicos C57BL , Línea Celular Tumoral , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ensayos Antitumor por Modelo de Xenoinjerto , SestrinasRESUMEN
BACKGROUND: Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is an effective therapeutic target for diseases such as cancer, diabetes, aging, and neurodegeneration. However, an efficient tool for monitoring mTORC1 inhibition in living cells or tissues is lacking. RESULTS: We developed a genetically encoded mTORC1 sensor called TORSEL. This sensor changes its fluorescence pattern from diffuse to punctate when 4EBP1 dephosphorylation occurs and interacts with eIF4E. TORSEL can specifically sense the physiological, pharmacological, and genetic inhibition of mTORC1 signaling in living cells and tissues. Importantly, TORSEL is a valuable tool for imaging-based visual screening of mTORC1 inhibitors. Using TORSEL, we identified histone deacetylase inhibitors that selectively block nutrient-sensing signaling to inhibit mTORC1. CONCLUSIONS: TORSEL is a unique living cell sensor that efficiently detects the inhibition of mTORC1 activity, and histone deacetylase inhibitors such as panobinostat target mTORC1 signaling through amino acid sensing.
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Exercise-induced activation of adenosine monophosphate-activated protein kinase (AMPK) and substrate phosphorylation modulate the metabolic capacity of mitochondria in skeletal muscle. However, the key effector(s) of AMPK and the regulatory mechanisms remain unclear. Here, we showed that AMPK phosphorylation of the folliculin interacting protein 1 (FNIP1) serine-220 (S220) controls mitochondrial function and muscle fuel utilization during exercise. Loss of FNIP1 in skeletal muscle resulted in increased mitochondrial content and augmented metabolic capacity, leading to enhanced exercise endurance in mice. Using skeletal muscle-specific nonphosphorylatable FNIP1 (S220A) and phosphomimic (S220D) transgenic mouse models as well as biochemical analysis in primary skeletal muscle cells, we demonstrated that exercise-induced FNIP1 (S220) phosphorylation by AMPK in muscle regulates mitochondrial electron transfer chain complex assembly, fuel utilization, and exercise performance without affecting mechanistic target of rapamycin complex 1-transcription factor EB signaling. Therefore, FNIP1 is a multifunctional AMPK effector for mitochondrial adaptation to exercise, implicating a mechanism for exercise tolerance in health and disease.
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Proteínas Quinasas Activadas por AMP , Proteínas Portadoras , Ratones , Animales , Fosforilación/fisiología , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Portadoras/metabolismo , Mitocondrias/metabolismo , Músculo Esquelético/metabolismoRESUMEN
The mechanistic target of rapamycin (mTOR) forms two distinct complexes: rapamycin-sensitive mTOR complex 1 (mTORC1) and rapamycin-insensitive mTORC2. mTORC2 primarily regulates cell survival by phosphorylating Akt, though the upstream regulation of mTORC2 remains less well-defined than that of mTORC1. In this study, we show that NOP14, a 40S ribosome biogenesis factor and a target of the mTORC1-S6K axis, plays an essential role in mTORC2 signaling. Knockdown of NOP14 led to mTORC2 inactivation and Akt destabilization. Conversely, overexpression of NOP14 stimulated mTORC2-Akt activation and enhanced cell proliferation. Fractionation and coimmunoprecipitation assays demonstrated that the mTORC2 complex was recruited to the rough endoplasmic reticulum through association with endoplasmic reticulum-bound ribosomes. In vivo, high levels of NOP14 correlated with poor prognosis in multiple cancer types. Notably, cancer cells with NOP14 high expression exhibit increased sensitivity to mTOR inhibitors, because the feedback activation of the PI3K-PDK1-Akt axis by mTORC1 inhibition was compensated by mTORC2 inhibition partly through NOP14 downregulation. In conclusion, our findings reveal a spatial regulation of mTORC2-Akt signaling and identify ribosome biogenesis as a potential biomarker for assessing rapalog response in cancer therapy.
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Proteínas Proto-Oncogénicas c-akt , Sirolimus , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Humanos , Línea Celular , Ribosomas/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Replicating SARS-CoV-2 has been shown to degrade HLA class I on target cells to evade the cytotoxic T-cell (CTL) response. HLA-I downregulation can be sensed by NK cells to unleash killer cell immunoglobulin-like receptor (KIR)-mediated self-inhibition by the cognate HLA-I ligands. Here, we investigated the impact of HLA and KIR genotypes and HLA-KIR combinations on COVID-19 outcome. We found that the peptide affinities of HLA alleles were not correlated with COVID-19 severity. The predicted poor binders for SARS-CoV-2 peptides belong to HLA-B subtypes that encode KIR ligands, including Bw4 and C1 (introduced by B*46:01), which have a small F pocket and cannot accommodate SARS-CoV-2 CTL epitopes. However, HLA-Bw4 weak binders were beneficial for COVID-19 outcome, and individuals lacking the HLA-Bw4 motif were at higher risk for serious illness from COVID-19. The presence of the HLA-Bw4 and KIR3DL1 combination had a 58.8% lower risk of developing severe COVID-19 (OR = 0.412, 95% CI = 0.187-0.904, p = 0.02). This suggests that HLA-Bw4 alleles that impair their ability to load SARS-CoV-2 peptides will become targets for NK-mediated destruction. Thus, we proposed that the synergistic responsiveness of CTLs and NK cells can efficiently control SARS-CoV-2 infection and replication, and NK-cell-mediated anti-SARS-CoV-2 immune responses being mostly involved in severe infection when the level of ORF8 is high enough to degrade HLA-I. The HLA-Bw4/KIR3DL1 genotype may be particularly important for East Asians undergoing COVID-19 who are enriched in HLA-Bw4-inhibitory KIR interactions and carry a high frequency of HLA-Bw4 alleles that bind poorly to coronavirus peptides.
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COVID-19 , Humanos , SARS-CoV-2 , Antígenos HLA-B/genética , Células Asesinas Naturales , Receptores KIR3DL1/genéticaRESUMEN
Despite mounting evidence linking pyroptotic cell death to tumor growth, the clinical significance and disease mechanism of pyroptosis in cancer remain uncertain. In this study, we established a unique gene signature (π signature) that can be used as a predictive and prognostic tool in pyroptosis-related cancer subtypes. We found that the 13 core pyroptosis genes exerted opposite prognostic effects in different cancer types, which were subgrouped as pyroptosis positively related cancer and pyroptosis negatively related cancer. Subsequently, π signature was identified separately from the hub genes in pyroptosis positively related cancer and pyroptosis negatively related cancer subtypes. It was shown that π signature was well correlated with patient survival, pathological stages, tumor lymphocyte infiltration, and immunotherapy response. π signature was also applied as a predictive tool for chemotherapy drug responses and used as an independent factor for patient overall survival prediction. In short, this elaborated genetic signature could help us understand the oncogenic mechanism and pave the way for further therapeutic strategies based on pyroptosis.
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Mitochondria are essential for maintaining skeletal muscle metabolic homeostasis during adaptive response to a myriad of physiologic or pathophysiological stresses. The mechanisms by which mitochondrial function and contractile fiber type are concordantly regulated to ensure muscle function remain poorly understood. Evidence is emerging that the Folliculin interacting protein 1 (Fnip1) is involved in skeletal muscle fiber type specification, function, and disease. In this study, Fnip1 was specifically expressed in skeletal muscle in Fnip1-transgenic (Fnip1Tg) mice. Fnip1Tg mice were crossed with Fnip1-knockout (Fnip1KO) mice to generate Fnip1TgKO mice expressing Fnip1 only in skeletal muscle but not in other tissues. Our results indicate that, in addition to the known role in type I fiber program, FNIP1 exerts control upon muscle mitochondrial oxidative program through AMPK signaling. Indeed, basal levels of FNIP1 are sufficient to inhibit AMPK but not mTORC1 activity in skeletal muscle cells. Gain-of-function and loss-of-function strategies in mice, together with assessment of primary muscle cells, demonstrated that skeletal muscle mitochondrial program is suppressed via the inhibitory actions of FNIP1 on AMPK. Surprisingly, the FNIP1 actions on type I fiber program is independent of AMPK and its downstream PGC-1α. These studies provide a vital framework for understanding the intrinsic role of FNIP1 as a crucial factor in the concerted regulation of mitochondrial function and muscle fiber type that determine muscle fitness.
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Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Mitocondrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animales , Femenino , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Mitocondrias Musculares/ultraestructura , Fibras Musculares Esqueléticas/ultraestructura , Especificidad de Órganos , Oxidación-Reducción , Estrés OxidativoRESUMEN
Growth signals, such as extracellular nutrients and growth factors, have substantial effects on genome integrity; however, the direct underlying link remains unclear. Here, we show that the mechanistic target of rapamycin (mTOR)-ribosomal S6 kinase (S6K) pathway, a central regulator of growth signalling, phosphorylates RNF168 at Ser60 to inhibit its E3 ligase activity, accelerate its proteolysis and impair its function in the DNA damage response, leading to accumulated unrepaired DNA and genome instability. Moreover, loss of the tumour suppressor liver kinase B1 (LKB1; also known as STK11) hyperactivates mTOR complex 1 (mTORC1)-S6K signalling and decreases RNF168 expression, resulting in defects in the DNA damage response. Expression of a phospho-deficient RNF168-S60A mutant rescues the DNA damage repair defects and suppresses tumorigenesis caused by Lkb1 loss. These results reveal an important function of mTORC1-S6K signalling in the DNA damage response and suggest a general mechanism that connects cell growth signalling to genome stability control.
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Proliferación Celular , Reparación del ADN , Neoplasias/enzimología , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células A549 , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Animales , Roturas del ADN de Doble Cadena , Femenino , Células HCT116 , Células HEK293 , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Neoplasias/genética , Neoplasias/patología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Carga Tumoral , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The autism spectrum disorders (ASDs) are a collection of human neurological disorders with heterogeneous etiologies. Hyperactivity of E3 ubiquitin (Ub) ligase UBE3A, stemming from 15q11-q13 copy number variations, accounts for 1%-3% of ASD cases worldwide, but the underlying mechanisms remain incompletely characterized. Here we report that the functionality of ALDH1A2, the rate-limiting enzyme of retinoic acid (RA) synthesis, is negatively regulated by UBE3A in a ubiquitylation-dependent manner. Excessive UBE3A dosage was found to impair RA-mediated neuronal homeostatic synaptic plasticity. ASD-like symptoms were recapitulated in mice by overexpressing UBE3A in the prefrontal cortex or by administration of an ALDH1A antagonist, whereas RA supplements significantly alleviated excessive UBE3A dosage-induced ASD-like phenotypes. By identifying reduced RA signaling as an underlying mechanism in ASD phenotypes linked to UBE3A hyperactivities, our findings introduce a new vista of ASD etiology and facilitate a mode of therapeutic development against this increasingly prevalent disease.
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Trastorno del Espectro Autista/metabolismo , Neuronas/metabolismo , Retinal-Deshidrogenasa/metabolismo , Tretinoina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Trastorno del Espectro Autista/tratamiento farmacológico , Preescolar , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Terapia Molecular Dirigida , Plasticidad Neuronal , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The alterations in cellular ubiquitin (Ub) homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. We recently reported that the macroautophagy/autophagy receptor SQSTM1/p62, functions as a novel Ub sensor to activate autophagy upon Ub+ stress (upregulation of the Ub level). First, SQSTM1 was found to undergo extensive ubiquitination and activate autophagy under Ub+ stress induced by prolonged Bortezomib (BTZ) treatment, Ub overexpression or by heat shock. Mechanistically, Ubiquitination of SQSTM1 disrupts its dimerization of the UBA domain, switching it from an auto-inhibitory conformation to recognize poly-ubiquitinated cargoes, promoting autophagic flux. Interestingly, Ub+ stress-responsive SQSTM1 ubiquitination is mediated by Ub conjugating enzymes, UBE2D2/3, in a unique E2-dependent manner. Our work has thus revealed a novel mechanism for how SQSTM1 senses cellular Ub stress conditions and regulates selective autophagy in response to diverse intrinsic or extrinsic challenges.
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Autofagia , Ubiquitina , Unión Proteica , Dominios Proteicos , Proteína Sequestosoma-1 , UbiquitinaciónRESUMEN
As a major diarrheagenic human pathogen, enterohemorrhagic Escherichia coli (EHEC) produce attaching and effacing (A/E) lesions, characterized by the formation of actin pedestals, on mammalian cells. A bacterial T3SS effector NleL from EHEC O157:H7 was recently shown to be a HECT-like E3 ligase in vitro, but its biological functions and host targets remain elusive. Here, we report that NleL is required to effectively promote EHEC-induced A/E lesions and bacterial infection. Furthermore, human c-Jun NH2-terminal kinases (JNKs) were identified as primary substrates of NleL. NleL-induced JNK ubiquitylation, particularly mono-ubiquitylation at the Lys 68 residue of JNK, impairs JNK's interaction with an upstream kinase MKK7, thus disrupting JNK phosphorylation and activation. This subsequently suppresses the transcriptional activity of activator protein-1 (AP-1), which modulates the formation of the EHEC-induced actin pedestals. Moreover, JNK knockdown or inhibition in host cells complements NleL deficiency in EHEC infection. Thus, we demonstrate that the effector protein NleL enhances the ability of EHEC to infect host cells by targeting host JNK, and elucidate an inhibitory role of ubiquitylation in regulating JNK phosphorylation.
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Adhesión Bacteriana , Escherichia coli Enterohemorrágica/fisiología , Infecciones por Escherichia coli/enzimología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Escherichia coli Enterohemorrágica/genética , Infecciones por Escherichia coli/genética , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/química , Proteínas Quinasas JNK Activadas por Mitógenos/genética , MAP Quinasa Quinasa 7/genética , MAP Quinasa Quinasa 7/metabolismo , Fosforilación , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Alterations in cellular ubiquitin (Ub) homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. Here we report that autophagy receptor p62/sequestosome-1 interacts with E2 Ub conjugating enzymes, UBE2D2 and UBE2D3. Endogenous p62 undergoes E2-dependent ubiquitylation during upregulation of Ub homeostasis, a condition termed as Ub+ stress, that is intrinsic to Ub overexpression, heat shock or prolonged proteasomal inhibition by bortezomib, a chemotherapeutic drug. Ubiquitylation of p62 disrupts dimerization of the UBA domain of p62, liberating its ability to recognize polyubiquitylated cargoes for selective autophagy. We further demonstrate that this mechanism might be critical for autophagy activation upon Ub+ stress conditions. Delineation of the mechanism and regulatory roles of p62 in sensing Ub stress and controlling selective autophagy could help to understand and modulate cellular responses to a variety of endogenous and environmental challenges, potentially opening a new avenue for the development of therapeutic strategies against autophagy-related maladies.
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Autofagia , Proteína Sequestosoma-1/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Animales , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Respuesta al Choque Térmico , Humanos , Ratones , Fosforilación , Fosfoserina/metabolismo , Poliubiquitina/metabolismo , Inhibidores de Proteasoma/farmacología , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Proteína Sequestosoma-1/química , Estrés Fisiológico , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
AKT activation requires phosphorylation of the activation loop (T308) by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and the hydrophobic motif (S473) by the mammalian target of rapamycin complex 2 (mTORC2). We recently observed that phosphorylation of the AKT hydrophobic motif was dramatically elevated, rather than decreased, in mTOR knockout heart tissues, indicating the existence of other kinase(s) contributing to AKT phosphorylation. Here we show that the atypical IκB kinase ε and TANK-binding kinase 1 (IKKε/TBK1) phosphorylate AKT on both the hydrophobic motif and the activation loop in a manner dependent on PI3K signaling. This dual phosphorylation results in a robust AKT activation in vitro. Consistently, we found that growth factors can induce AKT (S473) phosphorylation in Rictor(-/-) cells, and this effect is insensitive to mTOR inhibitor Torin1. In IKKε/TBK1 double-knockout cells, AKT activation by growth factors is compromised. We also observed that TBK1 expression is elevated in the mTOR knockout heart tissues, and that TBK1 is required for Ras-induced mouse embryonic fibroblast transformation. Our observations suggest a physiological function of IKKε/TBK1 in AKT regulation and a possible mechanism of IKKε/TBK1 in oncogenesis by activating AKT.
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Quinasa I-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Fibroblastos/enzimología , Células HEK293 , Células HeLa , Humanos , Quinasa I-kappa B/genética , Ratones , Ratones Noqueados , Mutación Missense , Miocardio/enzimología , Naftiridinas/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteína Asociada al mTOR Insensible a la Rapamicina , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Raf kinase trapping to Golgi (RKTG) is a potential tumor suppressor gene due to its negative roles in regulating Ras/Raf/MEK/ERK (extracellular signal-regulated kinase) pathway and GPCR (G protein-coupled receptor) Gßγ subunit signaling. Interestingly, RKTG-deficient mice are free of tumors, although they are prone to form skin cancer on carcinogen administration. On the other hand, p53 is a well-characterized tumor suppressor gene and p53 heterozygous mice develop sarcoma and other tumors starting from 12 months of age. In RKTG-null mouse embryonic fibroblasts, lypophosphatidic acid (LPA), but not EGF (epidermal growth factor), could stimulate hyperphosphorylation of AKT and GSK3ß, accompanied by increases in phosphorylation of p53 at Ser15 and accumulation of p53, as well as its target genes p21 and p16. Spontaneous skin cancer-like tumors were detected in about 25% of RKTG nullizygous and p53 heterozygous mice within 7 months of age. Hyperplasia and epithelial-mesenchymal transition (EMT) were observed in the tumor-overlying epidermis, in which LOH of p53 occurred and EMT features emerged. In p53-mutated A431 epithelial carcinoma cells, knockdown of RKTG led to enhancement of LPA-stimulated AKT and GSK3ß phosphorylation, together with increased accumulation of ß-catenin and appearance of EMT features that were antagonized by p53 overexpression. In HepG2 epithelial cells, LPA-stimulated AKT phosphorylation and EMT features reached maximum when both RKTG and p53 were simultaneously silenced. In summary, these results not only indicate that RKTG has an in vivo tumor suppressor function to cooperate with p53 in tumorigenesis but also suggest that p53 has an EMT checkpoint function and the loss of this function can combine with loss of RKTG to drive EMT and tumor progression.
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Transformación Celular Neoplásica/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transición Epitelial-Mesenquimal , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The target of rapamycin complex 2 (TORC2) is a key regulator of cell growth. Zinzalla et al. (2011) now provide evidence that TORC2 is activated by direct association with the ribosome, which may ensure that TORC2 activity is calibrated to match the cell's intrinsic growth capacity.
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Upon ligand binding, G-protein-coupled receptors (GPCRs) impart the signal to heterotrimeric G proteins composed of alpha, beta, and gamma subunits, leading to dissociation of the G alpha subunit from the G betagamma subunit. While the G alpha subunit is imperative for downstream signaling, the G betagamma subunit, in its own right, mediates a variety of cellular responses such as GPCR desensitization via recruiting GRK to the plasma membrane and AKT stimulation. Here we report a mode of spatial regulation of the G betagamma subunit through alteration in subcellular compartmentation. RKTG (Raf kinase trapping to Golgi apparatus) is a newly characterized membrane protein specifically localized at the Golgi apparatus. The N terminus of RKTG interacts with G beta and tethers G betagamma to the Golgi apparatus. Overexpression of RKTG impedes the interaction of G betagamma with GRK2, abrogates the ligand-induced change of subcellular distribution of GRK2, reduces isoproterenol-stimulated phosphorylation of the beta2-adrenergic receptor (beta 2AR), and alters beta 2AR desensitization. In addition, RKTG inhibits G betagamma- and ligand-mediated AKT phosphorylation that is enhanced in cells with downregulation of RKTG. Silencing of RKTG also alters GRK2 internalization and compromises ligand-induced G beta translocation to the Golgi apparatus. Taken together, our results reveal that RKTG can modulate GPCR signaling through sequestering G betagamma to the Golgi apparatus and thereby attenuating the functions of G betagamma.
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Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Aparato de Golgi/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Animales , Células Cultivadas , Chlorocebus aethiops , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Silenciador del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ligandos , Proteínas de la Membrana/genética , Ratones , Fosforilación , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Saccharomyces cerevisiae/genética , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
Raf kinase trapping to Golgi (RKTG) is a newly characterized negative regulator of the Ras-Raf-MEK-ERK signaling pathway via sequestrating Raf-1 to the Golgi apparatus. However, little is known about the physiological functions of RKTG in mitogenic pathway and carcinogenesis. Here, we describe a suppressive role of RKTG in skin carcinogenesis by analyzing chemical carcinogen-induced tumorigenesis. Epidermis hyperplasia and proliferation are increased in RKTG-deficient mice (RKTG(-/-)) after acute treatment with 7, 12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). Using a two-stage DMBA/TPA carcinogenesis protocol on mouse skin, the number and size of papillomas are increased in RKTG(-/-) mice, accompanied by shortened tumor latency and enhanced keratinocyte proliferation. The regression of the carcinogen-induced tumors is also prolonged in RKTG(-/-) mice. Consistently, the levels of Raf-1 and extracellular signal-regulated kinase phosphorylation in primary keratinocytes as well as skin tumors are elevated when RKTG is disrupted. Collectively, our results indicate that RKTG has a suppressive activity in chemical carcinogen-induced mitogenesis and tumor formation in mouse skin.
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Carcinógenos/toxicidad , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/prevención & control , Proteínas Supresoras de Tumor , Animales , Anticarcinógenos , Bromodesoxiuridina , Carcinógenos/antagonistas & inhibidores , División Celular , Queratinocitos/citología , Queratinocitos/fisiología , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Subcellular compartmentalization has become an important theme in cell signaling such as spatial regulation of Ras by RasGRP1 and MEK/ERK by Sef. Here, we report spatial regulation of Raf kinase by RKTG (Raf kinase trapping to Golgi). RKTG is a seven-transmembrane protein localized at the Golgi apparatus. RKTG expression inhibits EGF-stimulated ERK and RSK phosphorylation, blocks NGF-mediated PC12 cell differentiation, and antagonizes Ras- and Raf-1-stimulated Elk-1 transactivation. Through interaction with Raf-1, RKTG changes the localization of Raf-1 from cytoplasm to the Golgi apparatus, blocks EGF-stimulated Raf-1 membrane translocation, and reduces the interaction of Raf-1 with Ras and MEK1. In RKTG-null mice, the basal ERK phosphorylation level is increased in the brain and liver. In RKTG-deleted mouse embryonic fibroblasts, EGF-induced ERK phosphorylation is enhanced. Collectively, our results reveal a paradigm of spatial regulation of Raf kinase by RKTG via sequestrating Raf-1 to the Golgi apparatus and thereby inhibiting the ERK signaling pathway.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Quinasas raf/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Células Cultivadas , Femenino , Eliminación de Gen , Aparato de Golgi/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas de la Membrana , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Unión Proteica , Ratas , Quinasas raf/genéticaRESUMEN
Germline mutations of the serine/threonine kinase LKB1 (also known as STK11) lead to Peutz-Jeghers syndrome (PJS) that is associated with increased incidence of malignant cancers. However, the tumor suppressor function of LKB1 has not been fully elucidated. We applied yeast two-hybrid screening and identified that a novel WD-repeat protein WDR6 was able to interact with LKB1. Immunofluorescence staining revealed that WDR6 was localized in cytoplasm, similar to the localization of LKB1. Expression of LKB1 was able to inhibit colony formation of Hela cells. Interestingly, coexpression of WDR6 with LKB1 enhanced the inhibitory effect of LKB1 on Hela cell proliferation. Consistently, WDR6 was able to synergize with LKB1 in cell cycle G1 arrest in Hela cells. Coexpression of WDR6 and LKB1 was able to induce a cyclin-dependent kinase (CDK) inhibitor p27(Kip1). Furthermore, the stimulatory effect of LKB1 on p27(Kip1) promoter activity was significantly elevated by coexpression with WDR6. Collectively, these results provided initial evidence that WDR6 is implicated in the cell growth inhibitory pathway of LKB1 via regulation of p27(Kip1).