Differential Modulation of TCF/LEF-1 Activity by the Soluble LRP6-ICD

Brandon Beagle,Gail V W Johnson,Sudha Agarwal

doi:10.1371/journal.pone.0011821

Brandon Beagle, Gail V W Johnson + Show 1 more

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https://doi.org/10.1371/journal.pone.0011821

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Abstract

The canonical Wnt/β-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathway that controls numerous biological processes including proliferation and differentiation. As such, transcriptional activity of the Wnt pathway is tightly regulated and/or modulated by numerous proteins at the level of the membrane, cytosol and/or nucleus. In the nucleus, transcription of Wnt target genes by TCF/LEF-1 is repressed by the long Groucho/TLE co-repressor family. However, a truncated member of the Groucho/TLE family, amino terminal enhancer of Split (AES) can positively modulate TCF/LEF-1 activity by antagonizing long Groucho/TLE members in a dominant negative manner. We have previously shown the soluble intracellular domain of the LRP6 receptor, a receptor required for activation of the Wnt pathway, can positively regulate transcriptional activity within the Wnt pathway. In the current study, we show the soluble LRP6 intracellular domain (LRP6-ICD) can also translocate to the nucleus in CHO and HEK 293T cells and in contrast to cytosolic LRP6-ICD; nuclear LRP6-ICD represses TCF/LEF-1 activity. In agreement with previous reports, we show AES enhances TCF/LEF-1 mediated reporter transcription and further we demonstrate that AES activity is spatially regulated in HEK 293T cells. LRP6-ICD interacts with AES exclusively in the nucleus and represses AES mediated TCF/LEF-1 reporter transcription. These results suggest that LRP6-ICD can differentially modulate Wnt pathway transcriptional activity depending upon its subcellular localization and differential protein-protein interactions.

Highlights

The canonical Wnt/b-catenin (Wnt) pathway is a ubiquitous transcriptional regulatory pathway that affects the expression of over 1800 genes involved in at least 36 different pathways [1,2,3,4,5]
To make the GFP tagged LRP6-intracellular domain (ICD) constructs with a nuclear localization signal (NLS) or nuclear export signal (NES), wild type GFP-LRP6-ICD or GFP-LRP6ICD65 m were used as templates for PCR amplification
Immunocytochemistry (Fig. 2B) show that both wild type LRP6-ICD and LRP6-ICD65 m localize within DAPI stained nuclei which confirms the fractionation data showing nuclear localization of LRP6-ICD does not require GSK3b phosphorylation

Summary

Introduction

The canonical Wnt/b-catenin (Wnt) pathway is a ubiquitous transcriptional regulatory pathway that affects the expression of over 1800 genes involved in at least 36 different pathways [1,2,3,4,5]. Phosphorylation promotes b-catenin degradation resulting in low basal levels of cytosolic b-catenin preventing bcatenin nuclear translocation and activation of the Wnt responsive transcription factor TCF/LEF-1 [2,3,8,9,10]. In the absence of significant nuclear b-catenin, DNA bound TCF/LEF-1 proteins repress expression of Wnt target genes by interacting with the long Groucho/TLE family of co-repressors [8,9,10,11,12,13,14]. The Q domain mediates interaction with transcription factors such as TCF/LEF-1 as well as tetramerization of Groucho/ TLE members, which is essential for its repressor function and interaction with TCF/LEF-1 [8,10,11,12,14,15,16,17,18,19,20]. The GP domain has a critical repressor function as it is essential for interaction between long Groucho/TLE’s and co-repressor histone deacetylases (HDACs) [8,10,11,15,16,20,21]

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