Regulation of TCF3 by Wnt-Dependent Phosphorylation during Vertebrate Axis Specification

Abstract

A commonly accepted model of Wnt/β-catenin signaling involves target gene activation by a complex of β-catenin with a TCF family member. TCF3 is a transcriptional repressor that has been implicated in Wnt signaling and plays key roles in embryonic axis specification and stem cell differentiation. Here we demonstrate that Wnt proteins stimulate TCF3 phosphorylation in gastrulating Xenopus embryos and mammalian cells. This phosphorylation event involves β-catenin-mediated recruitment of homeodomain-interacting protein kinase 2 (HIPK2) to TCF3 and culminates in the dissociation of TCF3 from a target gene promoter. Mutated TCF3 proteins resistant to Wnt-dependent phosphorylation function as constitutive inhibitors of Wnt-mediated activation of Vent2 and Cdx4 during anteroposterior axis specification. These findings reveal an alternative in vivo mechanism of Wnt signaling that involves TCF3 phosphorylation and subsequent derepression of target genes and link this molecular event to a specific developmental process.

Wnt signaling through T-cell factor phosphorylation

“…In mammalian cells, Wnt1 can promote the phosphorylation of TCF4 [77], but there are conflicting reports regarding the ability of Wnt5a to stimulate LEF-1 and TCF-4 phosphorylation [77,78]. In Xenopus embryos and mammalian cells, we find that TCF3, TCF4 and LEF1 are phosphorylated in response to Wnt8 and Wnt3a, both in vitro and in vivo [79,80] (Figure 1). TCF3 constructs with mutated phosphorylation sites function as constitutive transcriptional repressors, indicating the essential role of this phosphorylation for signaling [79].…”

“…In Xenopus embryos and mammalian cells, we find that TCF3, TCF4 and LEF1 are phosphorylated in response to Wnt8 and Wnt3a, both in vitro and in vivo [79,80] (Figure 1). TCF3 constructs with mutated phosphorylation sites function as constitutive transcriptional repressors, indicating the essential role of this phosphorylation for signaling [79]. Thus, TCF phosphorylation appears to be a conserved mechanism operating in parallel with β-catenin stabilization to control Wnt target gene activation [80].…”

“…Interestingly, both HIPK2 and NLK have been found to control anteroposterior axis specification in Xenopus and zebrafish embryos [79,85]. The two NLK phosphorylation sites on LEF-1 [78] correspond to a subset of the Wnt8-dependent, HIPK2 phosphorylation sites within TCF3, while two additional clusters of phosphorylation sites appear to be specific for HIPK2 [79].…”

“…Other studies have reported both positive and negative effects of HIPK on Wnt/β-catenin signaling in mouse embryo fibroblasts [101,102], Drosophila and Xenopus embryos [103,104], but the underlying mechanisms remain to be fully elucidated. Linking HIPK2 more directly to TCF regulation, a recent study has shown that HIPK2 acts to antagonize TCF3 activity, thereby promoting ventroposterior development in Xenopus [79]. HIPK2 is required for Wnt8-dependent TCF3 phosphorylation, which results in the removal of TCF3 from target promoters culminating in target gene activation [79].…”

“…Linking HIPK2 more directly to TCF regulation, a recent study has shown that HIPK2 acts to antagonize TCF3 activity, thereby promoting ventroposterior development in Xenopus [79]. HIPK2 is required for Wnt8-dependent TCF3 phosphorylation, which results in the removal of TCF3 from target promoters culminating in target gene activation [79].…”