title: Four amino acids within a tandem QxVx repeat in a predicted extended α-helix of the Smad-binding domain of Sip1 are necessary for binding to activated Smad proteins.
abstract: The zinc finger transcription factor Smad-interacting protein-1 (Sip1; Zeb2, Zfhx1b) plays an important role during vertebrate embryogenesis in various tissues and differentiating cell types, and during tumorigenesis. Previous biochemical analysis suggests that interactions with several partner proteins, including TGFβ family receptor-activated Smads, regulate the activities of Sip1 in the nucleus both as a DNA-binding transcriptional repressor and activator. Using a peptide aptamer approach we mapped in Sip1 its Smad-binding domain (SBD), initially defined as a segment of 51 amino acids, to a shorter stretch of 14 amino acids within this SBD. Modelling suggests that this short SBD stretch is part of an extended α-helix that may fit the binding to a hydrophobic corridor within the MH2 domain of activated Smads. Four amino acids (two polar Q residues and two non-polar V residues) that form the tandem repeat (QxVx)2 in this 14-residue stretch were found to be crucial for binding to both TGFβ/Nodal/Activin-Smads and BMP-Smads. A full-length Sip1 with collective mutation of these Q and V residues (to A) no longer binds to Smads, while it retains its binding activity to its cognate bipartite target DNA sequence. This missense mutant Sip1(AxAx)2 provides a new molecular tool to identify SBD (in)dependent target genes in Sip1-controlled TGFβ and/or BMP (de)regulated cellular, developmental and pathological processes.
authors.affiliation: Laboratory of Molecular Biology (Celgen), Department of Development and Regeneration, Katholieke Universiteit Leuven, Leuven, Belgium ; Department of Cell Biology, Erasmus University Medical Center, Rotterdam, The Netherlands.
authors.forename: Andrea
authors.initials: A
authors.lastname: Conidi
authors.forename: Veronique
authors.initials: V
authors.lastname: van den Berghe
authors.forename: Kris
authors.initials: K
authors.lastname: Leslie
authors.forename: Agata
authors.initials: A
authors.lastname: Stryjewska
authors.forename: Hua
authors.initials: H
authors.lastname: Xue
authors.forename: Ye-Guang
authors.initials: YG
authors.lastname: Chen
authors.forename: Eve
authors.initials: E
authors.lastname: Seuntjens
authors.forename: Danny
authors.initials: D
authors.lastname: Huylebroeck
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D052158:Aptamers, Peptide / Q000378:metabolism; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D007395:Interneurons / Q000166:cytology / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D051785:Smad Proteins / Q000378:metabolism*; D055672:Static Electricity; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D000596:Amino Acids; D052158:Aptamers, Peptide; D008024:Ligands; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; C491561:Sip1 protein, mouse; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D004247:DNA