Functional interaction between Smad, CREB binding protein, and p68 RNA helicase

• Published in: Biochemical and biophysical research communications Vol. 324; no. 1; pp. 70 - 76
• Main Authors: Warner, Dennis R., Bhattacherjee, Vasker, Yin, Xiaolong, Singh, Saurabh, Mukhopadhyay, Partha, Pisano, M. Michele, Greene, Robert M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05-11-2004
• Subjects: Animals; CBP; Cell Line; CREB-Binding Protein; DEAD-box RNA Helicases; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Embryo, Mammalian - anatomy & histology; Embryo, Mammalian - physiology; Embryonic Structures - anatomy & histology; Embryonic Structures - physiology; Gene Expression Regulation, Developmental; Gene Library; Genes, Reporter; Helicase; Mice; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; p68; Protein Binding; Protein Kinases - genetics; Protein Kinases - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; RNA Helicases - genetics; RNA Helicases - metabolism; Signal Transduction - physiology; Smad; Smad3 Protein; TGFβ; Trans-Activators - genetics; Trans-Activators - metabolism; Transcription; Transcriptional Activation; Transforming Growth Factor beta - metabolism; Two-hybrid; Two-Hybrid System Techniques; CBP; Helicase; p68; Transcription; TGFβ; Smad; Two-hybrid
• DOI: 10.1016/j.bbrc.2004.09.017
• EISSN: 1090-2104
• ISSN: 0006-291X

The transforming growth factors β control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region. TGFβ superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFβ-responsive genes. Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN. Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell- and tissue-specific. In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue. The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays. Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFβ-stimulated reporter gene transcription. Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression. Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP. This offers a means of enhancing TGFβ-mediated cellular responses in developing orofacial tissue.