Nuclear Speckle-Associated Protein Pnn/DRS Binds to the Transcriptional Corepressor CtBP and Relieves CtBP- Mediated Repression of the E-Cadherin Gene

Abstract

Previously, we have shown that pinin/DRS (Pnn), a 140-kDa nuclear and cell adhesion-related phosphoprotein, is involved in the regulation of cell adhesion and modulation of the activity of multiple tumor suppressor genes. In the nucleus Pnn is concentrated in the "nuclear speckles," zones of accumulation of transcriptional and mRNA splicing factors, where Pnn is involved in mRNA processing. Alternatively, other roles of Pnn in gene regulation have not yet been established. By utilizing in vitro pull-down assays, in vivo interaction studies, and immunofluorescence in combination with overexpression and RNA interference experiments, we present evidence that Pnn interacts with the known transcriptional corepressor CtBP1. As a consequence of this interaction Pnn was capable of relieving the CtBP1-mediated repression of E-cadherin promoter activity. Our results suggest that the interaction of Pnn with the corepressor CtBP1 may modulate repression of transcription by CtBP1. This interaction may reflect the existence of coupling factors involved in CtBP-mediated transcriptional regulation and mRNA processing events.We studied a molecule, pinin (Pnn/DRS/memA), a 140-kDa phosphoprotein associated with the desmosome and localized in the nucleus of various tissues and cultured cell lines (4, 40-42, 52, 53, 62), which seems to play a key role in the establishment and maintenance of epithelia (53, 56). The expression of exogenous Pnn in transformed cells dramatically altered the recipient cells' morphology, driving them to a more epithelial phenotype. Expression of Pnn was linked to the expression of genes such as E-cadherin, p21 cip/waf , MIC-1, and Rho-A, which impact epithelial adhesion, proliferation, and cell motility (55). These observations suggest that Pnn may play an integral role in epithelial-cell-specific gene expression.Transfection experiments revealed that the majority of expressed Pnn is found within the nucleus, exhibiting a diffuse nucleoplasmic and nuclear speckle distribution. However, little, if any, exogenous Pnn was localized to cell-cell adhesion sites. This raises the possibility that Pnn may be exerting its effect predominantly via interaction with nuclear components, perhaps involving mRNA transcription and processing (48,62). The proposed functions of nuclear speckles include a role as storage compartments for molecular components involved in gene transcription, such as a subpopulation of polymerase II, and mRNA processing machinery, such as the SR family of proteins. SR proteins distinguished by their serine/argininerich motifs are required for constitutive mRNA splicing and regulation of alternative splice site selection (6,30,36,37,47,58). Previous two-hybrid and colocalization experiments revealed that Pnn indeed binds to, and colocalizes with, the SR protein family members SRp75 and SRm300, known components of the spliceosome machinery, and a novel SR protein (9,47,62,65). In addition, Pnn was demonstrated to contribute to the alternative splice site selection in splicing reporter assays, sp...

Role of Pinin in neural crest, dorsal dermis, and axial skeleton development and its involvement in the regulation of Tcf/Lef activity in mice

“…Recently, PNN has been shown to directly affect the expression of E-cadherin in a CtBPdependent manner in cell culture system (Alpatov et al, 2004), supporting the role of PNN in cell-cell adhesion. Most interestingly, Pnn hypomorphic mice did not display obvious alterations in cell-cell adhesion nor skin barrier formation, as determined by histological analysis and X-Gal penetration assay (data not shown), suggesting that their in vivo phenotypes did not stem primarily from the perturbation of intercellular adhesion.…”

“…However, the potential that PNN modulates Wnt signaling is supported by recent data pertaining to its asso-ciation to transcriptional corepressor CtBP. Our laboratory has previously demonstrated that PNN binds to CtBP (Alpatov et al, 2004). CtBP proteins have long been implicated in the regulation of Wnt signaling by direct interactions with a subset of TCF family members to repress the expression of Wnt target genes (Brannon et al, 1999;Chinnadurai, 2002;Valenta et al, 2003).…”

“…Pinin, a nuclear and desmosome-associated protein, has been shown to play multiple roles in intercellular adhesion, tumor suppression, transcriptional regulation, and pre-mRNA splicing (Shi et al, 2001;Zimowska et al, 2003;Alpatov et al, 2004;Joo et al, 2005). Prior in vitro studies demonstrated that PNN overexpression results in the enhanced epithelial phenotype characterized by increased cell-cell adhesion and decreased motility (Ouyang and Sugrue, 1996;Shi et al, 2000bShi et al, , 2001).…”

“…Notably, the increased expression of p21 Cip1/Waf1 upon PNN overexpression was determined to have resulted from up-regulation of p21 promoter activity. Although the signaling pathway or the mechanism orchestrating these coordinated alterations in gene expression are still unknown, PNN was shown to directly interact with CtBP and have an effect on the activity of E-cadherin promoter (Alpatov et al, 2004). Because CtBP has been closely implicated in Wnt signaling (Chinnadurai, 2002;Hamada and Bienz, 2004;Sierra et al, 2006) and expression of E-cadherin is often negatively regulated by Wnt signaling in development and cancer (Jamora et al, 2003;Yook et al, 2005), PNN's ability to interact with CtBP, along with PNNmediated phenotypic changes in adhesion, proliferation, and motility, may suggest possible involvement of PNN in Wnt signaling.…”

Homeodomain-interacting protein kinase-2 mediates CtBP phosphorylation and degradation in UV-triggered apoptosis

“…Both modifications have been linked to the regulation of CtBP localization. Furthermore, adenoviral E1A, Epstein-Barr virus oncoprotein EBNA3A, and the nuclear speckle-associated protein Pnn͞ DRS have been shown to sequester CtBP and thereby relieve CtBP-mediated repression (21)(22)(23)(24).…”