RESUMEN
Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional â¼ 200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors.
Asunto(s)
Adenocarcinoma/clasificación , Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/clasificación , Carcinoma Ductal Pancreático/metabolismo , Metaboloma , Metabolómica , Adenocarcinoma/genética , Adenocarcinoma/patología , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proliferación Celular , Glucosa/metabolismo , Glutamina/metabolismo , Glucólisis/genética , Humanos , Concentración 50 Inhibidora , Lipogénesis/genética , Mesodermo/metabolismo , Mesodermo/patología , Metaboloma/genética , Reproducibilidad de los Resultados , Transcripción GenéticaRESUMEN
Transforming growth factor ß (TGFß) regulates many physiological processes and requires control mechanisms to safeguard proper and timely action. We have previously described how negative regulation of TGFß signaling is controlled by the serine/threonine kinase salt-inducible kinase 1 (SIK1). SIK1 forms complexes with the TGFß type I receptor and with the inhibitory Smad7 and down-regulates the type I receptor. We now demonstrate that TGFß induces SIK1 levels via a direct transcriptional mechanism that implicates the Smad proteins, and we have mapped a putative enhancer element on the SIK1 gene. We provide evidence that the ubiquitin ligase Smurf2 forms complexes and functionally cooperates with SIK1. Both the kinase activity of SIK1 and the ubiquitin ligase activity of Smurf2 are important for proper type I receptor turnover. We also show that knockdown of endogenous SIK1 and Smurf2 enhances physiological signaling by TGFß that leads to epithelial growth arrest. In conclusion, TGFß induces expression of Smad7, Smurf2, and SIK1, the products of which physically and functionally interlink to control the activity of this pathway.
Asunto(s)
Proteínas Serina-Treonina Quinasas/genética , Activación Transcripcional/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Neoplasias de la Mama , Células COS , Línea Celular Transformada , Chlorocebus aethiops , Regulación hacia Abajo/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Células HEK293 , Humanos , Queratinocitos/citología , Visón , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Mucosa Respiratoria/citología , Proteína smad7/genética , Proteína smad7/metabolismoRESUMEN
Signal transduction by transforming growth factor beta (TGFbeta) coordinates physiological responses in diverse cell types. TGFbeta signals via type I and type II receptor serine/threonine kinases and intracellular Smad proteins that regulate transcription. Strength and duration of TGFbeta signaling is largely dependent on a negative-feedback program initiated during signal progression. We have identified an inducible gene target of TGFbeta/Smad signaling, the salt-inducible kinase (SIK), which negatively regulates signaling together with Smad7. SIK and Smad7 form a complex and cooperate to down-regulate the activated type I receptor ALK5. We further show that both the kinase and ubiquitin-associated domain of SIK are required for proper ALK5 degradation, with ubiquitin functioning to enhance SIK-mediated receptor degradation. Loss of endogenous SIK results in enhanced gene responses of the fibrotic and cytostatic programs of TGFbeta. We thus identify in SIK a negative regulator that controls TGFbeta receptor turnover and physiological signaling.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Línea Celular , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Humanos , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de Proteína , Receptor Tipo I de Factor de Crecimiento Transformador beta , Proteína smad7/metabolismo , Ubiquitina/metabolismoRESUMEN
We have previously shown that the T-cell protein tyrosine phosphatase (TC-PTP) dephosphorylates the platelet-derived growth factor (PDGF) beta-receptor. Here, we show that the increased PDGF beta-receptor phosphorylation in TC-PTP knockout (ko) mouse embryonic fibroblasts (MEFs) occurs primarily on the cell surface. The increased phosphorylation is accompanied by a TC-PTP-dependent, monensin-sensitive delay in clearance of cell surface PDGF beta-receptors and delayed receptor degradation, suggesting PDGF beta-receptor recycling. Recycled receptors could also be directly detected on the cell surface of TC-PTP ko MEFs. The effect of TC-PTP depletion was specific for the PDGF beta-receptor, because PDGF alpha-receptor homodimers were cleared from the cell surface at the same rate in TC-PTP ko MEFs as in wild-type MEFs. Interestingly, PDGF alphabeta-receptor heterodimers were recycling. Analysis by confocal microscopy revealed that, in TC-PTP ko MEFs, activated PDGF beta-receptors colocalized with Rab4a, a marker for rapid recycling. In accordance with this, transient expression of a dominant-negative Rab4a construct increased the rate of clearance of cell surface receptors on TC-PTP ko MEFs. Thus, loss of TC-PTP specifically redirects the PDGF beta-receptor toward rapid recycling, which is the first evidence of differential trafficking of PDGF receptor family members.
Asunto(s)
Proteínas Tirosina Fosfatasas/deficiencia , Proteínas Tirosina Fosfatasas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Vesículas Citoplasmáticas/metabolismo , Dimerización , Fibroblastos/citología , Ligandos , Ratones , Ratones Noqueados , Fosforilación , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteína Tirosina Fosfatasa no Receptora Tipo 2 , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rab4/metabolismoRESUMEN
Proteins containing ubiquitin-binding domains (UBDs) interact with ubiquitinated targets and regulate diverse biological processes, including endocytosis, signal transduction, transcription and DNA repair. Many of the UBD-containing proteins are also themselves monoubiquitinated, but the functional role and the mechanisms that underlie this modification are less well understood. Here, we demonstrate that monoubiquitination of the endocytic proteins Sts1, Sts2, Eps15 and Hrs results in intramolecular interactions between ubiquitin and their UBDs, thereby preventing them from binding in trans to ubiquitinated targets. Permanent monoubiquitination of these proteins, mimicked by the fusion of ubiquitin to their carboxyl termini, impairs their ability to regulate trafficking of ubiquitinated receptors. Moreover, we mapped the in vivo monoubiquitination site in Sts2 and demonstrated that its mutation enhances the Sts2-mediated effects of epidermal-growth-factor-receptor downregulation. We propose that monoubiquitination of ubiquitin-binding proteins inhibits their capacity to bind to and control the functions of ubiquitinated targets in vivo.
Asunto(s)
Proteínas Portadoras/fisiología , Ubiquitina/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Receptores ErbB/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisina/genética , Lisina/metabolismo , Proteínas de la Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Proteínas Tirosina Fosfatasas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Transferrina/metabolismo , Ubiquitina/metabolismoRESUMEN
The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Células COS , División Celular , Línea Celular , Transformación Celular Neoplásica , Cricetinae , ADN Complementario/metabolismo , Dimerización , Regulación hacia Abajo , Endocitosis , Receptores ErbB/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Ligandos , Proteínas de la Membrana , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Fosfoglicerato Mutasa/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas , Proteínas Proto-Oncogénicas c-cbl , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Timidina/metabolismo , Factores de Tiempo , Dominios Homologos srcRESUMEN
CIN85 is a multidomain adaptor protein involved in Cbl-mediated down-regulation of epidermal growth factor (EGF) receptors. CIN85 src homology 3 domains specifically bind to a proline-arginine (PxxxPR) motif in Cbl, and this association seems to be important for EGF receptor endocytosis. Here, we report identification of novel CIN85 effectors, all containing one or more PxxxPR motifs, that are indispensable for their mutual interactions. These effectors include phosphatidyl-inositol phosphatases SHIP-1 and synaptojanin 2B1, Arf GTPase-activating proteins ASAP1 and ARAP3, adaptor proteins Hip1R and STAP1, and a Rho exchange factor, p115Rho GEF. Acting as a molecular scaffold, CIN85 clusters its effectors and recruits them to high-molecular-weight complexes in cytosolic extracts of cells. Further characterization of CIN85 binding to ASAP1 revealed that formation of the complex is independent on cell stimulation. Overexpression of ASAP1 increased EGF receptor recycling, whereas ASAP1 containing mutated PxxxPR motif failed to promote this event. We propose that CIN85 functions as a scaffold molecule that binds to numerous endocytic accessory proteins, thus controlling distinct steps in trafficking of EGF receptors along the endocytic and recycling pathways.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptores ErbB/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Secuencias de Aminoácidos , Sitios de Unión , Línea Celular , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/fisiología , Humanos , Espacio Intracelular/ultraestructura , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Receptores de Transferrina/genética , Transferrina/análisisRESUMEN
CIN85 is a multidomain scaffold protein involved in downregulation of receptor tyrosine kinases. Here we show that disabled-2 (Dab2), an endocytic adaptor molecule implicated in clathrin-coat assembly, associates with CIN85 in mammalian cells. All three SH3 domains of CIN85 were able to bind to the PKPAPR peptide in the carboxyl-terminal part of Dab2, possibly enabling CIN85 to simultaneously interact with multiple Dab2 molecules. CIN85 association with Dab2 is essential for its recruitment to clathrin coat and appears to be modulated by growth factor stimulation. Dab2 and clathrin dissociated from CIN85 following growth factor treatment, enabling other molecules, such as Cbl, to bind to CIN85. Taken together, our data indicate a dynamic interplay between CIN85 and its effectors during endocytosis of receptor tyrosine kinases.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Vesículas Cubiertas por Clatrina , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Sitios de Unión , Línea Celular , Secuencia Conservada , Endocitosis , Genes Supresores de Tumor , Sustancias de Crecimiento/farmacología , Humanos , Ratones , Unión Proteica , Transfección , Proteínas Supresoras de Tumor , Dominios Homologos srcRESUMEN
CIN85 is a multidomain adaptor protein implicated in Cbl-mediated down-regulation of receptor tyrosine kinases. CIN85 binding to Cbl is increased after growth factor stimulation and is critical for targeting receptor tyrosine kinases to clathrin-mediated endocytosis. Here we report the identification of a novel polyproline-arginine motif (PXXXPR), specifically recognized by the SH3 domains of CIN85 and its homologue CMS/CD2AP. This motif was indispensable for CIN85 binding to Cbl/Cbl-b, to other CIN85 SH3 domains' effectors, and for mediating an intramolecular interaction between the SH3-A domain and the proline-rich region of CIN85. Individual SH3 domains of CIN85 bound to PXXXPR peptides of Cbl/Cbl-b with micromolar affinities, whereas an extended structure of two or three SH3 domains bound with higher stoichiometry and increased affinity to the same peptides. This enabled full size CIN85 to simultaneously interact with multiple Cbl molecules, promoting their clustering in mammalian cells. The ability of CIN85 to cluster Cbl was important for ligand-induced stabilization of CIN85.Cbl.epidermal growth factor receptor complexes, as well as for epidermal growth factor receptor degradation in the lysosome. Thus, specific interactions of CIN85 SH3 domains with the PXXXPR motif in Cbl play multiple roles in down-regulation of receptor tyrosine kinases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Receptores ErbB/metabolismo , Proteínas Oncogénicas de Retroviridae/química , Proteínas Oncogénicas de Retroviridae/metabolismo , Ubiquitina-Proteína Ligasas , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Arginina/química , Sitios de Unión , Células CHO , Proteínas Portadoras/genética , Línea Celular , Secuencia de Consenso , Cricetinae , Regulación hacia Abajo , Endocitosis , Receptores ErbB/genética , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Proteína Oncogénica v-cbl , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Prolina/química , Proteínas Proto-Oncogénicas c-cbl , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Oncogénicas de Retroviridae/genética , Homología de Secuencia de Aminoácido , Dominios Homologos srcRESUMEN
The Cbl family of ubiquitin ligases in mammals contains three members, Cbl, Cbl-b, and Cbl-3, that are involved in down-regulation of receptor tyrosine kinases (RTKs) by mediating receptor ubiquitination and degradation. More recently, a novel pathway has been identified whereby Cbl promotes internalization of EGF receptor via a CIN85/endophilin pathway that is functionally separable from the ubiquitin ligase activity of Cbl (1). Here we show that Cbl-b, but not Cbl-3, utilize the same mechanism to down-regulate multiple RTKs. CIN85 was shown to bind to the minimal binding domain identified in the carboxyl terminus of Cbl-b. Ligand-induced phosphorylation of Cbl-b further increased their interactions and led to a rapid and sustained recruitment of CIN85 in the complex with EGF or PDGF receptors. Inhibition of binding between CIN85 and Cbl-b was sufficient to impair Cbl-b-mediated internalization of EGF receptors, while being dispensable for Cbl-b-directed polyubiquitination of EGF receptors. Moreover, CIN85 and Cbl/Cbl-b were constitutively associated with activated PDGF, EGF, or c-Kit receptors in several tumor cell lines. Our data reveal a common pathway utilized by Cbl and Cbl-b that may have an important and redundant function in negative regulation of ligand-activated as well as oncogenically activated RTKs in vivo.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Regulación hacia Abajo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ubiquitina-Proteína Ligasas , Células 3T3 , Animales , Células CHO , Línea Celular , Línea Celular Transformada , Clonación Molecular , Cricetinae , Endocitosis , Factor de Crecimiento Epidérmico/metabolismo , Células HeLa , Humanos , Ligandos , Ratones , Microscopía Fluorescente , Modelos Biológicos , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-cbl , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factores de Tiempo , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Cbl is a multi-adaptor protein involved in ligand-induced downregulation of receptor tyrosine kinases. It is thought that Cbl-mediated ubiquitination of active receptors is essential for receptor degradation and cessation of receptor-induced signal transduction. Here we demonstrate that Cbl additionally regulates epidermal growth factor (EGF) receptor endocytosis. Cbl rapidly recruits CIN85 (Cbl-interacting protein of 85K; ref. 6) and endophilins (regulatory components of clathrin-coated vesicles) to form a complex with activated EGF receptors, thus controlling receptor internalization. CIN85 was constitutively associated with endophilins, whereas CIN85 binding to the distal carboxy terminus of Cbl was increased on EGF stimulation. Inhibition of these interactions was sufficient to block EGF receptor internalization, delay receptor degradation and enhance EGF-induced gene transcription, without perturbing Cbl-directed receptor ubiquitination. Thus, the evolutionary divergent C terminus of Cbl uses a mechanism that is functionally separable from the ubiquitin ligase activity of Cbl to mediate ligand-dependent downregulation of receptor tyrosine kinases.