RESUMEN
Autophagy is classified as non-selective or selective depending on the types of degrading substrates. Endoplasmic reticulum (ER)-phagy is a form of selective autophagy for transporting the ER-resident proteins to autolysosomes. FAM134B, a member of the family with sequence similarity 134, is a well-known ER-phagy receptor. Dysfunction of FAM134B results in several diseases including viral infection, inflammation, neurodegenerative disorder and cancer, indicating that FAM134B has crucial roles in various kinds of intracellular functions. However, how FAM134B-mediated ER-phagy regulates intracellular functions is not well understood. In this study, we found that FAM134B knockdown in mammalian cells accelerated cell proliferation. FAM134B knockdown increased the protein amount of STIM1, an ER Ca2+ sensor protein mediating the store-operated Ca2+ entry (SOCE) involved in G1 to S phase transition. FAM134B bound to STIM1 through its C-terminal cytosolic region. FAM134B knockdown reduced transport of STIM1 from the ER to autolysosomes. Finally, FAM134B knockdown accelerated G1 to S phase transition. These results suggest that FAM134B is involved in cell proliferation possibly through degradation of STIM1 via ER-phagy.
RESUMEN
Endoplasmic reticulum (ER) tubules are interconnected by three-way junctions, resulting in the formation of a tubular ER network. Lunapark (Lnp) localizes to and stabilizes the three-way junctions. The N-terminal cytoplasmic domain in Lnp has a ubiquitin ligase activity. However, the molecular mechanism of how the ubiquitin ligase activity of Lnp is involved in the formation of the tubular ER network remains unknown. In this study, we examined whether the ER membrane proteins responsible for the formation of the tubular ER network are ubiquitinated by Lnp. We found that atlastin-2 (ATL2), an isoform of the ATL family mediating the generation of the three-way junctions by connecting the ER tubules, is a novel substrate for ubiquitination by Lnp. The localization of Lnp at the three-way junctions is important for ubiquitination of ATL2. Lysine 56, 57, 282 and 302 are the potential ubiquitination sites by Lnp. Silencing ATL2 decreased the number of the three-way junctions, and the expression of the ATL2 mutant in which the lysine residues are substituted with arginine failed to rescue the decrease of the three-way junctions in the ATL2 knocked-down cells. These results suggest that Lnp ubiquitinates ATL2 at the three-way junctions for the proper tubular ER network formation.
Asunto(s)
Lisina , Proteínas de la Membrana , Arginina/metabolismo , Retículo Endoplásmico/metabolismo , Ligasas/análisis , Ligasas/metabolismo , Lisina/metabolismo , Proteínas de la Membrana/química , Ubiquitinas/análisis , Ubiquitinas/metabolismoRESUMEN
Endoplasmic reticulum (ER) tubules connect each other by three-way junctions, resulting in a tubular ER network. Oligomerization of three-way junction protein lunapark (Lnp) is important for its localization and the three-way junction stability. On the other hand, Lnp has an N-terminal ubiquitin ligase activity domain, which is also important for the three-way junction localization. To understand the mode of action of Lnp, we isolated Cullin-associated and neddylation-dissociated 1 (CAND1), a regulator of Skp1-Cul1-F-box (SCF) ubiquitin ligase, as a Lnp-binding protein by affinity chromatography. CAND1 and Lnp form a higher-molecular-weight complex in vitro, while they do not co-localize at the three-way junctions. CAND1 reduces the auto-ubiquitination activity of Lnp. CAND1 knockdown enhances proteasomal degradation of Lnp and reduces the tubular ER network in mammalian cells. These results suggest that CAND1 has the potency to promote the formation of the higher-molecular-weight complex with Lnp and reduce the auto-ubiquitination activity of Lnp, thereby regulating the three-way junction stability of the tubular ER network.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Mapas de Interacción de Proteínas , Factores de Transcripción/metabolismo , Animales , Sitios de Unión/genética , Western Blotting , Células COS , Chlorocebus aethiops , Humanos , Proteínas de la Membrana/genética , Mutación , Unión Proteica , Interferencia de ARN , Factores de Transcripción/genética , UbiquitinaciónRESUMEN
How cancer disseminates and metastasizes remains an outstanding open question. Emerging evidence indicates that membrane trafficking is frequently harnessed by tumors of epithelial origin to acquire a mesenchymal program of invasiveness. However, the critical molecular hubs used by cancer cells this context have only began to be elucidated. Here, we discussed the results of a recent phenotypic screening that led to the identification of the small GTPase RAB2A, not previously involved in cancer dissemination, as pivotal for the acquisition of pericellular proteolysis, cell dissemination and distant metastatic spreading of human breast cancer. At the cellular levels, RAB2A controls both canonical polarized Golgi-to-Plasma membrane trafficking of the junctional protein E-cadherin, and post-endocytic trafficking of the membrane-bound metalloprotease, MT1-MMP. This finding reveals an unexpected plasticity in the control of diverse trafficking routes exerted by RAB2A through canonical (Golgi stacking) and non-canonical (late endosome recycling) functional interactions, contributing to break established membrane trafficking dogma on the rigorous molecular distinction between polarized Golgi and post endocytic routes. Finally, they suggest that epithelial cancers may specifically select for those molecules that enable them to control multiple trafficking routes, in turn essential for the regulation of activities necessary for acquisition of mesenchymal traits.
Asunto(s)
Membrana Celular/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Proteínas de Unión al GTP rab/metabolismo , Animales , Cadherinas/metabolismo , Humanos , Invasividad Neoplásica , Transporte de ProteínasRESUMEN
The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER-to-Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post-endocytic trafficking of membrane-bound MT1-MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E-cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Aparato de Golgi/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Biomarcadores de Tumor , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Endosomas/metabolismo , Exocitosis , Matriz Extracelular/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Silenciador del Gen , Proteínas de Homeodominio/metabolismo , Humanos , Invasividad Neoplásica , Pronóstico , Transporte de Proteínas , Proteolisis , Recurrencia , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
The role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-ß1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors.
Asunto(s)
Células Epiteliales/citología , Regulación Neoplásica de la Expresión Génica , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Actomiosina/metabolismo , Animales , Cadherinas/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/patología , Línea Celular Tumoral , Endocitosis , Femenino , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Antígenos de Histocompatibilidad Menor , Trasplante de Neoplasias , Receptor ErbB-2/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND: Integrin-mediated adhesion of cells to the extracellular matrix (ECM) relies on the dynamic formation of focal adhesions (FAs), which are biochemical and mechanosensitive platforms composed of a large variety of cytosolic and transmembrane proteins. During migration, there is a constant turnover of ECM contacts that initially form as nascent adhesions at the leading edge, mature into FAs as actomyosin tension builds up, and are then disassembled at the cell rear, thus allowing for cell detachment. Although the mechanisms of FA assembly have largely been defined, the molecular circuitry that regulates their disassembly still remains elusive. RESULTS: Here, we show that RN-tre, a GTPase-activating protein (GAP) for Rabs including Rab5 and Rab43, is a novel regulator of FA dynamics and cell migration. RN-tre localizes to FAs and to a pool of Rab5-positive vesicles mainly associated with FAs undergoing rapid remodeling. We found that RN-tre inhibits endocytosis of ß1, but not ß3, integrins and delays the turnover of FAs, ultimately impairing ß1-dependent, but not ß3-dependent, chemotactic cell migration. All of these effects are mediated by its GAP activity and rely on Rab5. CONCLUSIONS: Our findings identify RN-tre as the Rab5-GAP that spatiotemporally controls FA remodeling during chemotactic cell migration.
Asunto(s)
Movimiento Celular/genética , Endocitosis/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Adhesión Celular/fisiología , Células Cultivadas , Doxiciclina/farmacología , Endocitosis/fisiología , Adhesiones Focales/metabolismo , Proteínas Activadoras de GTPasa/genética , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Ratones , Ratones Noqueados , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal/fisiología , Proteínas de Unión al GTP rab/biosíntesis , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab5/biosíntesisRESUMEN
Mechanisms controlling development, growth, and metabolism are coordinated in response to changes in environmental conditions, enhancing the likelihood of survival to reproductive maturity. Much remains to be learned about the molecular basis underlying environmental influences on these processes. C. elegans larvae enter a developmentally dormant state called L1 diapause when hatched into nutrient-poor conditions. The nematode pten homologue daf-18 is essential for maintenance of survival and germline stem cell quiescence during this period (Fukuyama et al., 2006; Sigmond et al., 2008), but the details of the signaling network(s) in which it functions remain to be elucidated. Here, we report that animals lacking both aak-1 and aak-2, which encode the two catalytic α subunits of AMP-activated protein kinase (AMPK), show reduced viability and failure to maintain mitotic quiescence in germline stem cells during L1 diapause. Furthermore, failure to arrest germline proliferation has a long term consequence; aak double mutants that have experienced L1 diapause develop into sterile adults when returned to food, whereas their continuously fed siblings are fertile. Both aak and daf-18 appear to maintain germline quiescence by inhibiting activity of the common downstream target, TORC1 (TOR Complex 1). In contrast, rescue of the lethality phenotype indicates that aak-2 acts not only in the intestine, as does daf-18, but also in neurons, likely promoting survival by preventing energy deprivation during L1 diapause. These results not only provide evidence that AMPK contributes to survival during L1 diapause in a manner distinct from that by which it controls dauer diapause, but they also suggest that AMPK suppresses TORC1 activity to maintain stem cell quiescence.
RESUMEN
Yes-associated protein (YAP) is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes. Although cytoplasmic retention of YAP is known to be mediated by Hippo pathway-dependent phosphorylation, post-translational modifications that regulate YAP in the nucleus remain unclear. Here we report the discovery of a novel cycle of acetylation/deacetylation of nuclear YAP induced in response to S(N)2 alkylating agents. We show that after treatment of cells with the S(N)2 alkylating agent methyl methanesulfonate, YAP phosphorylation mediated by the Hippo pathway is markedly reduced, leading to nuclear translocation of YAP and its acetylation. This YAP acetylation occurs on specific and highly conserved C-terminal lysine residues and is mediated by the nuclear acetyltransferases CBP (CREB binding protein) and p300. Conversely, the nuclear deacetylase SIRT1 is responsible for YAP deacetylation. Intriguingly, we found that YAP acetylation is induced specifically by S(N)2 alkylating agents and not by other DNA-damaging stimuli. These results identify a novel YAP acetylation cycle that occurs in the nucleus downstream of the Hippo pathway. Intriguingly, our findings also indicate that YAP acetylation is involved in responses to a specific type of DNA damage.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hipocalcina/metabolismo , Fosfoproteínas/metabolismo , Acetilación , Alquilantes/farmacología , Daño del ADN , Relación Dosis-Respuesta a Droga , Células HEK293 , Células HeLa , Humanos , Lisina/química , Modelos Biológicos , Interferencia de ARN , Transducción de Señal , Sirtuina 1/metabolismo , Factores de Transcripción , Transcripción Genética , Proteínas Señalizadoras YAP , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The Rab family of small guanosine triphosphatases (GTPases) plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs). Ras and Rab interactor (or Ras interaction/interference)-like (RINL), which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM) domain-containing (Anks) protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Proteolisis , Receptor EphA8/metabolismo , Proteínas de Unión al GTP rab/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Familia de Multigenes , Receptor EphA8/genética , Transducción de Señal/genética , Spodoptera , Transfección , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.
Asunto(s)
Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Portadoras/genética , Activación Enzimática/fisiología , Factores de Intercambio de Guanina Nucleótido/genética , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Células HEK293 , Células HeLa , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo , Red trans-Golgi/genética , Red trans-Golgi/metabolismoRESUMEN
Cytoplasmic ribonucleoprotein granules, known as processing bodies (P-bodies), contain a common set of conserved RNA-processing enzymes, and mRNAs with AU-rich elements (AREs) are delivered to P-bodies for translational silencing. Although the dynamics of P-bodies is physically linked to cytoskeletal network, it is unclear how small GTPases are involved in the P-body regulation and the ARE-mRNA metabolism. We found here that glucose depletion activates RhoA GTPase and alters the P-body dynamics in HeLa cells. These glucose-depleted effects are reproduced by the overexpression of the RhoA-subfamily GTPases and conversely abolished by the inhibition of RhoA activation. Interestingly, both RhoA activation and glucose depletion inhibit the mRNA accumulation and degradation. These findings indicate that RhoA participates in the stress-induced rearrangement of P-bodies and the release of nucleated ARE-mRNAs for their stabilization.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Adenina/análisis , Animales , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/metabolismo , Glucosa/fisiología , Células HeLa , Humanos , Ratones , Células 3T3 NIH , ARN Mensajero/química , Ribonucleoproteínas/análisis , Tristetraprolina/metabolismo , Uracilo/análisisRESUMEN
Many studies have shown that it is possible to use culture conditions to direct the differentiation of murine embryonic stem (ES) cells into a variety of cell types, including cardiomyocytes and neurons. However, the molecular mechanisms that control lineage commitment decisions by ES cells remain poorly understood. In this study, we investigated the role of the 3 major mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) in ES cell lineage commitment and showed that the p38 MAPK-specific inhibitor SB203580 blocks the spontaneous differentiation of ES cells into cardiomyocytes and instead induces the differentiation of these ES cells into neurons. Robust p38 MAPK activity between embryoid body culture days 3 and 4 is crucial for cardiomyogenesis of ES cells, and specific inhibition of p38 MAPK activity at this time results in ES cell differentiation into neurons rather than cardiomyocytes. At the molecular level, inhibition of p38 MAPK activity suppresses the expression of bmp-2 mRNA, whereas treatment of ES cells with bone morphogenetic protein 2 (BMP-2) inhibits the neurogenesis induced by SB203580. Further, luciferase reporter assays and chromatin immunoprecipitation experiments showed that BMP-2 expression in ES cells is regulated directly by the transcription factor myocyte enhancer factor 2C, a well-known substrate of p38 MAPK. Our findings reveal the molecular mechanism by which p38 MAPK activity in ES cells drives their commitment to differentiate preferentially into cardiomyocytes, and the conditions under which these same cells might develop into neurons.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Células Madre Embrionarias/fisiología , Miocitos Cardíacos/fisiología , Factores Reguladores Miogénicos/metabolismo , Neuronas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Proteína Morfogenética Ósea 2/genética , Diferenciación Celular/fisiología , Linaje de la Célula , Células Cultivadas , Células Madre Embrionarias/citología , Inhibidores Enzimáticos/metabolismo , Humanos , Imidazoles/metabolismo , Factores de Transcripción MEF2 , Ratones , Miocitos Cardíacos/citología , Factores Reguladores Miogénicos/genética , Neuronas/citología , Piridinas/metabolismo , Transcripción Genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) belongs to the MAPK (mitogen-activated protein kinase) family and is important in many biological contexts. JNK activation is regulated by phosphorylation of specific tyrosine and threonine residues sequentially catalysed by MKK4 and MKK7, which are both dual-specificity MAPKKs (MAPK kinases). Previously, we reported that tyrosine-phosphorylation of JNK by MKK4 precedes threonine-phosphorylation by MKK7, and that both are required for synergistic JNK activation. In the present study, we identify the actin-binding protein-280 (Filamin A) as a presumed 'binder' protein that can bind to MKK7, as well as to MKK4, connecting them in close proximity. We show that Filamin family members A, B and C interact with MKK4 and MKK7, but not with JNK. Filamin A binds to an N-terminal region (residues 31-60) present in the MKK7gamma and MKK7beta splice isoforms, but cannot bind to MKK7alpha which lacks these amino acids. This same N-terminal region is crucial for the intracellular co-localization of MKK7gamma with actin stress fibres and Filamin A. Experiments using Filamin-A-deletion mutants revealed that the MKK7-binding region of Filamin A differs from its MKK4-binding region, and that MKK7gamma (but not MKK7alpha) can form a complex with Filamin A and MKK4. Finally, we used Filamin-A-deficient cells to show that Filamin A enhances MKK7 activation and is important for synergistic stress-induced JNK activation in vivo. Thus Filamin A is a novel member of the group of scaffold proteins whose function is to link two MAPKKs together and promote JNK activation.
Asunto(s)
Proteínas Contráctiles/metabolismo , MAP Quinasa Quinasa 4/metabolismo , MAP Quinasa Quinasa 7/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Estrés Fisiológico , Sitios de Unión , Línea Celular Tumoral , Activación Enzimática , Filaminas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Unión Proteica , Transducción de SeñalRESUMEN
Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.
Asunto(s)
Células Madre Embrionarias/metabolismo , Proteínas de Homeodominio/biosíntesis , Animales , Proteínas de Homeodominio/genética , RatonesRESUMEN
Bardet-Biedl syndrome (BBS) is a pleiotropically genetic disorder, whose etiology is linked to cilia. Mutations in the Arf/Arl-family GTPase Arl6 have been recently shown to be responsible for BBS type 3. Here we show that BBS mutations alter the guanine nucleotide-binding properties of Arl6. Specifically, substitution of 31st Threonine to Arginine selectively abrogates the GTP-binding ability of Arl6 without affecting GDP-binding/dissociating properties. Furthermore, all the BBS mutations in Arl6 result in low expression of the mutant proteins, which can be restored by the inhibition of the proteasome. These findings implicate that Arl6 mutants are destabilized and eliminated by the proteasome in cells, probably due to the altered nucleotide-binding properties.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Síndrome de Bardet-Biedl/enzimología , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación Missense , Factores de Ribosilacion-ADP/genética , Animales , Síndrome de Bardet-Biedl/genética , Línea Celular , Perros , Estabilidad de Enzimas/genética , Guanina/metabolismo , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
SP600125 is used as a specific inhibitor of c-Jun N-terminal kinase (JNK). We initially aimed to examine physiological roles of JNK in mast cells that play a central role in inflammatory and immediate allergic responses. We found that Fc receptor for IgE (FcepsilonRI)-induced degranulation (serotonin release) and cytokine gene expression [interleukin (IL)-6, tumour necrosis factor-alpha and IL-13] in bone marrow-derived mast cells, were almost completely inhibited by SP600125. However, the time course of FcepsilonRI-induced JNK activation did not correlate with that of serotonin release. Furthermore, FcepsilonRI-induced degranulation and cytokine gene expression were not impaired in a JNK activator, MKK7-deficient mast cells, in which JNK activation was lost. These results indicate that the inhibitory effects by SP600125 are not due to impaired JNK activation. Instead, we found that SP600125 markedly inhibited the FcepsilonRI-induced activation of phosphatidylinositol 3-kinase (PI3K) and Akt, the same as a PI3K inhibitor, wortmannin. Finally, we found that SP600125 specifically inhibits delta form of p110 catalytic subunit (p110delta) of PI3K. Thus, SP600125 exerts its influence on mast cell functions by inhibiting the kinase activity of PI3K, but not JNK.
Asunto(s)
Antracenos/farmacología , Degranulación de la Célula/efectos de los fármacos , Citocinas/genética , Mastocitos/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Receptores de IgE/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/enzimología , Señalización del Calcio/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Citocinas/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoenzimas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 7/deficiencia , MAP Quinasa Quinasa 7/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/fisiología , Ratones , Proteínas Tirosina Quinasas/metabolismo , Receptores Purinérgicos P1/metabolismo , Quinasa Syk , Familia-src Quinasas/metabolismoRESUMEN
The small GTPase Rab5 plays a key role in early endocytic pathway, and its activation requires guanine-nucleotide exchange factors (GEFs). Rab5-GEFs share a conserved VPS9 domain for the GEF action, and RIN3 containing additional domains, such as Src-homology 2, RIN-family homology (RH), and Ras-association (RA), was identified as a new Rab5-GEF. However, precise functions of the additional domains and the activation mechanism of RIN3 remain unknown. Here, we found tyrosine-phosphorylation signals are involved in the Rab5-GEF activation. Treatment of HeLa cells with pervanadate translocates RIN3 from cytoplasm to the Rab5-positive vesicles. This RIN3 translocation was applied to various mutants lacking each domain of RIN3. Our present results suggest that a Ras GTPase(s) activated by tyrosine-phosphorylation signals interacts with the inhibitory RA domain, resulting in an active conformation of RIN3 as a Rab5-GEF and that RIN-unique RH domain constitutes a Rab5-binding region for the progress of GEF action.
Asunto(s)
Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Vesículas Transportadoras/metabolismo , Tirosina/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Citoplasma/metabolismo , Inhibidores Enzimáticos/farmacología , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Células HeLa , Humanos , Fosforilación , Conformación Proteica , Estructura Terciaria de Proteína/genética , Transporte de Proteínas , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Eliminación de Secuencia , Tirosina/genética , Vanadatos/farmacología , Proteínas de Unión al GTP rab5/química , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Rab-family GTPases function as key regulators for membrane traffic. Among them, Rab45/RASEF is an atypical GTPase in that it contains a coiled-coil motif at the mid region and a distinct N-terminal EF-hand domain with C-terminal Rab-homology domain. Here, we provide the initial biochemical characterization and intracellular localization of human Rab45. Rab45 bound guanine nucleotide tri- and di-phosphates through the C-terminal Rab domain. Rab45 was capable of self-interacting, and the self-interaction required the mid region containing the coiled-coil motif. Rab45 expressed in HeLa cells was localized in a small patch in the perinuclear area of the cell, and the localization was regulated by the guanine nucleotide-bound states of Rab45. Interestingly, the mid region, together with Rab domain, appeared to be essential for the characteristic perinuclear localization of Rab45, indicating that the self-interaction may be involved in the intracellular localization of Rab45.
Asunto(s)
Secuencias de Aminoácidos , GTP Fosfohidrolasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Nucleótidos de Guanina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Mutación , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) responds to a variety of stress stimuli and controls cell fates such as cell cycle entrance, apoptosis and senescence. Stimuli such as ultraviolet irradiation and chemical reagents that damage genomic DNA induce the activation of the SAPK/JNK signaling pathway. However, it is unclear how the signal arising in the nucleus owing to DNA damage is transmitted to SAPK/JNK in the cytoplasm. Here, we report that the nuclear components Daxx and Ras-association domain family 1C (RASSF1C) link DNA damage to SAPK/JNK activation in HeLa cells. In response to DNA damage, Daxx localized in promyelocytic leukaemia-nuclear bodies (PML-NBs) undergoes ubiquitination and degradation. RASSF1C, a tumor suppressor and newly identified binding partner of Daxx, is constitutively anchored by Daxx in PML-NBs but is released from the nucleus when Daxx is degraded. This released RASSF1C translocates to cytoplasmic microtubules and participates in the activation of SAPK/JNK. Our data define a novel mechanism by which the Daxx-RASSF1C complex in PML-NBs couples nuclear DNA damage to the cytoplasmic SAPK/JNK signaling pathway.