RESUMEN
While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3'-kinase C2beta (PI3K-C2beta) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2beta-AKT survival pathway. ITSN associated with PI3K-C2beta on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2beta activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2beta and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Supervivencia Celular , Neuronas/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Endocitosis/fisiología , Activación Enzimática , Factor de Crecimiento Epidérmico/metabolismo , Epistasis Genética , Humanos , Ratones , Datos de Secuencia Molecular , Neuronas/citología , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
The ubiquitin-proteasome system (UPS) actively controls protein dynamics and local abundance via regulated protein degradation. This study investigates UPS' roles in the regulation of postsynaptic function and molecular composition in the Drosophila neuromuscular junction (NMJ) genetic system. To specifically impair UPS function postsynaptically, the UAS/GAL4 transgenic method was employed to drive postsynaptic expression of proteasome beta2 and beta6 subunit mutant proteins, which operate through a dominant negative mechanism to block proteasome function. When proteasome mutant subunits were constitutively expressed, excitatory junctional current (EJC) amplitudes were increased, demonstrating that postsynaptic proteasome function limits neurotransmission strength. Interestingly, the alteration in synaptic strength was calcium-dependent and miniature EJCs had significantly smaller mean amplitudes and more rapid mean decay rates. Postsynaptic levels of the Drosophila PSD-95/SAP97 homologue, discs large (DLG), and the GluRIIB-containing glutamate receptor were increased, but GluRIIA-containing receptors were unaltered. With acute postsynaptic proteasome inhibition using an inducible transgenic system, neurotransmission was similarly elevated with the same specific increase in postsynaptic GluRIIB abundance. These findings demonstrate postsynaptic proteasome regulation of glutamatergic synaptic function that is mediated through specific regulation of GluRIIB-containing glutamate receptors.
Asunto(s)
Ácido Glutámico/metabolismo , Unión Neuromuscular/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Sinapsis/enzimología , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Homólogo 1 de la Proteína Discs Large , Drosophila , Proteínas de Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Proteómica , Receptores de Glutamato/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Expansion of polyglutamine (polyQ) tracts within proteins underlies a number of neurodegenerative diseases, such as Huntington disease, Kennedy disease, and spinocerebellar ataxias. The resulting mutant proteins are unstable, forming insoluble aggregates that are associated with components of the ubiquitin system, including ubiquitin, ubiquitin-like proteins, and proteins that bind to ubiquitin. Given the presence of these ubiquitin-binding proteins in the insoluble aggregates, we examined whether heterologous expression of short motifs that bind ubiquitin, termed ubiquitin-interacting motifs (UIMs), altered the aggregation of polyQ-expanded huntingtin (Htt), the protein product of the Huntington disease gene. We found that a subset of UIMs associated with mutant Htt. The ability to interact with ubiquitin was necessary, but not sufficient, for interaction with mutant Htt. Furthermore, we found that expression of single, isolated UIMs inhibited aggregation of mutant Htt. These data suggest that isolated UIMs might serve as potential inhibitors of polyQ-aggregation in vivo.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Péptidos/metabolismo , Ubiquitina/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Línea Celular , Humanos , Proteína Huntingtina , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Péptidos/antagonistas & inhibidores , Péptidos/genética , Ubiquitina/antagonistas & inhibidoresRESUMEN
Epsin is an endocytic adaptor protein involved in the regulation of clathrin-dependent endocytosis. We and others have demonstrated that Epsin is ubiquitylated in cells and requires its ubiquitin interacting motifs (UIMs) for this modification. To further elucidate the mechanism of Epsin ubiquitylation, we initiated studies to identify the E3 ligase(s) that modifies Epsin. In this study, we discovered that the U-box ubiquitin ligase carboxyl-terminus of Hsc70 interacting protein (CHIP) ubiquitylated Epsin. Using an in vitro ubiquitylation assay, we demonstrate that CHIP specifically ubiquitylated Epsin in a UIM-dependent manner. Furthermore, overexpression of CHIP in cells increased Epsin ubiquitylation also in a UIM-dependent manner. Together, these data provide evidence that CHIP functions to ubiquitylate the endocytic protein Epsin.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Riñón/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Sitios de Unión , Línea Celular , Proteínas del Choque Térmico HSC70 , Proteínas HSP70 de Choque Térmico/química , Humanos , Riñón/embriología , Unión Proteica , Relación Estructura-Actividad , Proteínas de Transporte Vesicular/químicaRESUMEN
The ubiquitin-interacting motif (UIM) is a short peptide motif with the dual function of binding ubiquitin and promoting ubiquitylation. This motif is conserved throughout eukaryotes and is present in numerous proteins involved in a wide variety of cellular processes including endocytosis, protein trafficking, and signal transduction. We previously reported that the UIMs of epsin were both necessary and sufficient for its ubiquitylation. In this study, we found that many, but not all, UIM-containing proteins were ubiquitylated. When expressed as chimeric fusion proteins, most UIMs promoted ubiquitylation of the chimera. In contrast to previous studies, we found that UIMs do not exclusively promote monoubiquitylation but rather a mixture of mono-, multi-, and polyubiquitylation. However, UIM-dependent polyubiquitylation does not lead to degradation of the modified protein. UIMs also bind polyubiquitin chains of varying lengths and to different degrees, and this activity is required for UIM-dependent ubiquitylation. Mutational analysis of the UIM revealed specific amino acids that are important for both polyubiquitin binding and ubiquitin conjugation. Finally we provide evidence that UIM-dependent ubiquitylation inhibits the interaction of UIM-containing proteins with other ubiquitylated cellular proteins. Our results suggest a new model for the ubiquitylation of UIM-containing proteins.
Asunto(s)
Quinasa 1 de Quinasa de Quinasa MAP , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Ubiquitina/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Ataxina-3 , Sitios de Unión , Línea Celular , Secuencia Conservada , Embrión de Mamíferos , Endocitosis , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas del Choque Térmico HSP40 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Riñón , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares , Oligopéptidos , Fragmentos de Péptidos/genética , Péptidos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras , Transducción de Señal , Relación Estructura-Actividad , Transfección , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The covalent attachment of ubiquitin to proteins is an evolutionarily conserved signal for rapid protein degradation. However, additional cellular functions for ubiquitination are now emerging, including regulation of protein trafficking and endocytosis. For example, recent genetic studies suggested a role for ubiquitination in regulating epsin, a modular endocytic adaptor protein that functions in the assembly of clathrin-coated vesicles; however, biochemical evidence for this notion has been lacking. Epsin consists of an epsin NH(2)-terminal homology (ENTH) domain that promotes the interaction with phospholipids, several AP2 binding sites, two clathrin binding sequences, and several Eps15 homology (EH) domain binding motifs. Interestingly, epsin also possesses several recently described ubiquitin-interacting motifs (UIMs) that have been postulated to bind ubiquitin. Here, we demonstrate that epsin is predominantly monoubiquitinated and resistant to proteasomal degradation. The UIMs are necessary for epsin ubiquitination but are not the site of ubiquitination. Finally, we demonstrate that the isolated UIMs from both epsin and an unrelated monoubiquitinated protein, Eps15, are sufficient to promote ubiquitination of a chimeric glutathione-S-transferase (GST)-UIM fusion protein. Thus, our data suggest that UIMs may serve as a general signal for ubiquitination.