RESUMO
Transforming growth factor beta (TGF-beta) receptors phosphorylate Smad3 and induce its nuclear import so it can regulate gene transcription. Smad3 can return to the cytoplasm to propagate further cycles of signal transduction or to be degraded. We demonstrate that Smad3 is exported by a constitutive mechanism that is insensitive to leptomycin B. The Mad homology 2 (MH2) domain is responsible for Smad3 export, which requires the GTPase Ran. Inactive, GDP-locked RanT24N or nuclear microinjection of Ran GTPase activating protein 1 blocked Smad3 export. Inactivation of the Ran guanine nucleotide exchange factor RCC1 inhibited Smad3 export and led to nuclear accumulation of phosphorylated Smad3. A screen for importin/exportin family members that associate with Smad3 identified exportin 4, which binds a conserved peptide sequence in the MH2 domain of Smad3 in a Ran-dependent manner. Exportin 4 is sufficient for carrying the in vitro nuclear export of Smad3 in cooperation with Ran. Knockdown of endogenous exportin 4 completely abrogates the export of endogenous Smad3. A short peptide representing the minimal interaction domain in Smad3 effectively competes with Smad3 association to exportin 4 and blocks nuclear export of Smad3 in vivo. We thus delineate a novel nuclear export pathway for Smad3.
Assuntos
Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Proteína Smad3/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Sítios de Ligação/genética , Linhagem Celular , Células HeLa , Humanos , Técnicas In Vitro , Carioferinas/antagonistas & inibidores , Carioferinas/genética , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Proteína Smad3/química , Proteína Smad3/genética , TransfecçãoRESUMO
Smad proteins transduce transforming growth factor beta (TGF-beta) and bone morphogenetic protein (BMP) signals that regulate cell growth and differentiation. We have identified YY1, a transcription factor that positively or negatively regulates transcription of many genes, as a novel Smad-interacting protein. YY1 represses the induction of immediate-early genes to TGF-beta and BMP, such as the plasminogen activator inhibitor 1 gene (PAI-1) and the inhibitor of differentiation/inhibitor of DNA binding 1 gene (Id-1). YY1 inhibits binding of Smads to their cognate DNA elements in vitro and blocks Smad recruitment to the Smad-binding element-rich region of the PAI-1 promoter in vivo. YY1 interacts with the conserved N-terminal Mad homology 1 domain of Smad4 and to a lesser extent with Smad1, Smad2, and Smad3. The YY1 zinc finger domain mediates the association with Smads and is necessary for the repressive effect of YY1 on Smad transcriptional activity. Moreover, downregulation of endogenous YY1 by antisense and small interfering RNA strategies results in enhanced transcriptional responses to TGF-beta or BMP. Ectopic expression of YY1 inhibits, while knockdown of endogenous YY1 enhances, TGF-beta- and BMP-induced cell differentiation. In contrast, overexpression or knockdown of YY1 does not affect growth inhibition induced by TGF-beta or BMP. Accordingly, YY1 does not interfere with the regulation of immediate-early genes involved in the TGF-beta growth-inhibitory response, the cell cycle inhibitors p15 and p21, and the proto-oncogene c-myc. In conclusion, YY1 represses Smad transcriptional activities in a gene-specific manner and thus regulates cell differentiation induced by TGF-beta superfamily pathways.