Abstract
β-Catenin functions as a transcriptional regulator in Wnt signaling. Its function is regulated by a specific destruction system. Plakoglobin is a close homologue of β-catenin in mammalian cells and is regulated in a similar fashion. When β-catenin or plakoglobin is exogenously expressed in cells, endogenous β-catenin is stabilized, which complicates estimation of the transcriptional activities of exogenously expressed proteins. To facilitate the design of experiments aimed at investigating the transcriptional activities of β-catenin and plakoglobin, we utilized F9 cells in which we knocked out endogenous β-catenin and/or plakoglobin by gene deletion and exogenously expressed wild-type and mutant β-catenin and/or plakoglobin. We show that C-terminally deleted β-catenin, but not plakoglobin, has a strong dominant-negative effect on transcription without altering the nuclear accumulation of β-catenin. Moreover, we show that Wnt-3a activation of LEF/T-cell factor (TCF)-dependent transcription depends on β-catenin but not on plakoglobin. Using chimeras of β-catenin and plakoglobin, we demonstrate that plakoglobin has the potential to function in transcriptional regulation but is not responsible for Wnt-3a signaling in F9 cells. Our data show that preferential nuclear accumulation of β-catenin is not necessarily linked to its transcriptional activity. We also clearly demonstrate that plakoglobin is insufficient for LEF/TCF-dependent transcriptional activation by Wnt-3a in F9 cells.
ACKNOWLEDGMENTS
We are grateful to all laboratory members (Division of Cellular Interactions of the Institute of Molecular Embryology and Genetics at Kumamoto University) for their participation in helpful discussions. We also thank M. Matsuda for pCAG-CGFP; M. van de Wetering and H. Clevers for pTOPflash and pFOPflash; H. Niwa for pCAGGSneodelEcoRI; S. Takada for Wnt-3a-secreting L cells; C. Fujiwara, E. Morikawa, and T. Magarikaji for excellent technical assistance; and M. J. Wheelock for critical reading of the manuscript. A.N. thanks the late Shoichiro Tsukita for his encouragement at the initiation of this work.
Part of this work at the Department of Cellular Interactions of the Institute of Molecular Embryology and Genetics of Kumamoto University was supported by a grant to A.N. from the Core Research for Evolutional Science and Technology (CREST), Grants-in-Aid for Scientific Research and Cancer Research, and a Grant-in-Aid for 21st Century COE Research “Cell Fate Regulation Research and Education Unit” from the Ministry of Education, Culture, Sports, Science and Technology.