Biochemical and Biophysical Research Communications
Functional interaction between Smad, CREB binding protein, and p68 RNA helicase☆
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.