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.
Highlights
The Transforming Growth Factor type b (TGFb) system controls many cellular processes including proliferation versus differentiation,adhesion, epithelial-mesenchymal transition (EMT), and cell migration and accompanying cell shape changes [1,2,3,4]
The initially mapped Smad-binding domain (SBD) of Smad-interacting protein-1 (Sip1) was a 51 aa-long linear sequence encompassing aa437–487 of mouse Sip1. This SBD is necessary and sufficient to interact with the MH2 i.e. C-terminal domain of Smad1 in GST-based pull-down assays, with the MH2 domain of Smad2 and 3 and with the MH2 domain of Smad1, 5 and 8 in yeast two-hybrid assay, and in Bone Morphogenetic Proteins (BMPs) pathway-activated mammalian cells [12,31; Dzwonek, Conidi, van Grunsven, Huylebroeck and Verschueren, unpublished results)
We tested by co-immunoprecipitation whether these SBD-Trx proteins were able to bind to Smads
Summary
The Transforming Growth Factor type b (TGFb) system controls many cellular processes including proliferation versus differentiation, (de)adhesion, epithelial-mesenchymal transition (EMT), and cell migration and accompanying cell shape changes [1,2,3,4]. TGFb family signaling, including via Nodal and Bone Morphogenetic Proteins (BMPs), is crucial during embryogenesis in induction, patterning and morphogenesis, and for the regulation of stem/progenitor cells and their niche in the embryo and the adult animal [5,6]. In each of these normal processes, the signaling by this ligand-receptor system together with its intracellular signal transduction involving Smad proteins and proteinkinase based non-Smad signaling, is under tight control at multiple levels and by various mechanisms [7,8,9,10]. Understanding how each of these many Smad-interacting proteins (SIPs) precisely function when bound to Smads, and whether they can function Smad-independently, remains both in the Smad and SIP fields a relevant challenge
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