NARF, an nemo-like kinase (NLK)-associated ring finger protein regulates the ubiquitylation and degradation of T cell factor/lymphoid enhancer factor (TCF/LEF).
beta-Catenin is a key player in the Wnt signaling pathway, and interacts with cofactor T cell factor/lymphoid enhancer factor (TCF/LEF) to generate a transcription activator complex that activates Wnt-induced genes. We previously reported that Nemo-like kinase (NLK) negatively regulates Wnt signaling via phosphorylation of TCF/LEF. To further evaluate the physiological roles of NLK, we performed yeast two-hybrid screening to identify NLK-interacting proteins. From this screen, we isolated a novel RING finger protein that we term NARF (NLK associated RING finger protein). Here, we show that NARF induces the ubiquitylation of TCF/LEF in vitro and in vivo, and functions as an E3 ubiquitin-ligase that specifically cooperates with the E2 conjugating enzyme E2-25K. We found that NLK augmented NARF binding and ubiquitylation of TCF/LEF, and this required NLK kinase activity. The ubiquitylated TCF/LEF was subsequently degraded by the proteasome. Furthermore, NARF inhibited formation of the secondary axis induced by the ectopic expression of beta-catenin in Xenopus embryos. Collectively, our findings raise the possibility that NARF functions as a novel ubiquitin-ligase to suppress the Wnt-beta-catenin signaling.
Yamada
Misato
M
Department of Molecular Cell Biology, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, and SORST, JST, Chiyoda-ku, Tokyo 101-0062.
Ohnishi
Junji
J
Department of Molecular Cell Biology, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, and SORST, JST, Chiyoda-ku, Tokyo 101-0062.
Ohkawara
Bisei
B
Department of Molecular Cell Biology, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, and SORST, JST, Chiyoda-ku, Tokyo 101-0062.
Iemura
Shunichiro
S
National Institutes of Advanced Industrial Science and Technology, Biological Information Research Center (JBIRC), Kohtoh-ku, Tokyo 135-0064.
Satoh
Kiyotoshi
K
Department of Molecular Cell Biology, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, and SORST, JST, Chiyoda-ku, Tokyo 101-0062.
Hyodo-Miura
Junko
J
Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.
Kawachi
Kaoru
K
Department of Molecular Cell Biology, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, and SORST, JST, Chiyoda-ku, Tokyo 101-0062.
Natsume
Tohru
T
National Institutes of Advanced Industrial Science and Technology, Biological Information Research Center (JBIRC), Kohtoh-ku, Tokyo 135-0064.
Shibuya
Hiroshi
H
Department of Molecular Cell Biology, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, and SORST, JST, Chiyoda-ku, Tokyo 101-0062; Center of Excellence Program for Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Chiyoda, Tokyo 101-0062. Electronic address: shibuya.mcb@mri.tmd.ac.jp.
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis*; D009865:Oocytes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051820:TCF Transcription Factors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D014981:Xenopus; D051176:beta Catenin / Q000378:metabolism
C400022:NARF protein, human; D009687:Nuclear Proteins; D051820:TCF Transcription Factors; D025801:Ubiquitin; D051176:beta Catenin