RESUMEN
Smad3 and Smad4 are sequence-specific DNA-binding factors that bind to their consensus DNA-binding sites in response to transforming growth factor beta (TGFbeta) and activate transcription. Recent evidence implicates Smad3 and Smad4 in the transcriptional activation of consensus AP-1 DNA-binding sites that do not interact with Smads directly. Here, we report that Smad3 and Smad4 can physically interact with AP-1 family members. In vitro binding studies demonstrate that both Smad3 and Smad4 bind all three Jun family members: JunB, cJun, and JunD. The Smad interacting region of JunB maps to a C-terminal 20-amino acid sequence that is partially conserved in cJun and JunD. We show that Smad3 and Smad4 also associate with an endogenous form of cJun that is rapidly phosphorylated in response to TGFbeta. Providing evidence for the importance of this interaction between Smad and Jun proteins, we demonstrate that Smad3 is required for the activation of concatamerized AP-1 sites in a reporter construct that has previously been characterized as unable to bind Smad proteins directly. Together, these data suggest that TGFbeta-mediated transcriptional activation through AP-1 sites may involve a regulated interaction between Smads and AP-1 transcription factors.
Asunto(s)
Proteínas de Unión al ADN/genética , Transactivadores/genética , Factor de Transcripción AP-1/genética , Activación Transcripcional , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Animales , Células COS , Datos de Secuencia Molecular , Transducción de Señal/genética , Proteína smad3 , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Transcriptional regulation by transforming growth factor beta (TGF-beta) is a complex process which is likely to involve cross talk between different DNA responsive elements and transcription factors to achieve maximal promoter activation and specificity. Here, we describe a concurrent requirement for two discrete responsive elements in the regulation of the c-Jun promoter, one a binding site for a Smad3-Smad4 complex and the other an AP-1 binding site. The two elements are located 120 bp apart in the proximal c-Jun promoter, and each was able to independently bind its corresponding transcription factor complex. The effects of independently mutating each of these elements were nonadditive; disruption of either sequence resulted in complete or severe reductions in TGF-beta responsiveness. This simultaneous requirement for two distinct and independent DNA binding elements suggests that Smad and AP-1 complexes function synergistically to mediate TGF-beta-induced transcriptional activation of the c-Jun promoter.
