The PDZ protein tax-interacting protein-1 inhibits beta-catenin transcriptional activity and growth of colorectal cancer cells.
Wnt signaling is essential during development while deregulation of this pathway frequently leads to the formation of various tumors including colorectal carcinomas. A key component of the pathway is beta-catenin that, in association with TCF-4, directly regulates the expression of Wnt-responsive genes. To identify novel binding partners of beta-catenin that may control its transcriptional activity, we performed a mammalian two-hybrid screen and isolated the Tax-interacting protein (TIP-1). The in vivo complex formation between beta-catenin and TIP-1 was verified by coimmunoprecipitation, and a direct physical association was revealed by glutathione S-transferase pull-down experiments in vitro. By using a panel of deletion mutants of both proteins, we demonstrate that the interaction is mediated by the PDZ (PSD-95/DLG/ZO-1 homology) domain of TIP-1 and requires primarily the last four amino acids of beta-catenin. TIP-1 overexpression resulted in a dose-dependent decrease in the transcriptional activity of beta-catenin when tested on the TOP/FOPFLASH reporter system. Conversely, siRNA-mediated knock-down of endogenous TIP-1 slightly increased endogenous beta-catenin transactivation function. Moreover, we show that overexpression of TIP-1 reduced the proliferation and anchorage-independent growth of colorectal cancer cells. These data suggest that TIP-1 may represent a novel regulatory element in the Wnt/beta-catenin signaling pathway.
Kanamori
Mutsumi
M
Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
Sandy
Peter
P
Marzinotto
Stefania
S
Benetti
Roberta
R
Kai
Chikatoshi
C
Hayashizaki
Yoshihide
Y
Schneider
Claudio
C
Suzuki
Harukazu
H
D000362:Agar / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins; D002455:Cell Division; D002460:Cell Line; D015179:Colorectal Neoplasms / Q000378:metabolism*; D006224:Cricetinae; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D005972:Glutaminase; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000502:physiology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques; D051176:beta Catenin
C495270:CTNNB1 protein, human; C495267:CTNNB1 protein, mouse; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D034741:RNA, Small Interfering; C421249:TAX1BP3 protein, human; D015534:Trans-Activators; D051176:beta Catenin; D000362:Agar; D008156:Luciferases; D005982:Glutathione Transferase; D005972:Glutaminase