RESUMEN
Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met-specific branching morphogenesis. It associates directly with c-Met via the c-Met-binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met-binding site is localized to a 13-amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met-binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1-specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Proteína Adaptadora GRB2 , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas/fisiología , Datos de Secuencia Molecular , Morfogénesis/fisiología , Proteínas Nucleares/metabolismo , Fosforilación , Estructura Terciaria de Proteína/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Protozoarias/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Técnicas del Sistema de Dos HíbridosRESUMEN
Signalling through the transmembrane receptor Notch is triggered by ligand binding, which induces the proteolytic cleavage of the Notch protein. This cleavage generates an intracellular fragment of the Notch protein (Notch-IC), which translocates into the nucleus and modifies transcription of target genes through its association with the RBP-J protein. Thus, the isolated Notch-IC protein represents the constitutively activated receptor. We have performed a deletion analysis of Notch IC in order to identify the transferable transactivation domain of Notch-IC and the minimal domain of Notch-IC required for RBP-J dependent transactivational activation. Functionally, Notch-IC has been linked to cell fate decision in development and oncogenesis in vivo. In vitro, Notch-IC can cooperate in neoplastic transformation of baby rat kidney cells with the adenoviral E1A protein. We have defined the minimal domain of Notch-IC required for E1A cotransformation. This domain, consisting of the ankyrin repeats of Notch-IC only, can neither activate RBP-J dependent transcription nor does it carry a transactivation domain. Therefore, the ankyrin repeat domain of Notch-IC might trigger novel pathways relevant for transformation but unrelated to RBP-J signalling.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/fisiología , Proteínas de la Membrana/fisiología , Proteínas Nucleares , Transducción de Señal/fisiología , Activación Transcripcional , Proteínas E1A de Adenovirus/fisiología , Animales , Células COS , Chlorocebus aethiops , Cocarcinogénesis , Proteínas de Unión al ADN/química , Regulación de la Expresión Génica , Genes Sintéticos , Células HeLa , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Ratas , Receptores Notch , Proteínas Recombinantes de Fusión/fisiología , Secuencias Repetitivas de AminoácidoRESUMEN
Epstein-Barr virus nuclear antigen 2 (EBNA2) and the Notch protein both function within the nucleus as transcriptional adaptor proteins. EBNA2 plays a key role during the immortalization of primary B-cells by Epstein-Barr virus (EBV). Notch proteins are involved in lymphomagenesis as well as in multiple cell fate decisions during tissue differentiation and development. Both, EBNA2 and Notch interact with the DNA binding protein RBP-J and thereby gain access to the promoter of their target genes. In order to identify regions within the J recombination signal sequence binding protein (RBP-J), that are relevant for either the Notch or the EBNA2 interaction, we have performed a mutational analysis of RBP-J. A library of RBP-J mutants was screened by a reverse two-hybrid system for alleles that fail to bind to either EBNA2 or Notch. The sequence analysis of these alleles reveals that a limited and particularly distinct number of amino-acid positions are relevant for either interaction only. Given the important role of RBP-J in B-cell immortalization, the EBNA2/RBP-J protein-protein interaction could be a candidate target for therapeutic intervention in EBV related diseases.