RESUMO
Transforming growth factor-ß (TGF-ß) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-ß signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-ß signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-ß signaling, and knockdown of endogenous RB1CC1 attenuated TGF-ß-induced expression of target genes as well as TGF-ß-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-ß signaling by restricting substrate specificity of Arkadia.
Assuntos
Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Crescimento Transformador beta/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Transforming growth factor (TGF)-beta induces various cellular responses principally through Smad-dependent transcriptional regulation. Activated Smad complexes cooperate with transcription factors in regulating a group of target genes. The target genes controlled by the same Smad-cofactor complexes are denoted a synexpression group. We found that an Id-like helix-loop-helix protein, human homologue of Maid (HHM), is a synexpression group-restricted regulator of TGF-beta signalling. HHM suppressed TGF-beta-induced growth inhibition and cell migration but not epithelial-mesenchymal transition. In addition, HHM inhibited TGF-beta-induced expression of plasminogen activator inhibitor-type 1 (PAI-1), PDGF-B, and p21(WAF), but not Snail. We identified a basic-helix-loop-helix protein, Olig1, as one of the Smad-binding transcription factors affected by HHM. Olig1 interacted with Smad2/3 in response to TGF-beta stimulation, and was involved in transcriptional activation of PAI-1 and PDGF-B. HHM, but not Id proteins, inhibited TGF-beta signalling-dependent association of Olig1 with Smad2/3 through physical interaction with Olig1. HHM thus appears to regulate a subset of TGF-beta target genes including the Olig1-Smad synexpression group. HHM is the first example of a cellular response-selective regulator of TGF-beta signalling with clearly determined mechanisms.
Assuntos
Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-sis/metabolismo , Interferência de RNA , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Fator de Crescimento Transformador beta/genéticaRESUMO
Inhibitory Smad, Smad7, is a potent inhibitor of TGF-beta (transforming growth factor-beta) superfamily signalling. By binding to activated type I receptors, it prevents the activation of R-Smads (receptor-regulated Smads). To identify new components of the Smad pathway, we performed yeast two-hybrid screening using Smad7 as bait, and identified NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) as a direct binding partner of Smad7. NEDD4-2 is structurally similar to Smurfs (Smad ubiquitin regulatory factors) 1 and 2, which were identified previously as E3 ubiquitin ligases for R-Smads and TGF-beta superfamily receptors. NEDD4-2 functions like Smurfs 1 and 2 in that it associates with TGF-beta type I receptor via Smad7, and induces its ubiquitin-dependent degradation. Moreover, NEDD4-2 bound to TGF-beta-specific R-Smads, Smads 2 and 3, in a ligand-dependent manner, and induced degradation of Smad2, but not Smad3. However, in contrast with Smurf2, NEDD4-2 failed to induce ubiquitination of SnoN (Ski-related novel protein N), although NEDD4-2 bound to SnoN via Smad2 more strongly than Smurf2. We showed further that overexpressed NEDD4-2 prevents transcriptional activity induced by TGF-beta and BMP, whereas silencing of the NEDD4-2 gene by siRNA (small interfering RNA) resulted in enhancement of the responsiveness to TGF-beta superfamily cytokines. These data suggest that NEDD4-2 is a member of the Smurf-like C2-WW-HECT (WW is Trp-Trp and HECT is homologous to the E6-accessory protein) type E3 ubiquitin ligases, which negatively regulate TGF-beta superfamily signalling through similar, but not identical, mechanisms to those used by Smurfs.
Assuntos
Receptores de Ativinas Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Camundongos , Ubiquitina-Proteína Ligases Nedd4 , Ligação Proteica , Proteínas Serina-Treonina Quinases , Transporte Proteico , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Proteína Smad2 , Proteína Smad3 , Proteína Smad6 , Proteína Smad7 , Transativadores/genética , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genéticaRESUMO
Members of the transforming growth factor-beta (TGF-beta) superfamily bind to two different serine/threonine kinase receptors, i.e. type I and type II receptors. Upon ligand binding, type I receptors specifically activate intracellular Smad proteins. R-Smads are direct substrates of type I receptors; Smads 2 and 3 are specifically activated by activin/nodal and TGF-beta type I receptors, whereas Smads 1, 5 and 8 are activated by BMP type I receptors. Nearly 30 proteins have been identified as members of the TGF-beta superfamily in mammals, and can be classified based on whether they activate activin/TGF-beta-specific R-Smads (AR-Smads) or BMP-specific R-Smads (BR-Smads). R-Smads form complexes with Co-Smads and translocate into the nucleus, where they regulate the transcription of target genes. AR-Smads bind to various proteins, including transcription factors and transcriptional co-activators or co-repressors, whereas BR-Smads interact with other proteins less efficiently than AR-Smads. Id proteins are induced by BR-Smads, and play important roles in exhibiting some biological effects of BMPs. Understanding the mechanisms of TGF-beta superfamily signalling is thus important for the development of new ways to treat various clinical diseases in which TGF-beta superfamily signalling is involved.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/classificação , Proteínas de Ligação a DNA/genética , Humanos , Modelos Moleculares , Família Multigênica , Filogenia , Estrutura Terciária de Proteína , Proteínas Smad , Transativadores/química , Transativadores/classificação , Transativadores/genéticaRESUMO
Arkadia was originally identified as a protein that enhances signalling activity of Nodal and induces mammalian nodes during early embryogenesis; however, the mechanisms by which Arkadia affects transforming growth factor-beta (TGF-beta) superfamily signalling have not been determined. Here we show that Arkadia is widely expressed in mammalian tissues, and that it enhances both TGF-beta and bone morphogenetic protein (BMP) signalling. Arkadia physically interacts with inhibitory Smad, Smad7, and induces its poly-ubiquitination and degradation. In contrast to Smurf1, which interacts with TGF-beta receptor complexes through Smad7 and degrades them, Arkadia fails to associate with TGF-beta receptors. In contrast to Smad7, expression of Arkadia is down-regulated by TGF-beta. Silencing of the Arkadia gene resulted in repression of transcriptional activities induced by TGF-beta and BMP, and accumulation of the Smad7 protein. Arkadia may thus play an important role as an amplifier of TGF-beta superfamily signalling under both physiological and pathological conditions.