Interaction of the BMPR-IA tumor suppressor with a developmentally relevant splicing factor☆
Section snippets
Materials and methods
Yeast two-hybrid. The Proquest Yeast Two-Hybrid kit with Gateway technology (Invitrogen) was used as described by the manufacturer. Briefly, the intracellular domain of BMPR-IA was amplified out of the hALK3 expression vector (a kind gift of Dr. Peter ten-Dijke) by polymerase chain reaction (PCR). The resulting PCR product was cloned into the Gal4 DNA binding domain yeast expression vector pDEST32. The resulting expression vector was cotransformed into MaV203 competent yeast (Invitrogen) along
BMPR-IA yeast two-hybrid screening
To identify BMPR-IA interacting proteins, the intracellular domain (ICD) of BMPR-IA was used as “bait” in a yeast two-hybrid screen as described in Materials and methods. First, a human fetal brain library was screened. Of the 31 interacting proteins isolated from the human fetal brain library, 30 were identified as splicosome-associated protein subunit 4 (SAP49), also known as splicing factor 3b, subunit 4 (Sf3b4). Of the 30 SAP49 clones, there were 10 distinct clones (with different
Acknowledgments
We thank Christian Sirard for the Msx2-luciferase reporter; Peter ten-Dijke for the human BMPR-IA expression vector; Henry Yang for DNA sequencing; and Challice Bonifant, Jung-Sik Kim, and Carolyn Lee for comments on the manuscript. This work was supported by NIH Grants K01 CA87828 and R01 CA095736 (to T.W.), and the Lombardi Cancer Center Support Grant (P30 CA51008).
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Abbreviations: BMP, bone morphogenetic protein; BMPR-IA, bone morphogenetic protein receptor 1A; BD, binding domain; AD, activation domain; SAP49, splicoseome associated protein 49; TGF-β, transforming growth factor β; ICD, intracellular domain.