RESUMO
The Rad1-Rad9-Hus1 (9-1-1) complex serves a dual role as a DNA-damage sensor in checkpoint signaling and as a mediator in the DNA repair pathway. However, the intercellular mechanisms that regulate the 9-1-1 complex are poorly understood. Jab1, the fifth component of the COP9 signalosome complex, has a central role in the degradation of multiple proteins and is emerging as an important regulator in cancer development. Here, we tested the hypothesis that Jab1 controls the protein stability of the 9-1-1 complex via the proteosome pathway. We provide evidence that Jab1 physically associates with the 9-1-1 complex, and show that this association is mediated through direct interaction between Jab1 and Rad1, one of the subunits of the 9-1-1 complex. Importantly, Jab1 causes translocation of the 9-1-1 complex from the nucleus to the cytoplasm, mediating rapid degradation of the 9-1-1 complex via the 26 S proteasome. Furthermore, Jab1 significantly suppresses checkpoint signaling activation, DNA synthesis recovery from blockage and cell viability after replication stresses such as UV exposure, gamma radiation and treatment with hydroxyurea. These results suggest that Jab1 is an important regulator for the stability of protein 9-1-1 control in cells, which may provide novel information on the involvement of Jab1 in the checkpoint and DNA repair signaling in response to DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Exonucleases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeo Hidrolases/metabolismo , Transporte Ativo do Núcleo Celular , Complexo do Signalossomo COP9 , Proteínas de Ciclo Celular/genética , DNA/biossíntese , Exonucleases/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
Smad4, also known as deleted in pancreatic carcinoma locus 4 (DPC4), is a critical co-factor in signal transduction pathways activated by transforming growth factor (TGF)-beta-related ligands that regulate cell growth and differentiation. Mutations in Smad4/DPC4 have been identified in approximately 50% of pancreatic adenocarcinomas. Here we report that SCF(beta-TrCP1), a ubiquitin (E3) ligase, is a critical determinant for Smad4 protein degradation in pancreatic cancer cells. We found that F-box protein beta-TrCP1 in this E3 ligase interacted with Smad4 and that SCF(beta-TrCP1) inhibited TGF-beta biological activity in pancreatic cancer cells by decreasing Smad4 stability. Very low Smad4 protein levels in human pancreatic ductal adenocarcinoma cells were observed by immunohistochemistry. By analyzing pancreatic tumor-derived Smad4 mutants, we found that most point-mutated Smad4 proteins, except those within or very close to a mutation cluster region, exhibited higher interaction affinity with beta-TrCP1 and significantly elevated protein ubiquitination by SCF(beta-TrCP1). Furthermore, AsPC-1 and Caco-2, two cancer cell lines harboring Smad4 point mutations, exhibited rapid Smad4 protein degradation due to the effect of SCF(beta-TrCP1). Both Smad4 levels and TGF-beta signaling were elevated by retrovirus-delivered beta-TrCP1 siRNA in pancreatic cancer cells. Therefore, inhibition of Smad4-specific E3 ligase might be a target for therapeutic intervention in pancreatic cancer.
Assuntos
Adenocarcinoma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Transativadores/metabolismo , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal de Mama/metabolismo , Proteínas de Ligação a DNA/genética , Estabilidade de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/patologia , Mutação Puntual , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Ligases SKP Culina F-Box/farmacologia , Proteína Smad4 , Transativadores/genética , Fator de Crescimento Transformador beta/antagonistas & inibidores , Ubiquitina/metabolismoRESUMO
Antiestrogen compounds exhibit a variety of different effects in different tissues and are widely used for the treatment of osteoporosis, breast cancer, and other diseases. Upon examining the molecular mechanisms, we found that Smad4, a common signal transducer in the bone morphogenetic protein (BMP)/transforming growth factor-beta (TGF-beta) signaling pathway, functions as a transcription corepressor for human estrogen receptor alpha (ERalpha). Endogenous ERalpha was co-immunoprecipitated with Smad4, and the interaction was induced by antiestrogen ligands such as tamoxifen, raloxifene, and droloxifen, which was confirmed in chromatin immunoprecipitation assays. Smad4 and ERalpha form a complex when ERalpha binds to the estrogen-responsive element within the estrogen target gene promoter. Importantly, the expression of Smad4 inhibits both antiestrogen-induced luciferase activity and estrogen downstream target gene transcription in breast cancer cells. Mapping of the interaction domains indicates that the activation function 1 (AF1) domain of ERalpha is essential for its interaction with Smad4, while the MH1 domain and linker region of Smad4 are essential for the interaction. Our findings represent a novel mechanism that TGF-beta may regulate cell fate through Smad4-mediated cross-talk with estrogen.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Receptores de Estrogênio/antagonistas & inibidores , Proteínas Repressoras/fisiologia , Transativadores/fisiologia , Transcrição Gênica , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Antagonistas de Estrogênios , Receptor alfa de Estrogênio , Regulação da Expressão Gênica , Humanos , Receptor Cross-Talk , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Proteínas Repressoras/genética , Elementos de Resposta , Proteína Smad4 , Transativadores/genética , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Smad4 is a key intracellular mediator for the transforming growth factor-beta (TGF-beta) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 protein stability is unclear. We have identified SCF(beta-TrCP1), a ubiquitin (E3) ligase, as a critical determinant for the protein degradation of Smad4 protein. F-box protein beta-TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The beta-TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing beta-TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous beta-TrCP1 suppression increases the expression of Smad4 protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-beta-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCF(beta-TrCP1) abrogates TGF-beta function in vivo by decreasing Smad4 stability.