RESUMO
STAP-2 (signal transducing adaptor protein-2) is a recently identified adaptor protein that contains pleckstrin homology (PH) and Src homology 2-like domains, as well as a STAT3-binding motif in its C-terminal region. STAP-2 is also a substrate of breast tumor kinase (Brk). In breast cancers, Brk expression is deregulated and promotes STAT3-dependent cell proliferation. In the present study, manipulated STAP-2 expression demonstrated essential roles of STAP-2 in Brk-mediated STAT3 activation. STAP-2 interacts with both Brk and STAT3. In addition, small interfering RNA-mediated reduction of endogenous STAP-2 expression strongly decreased Brk-mediated STAT3 activation in T47D breast cancer cells. The PH domain of STAP-2 is involved in multiple steps: the binding between Brk and STAP-2, the activation and tyrosine phosphorylation of STAT3, and the activation of Brk. Notably, a STAP-2 PH-Brk fusion protein exhibited robust kinase activity and increased activation and tyrosine phosphorylation of STAT3. Finally, STAP-2 knockdown in T47D cells induced a significant decrease of proliferation, as strong as that of Brk or STAT3 knockdown. Taken together, our findings are likely to inform the development of a novel therapeutic strategy, as well as the determination of novel prognostic values, in breast carcinomas.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Fosforilação/genética , Proteínas Tirosina Quinases/genética , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/genéticaRESUMO
Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-kappaB signaling pathways through two C-terminal regions, CTAR1 and CTAR2. Previous studies have demonstrated that BS69, a multidomain cellular protein, regulates LMP1/CTAR2-mediated NF-kappaB activation by interfering with the complex formation between TRADD and LMP1/CTAR2. Here, we found that BS69 directly interacted with the LMP1/CTAR1 domain and regulated LMP1/CTAR1-mediated NF-kappaB activation and subsequent IL-6 production. Regarding the mechanisms involved, we found that BS69 directly interacted with TRAF3, a negative regulator of NF-kappaB activation. Furthermore, small-interfering RNA-mediated knockdown experiments revealed that TRAF3 was involved in the BS69-mediated suppression of LMP1/CTAR1-induced NF-kappaB activation.
Assuntos
Proteínas de Transporte/metabolismo , NF-kappa B/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Proteínas da Matriz Viral/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Correpressoras , Proteínas de Ligação a DNA , Humanos , Immunoblotting , Imunoprecipitação , Interleucina-6/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator 3 Associado a Receptor de TNF/genética , Proteínas da Matriz Viral/genéticaRESUMO
Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. STAP-2 is also known as breast tumor kinase (Brk) substrate (BKS). Our previous studies revealed that STAP-2 binds to signal transducer and activator of transcription 3 (STAT3) and STAT5, and regulates the signaling pathways downstream of them. In the present study, we identified tyrosine-250 (Tyr250) in STAP-2 as a major site of phosphorylation by Brk, using a series of STAP-2 YF mutants and anti-phospho-STAP-2 Tyr250 antibody. Furthermore, overexpression of the STAP-2 Y250F mutant protein affected Brk-mediated STAT3 activation. Importantly, small-interfering RNA-mediated reduction of endogenous STAP-2 expression decreased Brk-mediated STAT3 activation. Taken together, our findings demonstrate that STAP-2 is phosphorylated at Tyr250 by Brk, and plays an important role in Brk-mediated STAT3 activation.