RESUMEN
Signal-transducing adaptor protein-2 (STAP-2) is an adaptor molecule involved in several cellular signaling cascades. Here, we attempted to identify novel STAP-2 interacting molecules, and identified c-Cbl associated protein (CAP) as a binding protein through the C-terminal proline-rich region of STAP-2. Expression of STAP-2 increased the interaction between CAP and c-Cbl, suggesting that STAP-2 bridges these proteins and enhances complex formation. CAP/c-Cbl complex is known to regulate GLUT4 translocation in insulin signaling. STAP-2 overexpressed human hepatocyte Hep3B cells showed enhanced GLUT4 translocation after insulin treatment. Elevated levels of Stap2 mRNA have been observed in 3T3-L1 cells and mouse embryonic fibroblasts (MEFs) during adipocyte differentiation. The differentiation of 3T3-L1 cells into adipocytes was highly promoted by retroviral overexpression of STAP-2. In contrast, STAP-2 knockout (KO) MEFs exhibited suppressed adipogenesis. The increase in body weight with high-fat diet feeding was significantly decreased in STAP-2 KO mice compared to WT animals. These data suggest that the expression of STAP-2 correlates with adipogenesis. Thus, STAP-2 is a novel regulatory molecule that controls insulin signal transduction by forming a c-Cbl/STAP-2/CAP ternary complex.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Diferenciación Celular , Insulina , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adipocitos/metabolismo , Fibroblastos/metabolismo , Insulina/metabolismo , Transducción de Señal , Diferenciación Celular/genéticaRESUMEN
Signal-transducing adaptor protein (STAP)-2 is a recently identified adaptor protein that contains Pleckstrin homology and Src homology 2-like domains, and is also known to be a substrate of breast tumor kinase (Brk). In a previous study, we found that STAP-2 upregulated Brk-mediated activation of signal transducer and activator of transcription (STAT) 3 in breast cancer cells. Here, we examined the involvement of STAP-2 in Brk-mediated STAT5 activation in breast cancer cells. Ectopic expression of STAP-2 induced Brk-mediated transcriptional activity of STAT5. Furthermore, STAP-2-knockdown in T47D breast cancer cells induced a marked decrease in proliferation that was as strong as that after Brk- or STAT5b-knockdown. Regarding the mechanism, the Pleckstrin homology domain of STAP-2 is likely to participate in the process by which Brk phosphorylates and activates STAT5. Taken together, our findings provide insights toward the development of novel therapeutic strategies as well as novel prognostic values in breast carcinomas.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Western Blotting , Neoplasias de la Mama , Proliferación Celular , Femenino , Humanos , Inmunoprecipitación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosforilación , Regiones Promotoras Genéticas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , ARN Interferente Pequeño/genética , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Fosforilación/genética , Proteínas Tirosina Quinasas/genética , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/genéticaRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , FN-kappa B/metabolismo , Factor 3 Asociado a Receptor de TNF/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Línea Celular , Línea Celular Tumoral , Proteínas Co-Represoras , Proteínas de Unión al ADN , Humanos , Immunoblotting , Inmunoprecipitación , Interleucina-6/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/fisiología , Factor 3 Asociado a Receptor de TNF/genética , Proteínas de la Matriz Viral/genéticaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Tirosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Humanos , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Fosforilación , Proteínas Tirosina Quinasas/genética , Tirosina/genéticaRESUMEN
Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates NF-kappaB signaling pathways through the two C-terminal regions, CTAR1 and CTAR2. BS69 has previously been shown to be involved in LMP1-induced c-Jun N-terminal kinase activation through CTAR2 by interacting with tumor necrosis factor (TNFR) receptor-associated factor 6. In the present study, our manipulation of BS69 expression clearly indicates that BS69 negatively regulates LMP1-mediated NF-kappaB activation and up-regulates IL-6 mRNA expression and IkappaB degradation. Our immunoprecipitation experiments suggest that BS69 decreases complex formation between LMP1 and TNFR-associated death domain protein (TRADD).