Abstract
RAP250 is a coactivator for nuclear receptors as well as other transcription factors. Recent studies have established RAP250 as an essential coactivator for many important biological processes, but its exact mechanism of action is not fully understood. To identify novel proteins that can associate with RAP250, we used a yeast two-hybrid system to screen cDNA libraries and identified the intracellular mediators of transforming growth factor-beta (TGF-beta) response Smad2 and Smad3 as direct interacting proteins. We show that the interaction between RAP250 and Smad2/3 is dependent upon the second LXXLL interaction motif in RAP250 and the MH2 domain in Smad2 and Smad3. Mouse embryonic fibroblasts lacking RAP250 have reduced expression of the TGF-beta target gene PAI-1 after stimulation by TGF-beta when compared with wild type cells. Furthermore, we demonstrate a cross-talk between TGF-beta and liver X receptors (LXR) signaling pathways and show that stimulation of cells with TGF-beta and LXR agonists have a synergistic effect on the expression of the LXR target gene ABCG1. Our data identify RAP250 as a new coactivator in the TGF-beta signaling pathway that binds Smad2 and Smad3. Our data also suggest that the interaction between RAP250, Smad2, and Smad3 constitutes an important bridging mechanism linking LXR and TGF-beta signaling pathways.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP Binding Cassette Transporter, Subfamily G, Member 1
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ATP-Binding Cassette Transporters / genetics
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ATP-Binding Cassette Transporters / metabolism
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Amino Acid Motifs / physiology
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Animals
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DNA, Complementary / genetics
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DNA-Binding Proteins / agonists
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DNA-Binding Proteins / metabolism
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Embryo, Mammalian / cytology
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Embryo, Mammalian / metabolism
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Fibroblasts / cytology
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Fibroblasts / metabolism
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Humans
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism*
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Lipoproteins / genetics
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Lipoproteins / metabolism
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Liver X Receptors
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Mice
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Mice, Knockout
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Nuclear Receptor Coactivators
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Orphan Nuclear Receptors
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Plasminogen Activator Inhibitor 1 / genetics
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Plasminogen Activator Inhibitor 1 / metabolism
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Receptors, Cytoplasmic and Nuclear / agonists
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Receptors, Cytoplasmic and Nuclear / metabolism
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Serpin E2
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Serpins / genetics
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Serpins / metabolism
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Signal Transduction / drug effects
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Signal Transduction / physiology*
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Smad2 Protein / genetics
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Smad2 Protein / metabolism*
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Smad3 Protein / genetics
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Smad3 Protein / metabolism*
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Transforming Growth Factor beta / pharmacology
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Two-Hybrid System Techniques
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U937 Cells
Substances
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ABCG1 protein, human
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ABCG1 protein, mouse
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ATP Binding Cassette Transporter, Subfamily G, Member 1
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ATP-Binding Cassette Transporters
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DNA, Complementary
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DNA-Binding Proteins
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Intracellular Signaling Peptides and Proteins
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Lipoproteins
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Liver X Receptors
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NCOA6 protein, human
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Ncoa6 protein, mouse
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Nuclear Receptor Coactivators
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Orphan Nuclear Receptors
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Plasminogen Activator Inhibitor 1
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Receptors, Cytoplasmic and Nuclear
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SERPINE1 protein, human
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SMAD2 protein, human
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SMAD3 protein, human
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Serpin E2
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Serpine2 protein, mouse
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Serpins
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Smad2 Protein
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Smad2 protein, mouse
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Smad3 Protein
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Smad3 protein, mouse
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Transforming Growth Factor beta