Defining the Roles of β-Catenin and Plakoglobin in LEF/T-Cell Factor-Dependent Transcription Using β-Catenin/Plakoglobin-Null F9 Cells

Abstract

␤-Catenin functions as a transcriptional regulator in Wnt signaling. Its function is regulated by a specific destruction system. Plakoglobin is a close homologue of ␤-catenin in mammalian cells and is regulated in a similar fashion. When ␤-catenin or plakoglobin is exogenously expressed in cells, endogenous ␤-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 ␤-catenin and plakoglobin, we utilized F9 cells in which we knocked out endogenous ␤-catenin and/or plakoglobin by gene deletion and exogenously expressed wild-type and mutant ␤-catenin and/or plakoglobin. We show that Cterminally deleted ␤-catenin, but not plakoglobin, has a strong dominant-negative effect on transcription without altering the nuclear accumulation of ␤-catenin. Moreover, we show that Wnt-3a activation of LEF/Tcell factor (TCF)-dependent transcription depends on ␤-catenin but not on plakoglobin. Using chimeras of ␤-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 ␤-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.

Anchoring Junctions As Drug Targets: Role in Contraceptive Development

“…Similar to ␤-catenin, plakoglobin can activate the Wnt signaling pathway, transduce signals to the nucleus, and activate lymphoid enhancer binding factor/T cell factor-dependent transcription (Simcha et al, 1998;Miravet et al, 2002;Maeda et al, 2004;Shimizu et al, 2008). It is noteworthy that plakoglobin, as well as E-cadherin, increased after Wnt-1 expression in PC-12 cells, and this strengthened cell-cell contacts (Bradley et al, 1993).…”

Cadherin-based adhesions in the apical endfoot are required for active Notch signaling to control neurogenesis in vertebrates

“…pCAG-dE(HA) was constructed from pCAG-EM2, a mouse E-cad expression vector (Shimizu et al, 2008), by replacing the HincII-XhoI fragment with a PCR product including HA-tag sequence and several restriction enzyme sites. pAcLCL-AFP was constructed by replacing the lacZ-cassette of pAcCATZ (Araki et al, 1995) with GFP by blunt ligation.…”

Plakoglobin regulates cell motility through Rho- and fibronectin-dependent Src signaling

“…PG is also found in the cytoplasm and nucleus (Green and Simpson, 2007;Schmidt and Koch, 2007), where it is able to act independently of its function in intercellular adhesion. Its adhesionindependent functions are still not well defined, but the data suggest that PG can regulate gene expression and protein stability (Aktary et al, 2010;Hakimelahi et al, 2000;Shimizu et al, 2008) in both a -catenin-dependent and -independent manner (Raurell et al, 2006;Teuliere et al, 2004;Yin and Green, 2004;Zhurinsky et al, 2000).…”

“…PG is also found in the cytoplasm and nucleus (Green and Simpson, 2007;Schmidt and Koch, 2007), where it is able to act independently of its function in intercellular adhesion. Its adhesionindependent functions are still not well defined, but the data suggest that PG can regulate gene expression and protein stability (Aktary et al, 2010;Hakimelahi et al, 2000;Shimizu et al, 2008) in both a -catenin-dependent and -independent manner (Raurell et al, 2006;Teuliere et al, 2004;Yin and Green, 2004;Zhurinsky et al, 2000).Recently we demonstrated that PG not only inhibits motility of keratinocytes in contact, but also inhibits Src-dependent single cell motility (Yin et al, 2005b). The observed changes in motility and altered cell morphology of PG -/-keratinocytes suggested to us that PG could be regulating cell-substrate interactions by modulating components of the extracellular matrix (ECM), its integrin receptors and/or the molecules involved in ECM-triggered motility cues.…”