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

doi:10.1016/j.bbrc.2004.09.017

Biochemical and Biophysical Research Communications

Volume 324, Issue 1, 5 November 2004, Pages 70-76

https://doi.org/10.1016/j.bbrc.2004.09.017 Get rights and content

Abstract

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.

Section snippets

Materials and methods

Yeast two-hybrid expression library and bait construct. The cDNA expression library and bait construction previously have been described in detail [7], [8]. Briefly, a cDNA expression library fused to the activation domain of Gal4 was prepared by Research Genetics (Huntsville, AL) using RNA isolated from embryonic maxillofacial tissue dissected from mid-gestation mouse embryos (ICR, Harlan, Indianapolis, IN) and cloned into pGADT7 (Clontech, Palo Alto, CA). The Smad 3 MH2 domain was amplified

Yeast two-hybrid assay

To identify novel Smad binding proteins expressed in developing orofacial tissue, a yeast two-hybrid screen was employed using the MH2 domain of the TGFβ-regulated Smad 3 (in pGBKT7) as bait to screen a mouse embryonic orofacial cDNA expression library prepared in pGADT7. Of approximately 5 × 10⁵ transformants from the initial screen, approximately 100 colonies proliferated on nutritionally restrictive medium (i.e., medium lacking histidine, tryptophan, adenine, and leucine) and expressed

Discussion

The TGFβ superfamily of secreted growth and differentiation factors controls diverse developmental processes such as embryonic axis determination, cellular proliferation/differentiation/apoptosis, and extracellular matrix production [1]. The role of TGFβs in adult tissues is underscored by the observation that the TGFβ signaling pathway is frequently dysregulated in tumors, leading to escape of the cells from the growth inhibitory action of TGFβ and tumor suppressors [18], [19]. TGFβ’s role in

Acknowledgments

The authors thank Dr. H. Brivanlou at Rockefeller University, New York, NY for pBluescript-Smad 7; Dr. M. Kato at the Japanese Foundation for Cancer Research, Tokyo, Japan, for pGEX4T1-Smad 3 ΔMH2; Dr. H. Heldin at the Ludwig Institute for Cancer Research, Uppsala, Sweden, for pcDNA-Smad 3; Dr. X. Lin at Baylor University, Houston, TX, for pGEX2TK- Smad 1; Dr. Y. Chen at Indiana University, Indianapolis, IN for pGEX4T2-Smad 2 and pGEX4T2-Smad 4; and Dr. R. Janknecht at the Mayo Clinic,

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^☆: Abbreviations: TGFβ, transforming growth factor β; p68, p68 RNA helicase; CBP, Creb binding protein; PCR, polymerase chain reaction; X-α-gal, 5-bromo-4-chloro-3-indolyl-α d-galactopyranoside; GST, glutathione S-transferase; RIPA, radioimmunoprecipitation assay; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

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

Abstract

Section snippets

Materials and methods

Yeast two-hybrid assay

Discussion

Acknowledgments

Cell

J. Biol. Chem.

Mol. Cell

Cell

Mol. Cell

Biochem. Biophys. Res. Commun.

FEBS Lett.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Brain Res. Bull.

J. Biol. Chem.

Molecular fingerprinting of TGF beta-treated embryonic maxillary mesenchymal cells

Orthod. Craniofac. Res.

Nuclear protein with sequence homology to translation initiation factor eIF-4A

Nature

Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor α

Mol. Cell. Biol.

Synergism between p68 RNA helicase and the transcriptional coactivators CBP and p300

Oncogene

TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4

EMBO J.