title: Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus.
abstract: Smad4 plays a pivotal role in all transforming growth factor beta (TGF-beta) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-beta signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-beta signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-beta signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm.
authors.affiliation: Laboratory of Developmental Signalling, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.
authors.forename: C E
authors.initials: CE
authors.lastname: Pierreux
authors.forename: F J
authors.initials: FJ
authors.lastname: Nicolás
authors.forename: C S
authors.initials: CS
authors.lastname: Hill
mesh_terms: D021581:Active Transport, Cell Nucleus; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D002451:Cell Compartmentation; D002467:Cell Nucleus; D003593:Cytoplasm; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005091:Exons; D051858:Forkhead Transcription Factors; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D019913:Nuclear Localization Signals; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D029867:Xenopus Proteins
chemical_list: D004268:DNA-Binding Proteins; C510350:FOXH1 protein, human; D051858:Forkhead Transcription Factors; C491117:Foxh1 protein, mouse; D009414:Nerve Growth Factors; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D021382:Protein Sorting Signals; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D029867:Xenopus Proteins; C490808:smad4.1 protein, Xenopus; C117263:smad4.2 protein, Xenopus