Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-J kappa/Su(H).
BACKGROUND
The mammalian transcription factor RBP-J kappa binds to the DNA sequence motif CGTGGGAA and is involved in the regulation of gene expression; for example, it plays a part in the transactivation of viral and cellular genes by Epstein-Barr virus nuclear antigen-2. The Drosophila homologue of RBP-J kappa is the product of the Suppressor of Hairless (Su(H)) gene. Su(H) is a neurogenic gene that acts downstream of Notch, which encodes a cell-surface receptor. Furthermore, in the mouse, the phenotypes of homozygous mutant Notch1 embryos are very similar to those of homozygous mutant RBP-J kappa embryos. Recent studies, using the yeast two-hybrid system, have led to the suggestion that the CDC10/ankyrin-like repeats of the Drosophila Notch protein interact with the Su(H) protein.


RESULTS
We searched for proteins that interact with mouse RBP-J kappa using the yeast two-hybrid system, and in this way identified a short intracellular region (mRAM23) of the mouse Notch1 protein that lacks any known sequence motif. In vitro interaction studies, using proteins fused to glutathione-S-transferase, showed that RBP-J kappa and Su(H) bind directly to the RAM23 regions of mouse Notch1 and Drosophila Notch, respectively. Immunoprecipitation analysis showed that RBP-J kappa and the mRAM23 region of mouse Notch1 also interact in vivo. Further studies, including site-directed mutagenesis experiments, narrowed down the region of mouse Notch1 that interacts with RBP-J kappa. The results indicate that this region is less than 50 amino-acid residues in length, and lies immediately downstream of the transmembrane region.


CONCLUSIONS
We show that the transcription factor RBP-J kappa/Su(H) interacts directly with a novel intracellular domain of the cell-surface receptor Notch. RBP-J kappa/Su(H) does not appear to interact with Notch via the CDC10/ankyrin repeats implicated in previous studies.
Tamura
K
K
Department of Medical Chemistry, Kyoto University Faculty of Medicine, Japan.
Taniguchi
Y
Y
Minoguchi
S
S
Sakai
T
T
Tun
T
T
Furukawa
T
T
Honjo
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029721:Drosophila Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D051880:Receptors, Notch; D012097:Repressor Proteins / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*; D015003:Yeasts
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D051880:Receptors, Notch; D012097:Repressor Proteins; C088731:Su(H) protein, Drosophila; D014157:Transcription Factors; D004247:DNA