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Biochemical and structural characterization of ?-catenin interactions with nonphosphorylated and CK2-phosphorylated Lef-1.

ABSTRACT: In the Wnt/?-catenin signaling pathway, ?-catenin activates target genes through its interactions with the T-cell factor/lymphoid enhancer-binding factor (TCF/Lef) family of transcription factors. The crystal structures of complexes between the ?-catenin armadillo domain and the Lef-1 N-terminal domain show that the overall conformation and many of the interactions are similar to other published structures of TCFs bound to ?-catenin. However, a second salt bridge in other TCF-?-catenin structures is absent in the structure of ?-catenin-Lef-1 complex, indicating that this feature is not obligatory for ?-catenin binding. Casein kinase II (CK2) has been shown to act as a positive regulator of Wnt signaling, and Lef-1 is a substrate of CK2. In vitro phosphorylation of purified Lef-1 was used to examine the effect of CK2 on the interaction of Lef-1 with ?-catenin. Mass spectrometry data show that CK2 phosphorylation of Lef-1 N-terminal domain results in a single phosphorylation site at Ser40. Isothermal titration calorimetry revealed that ?-catenin binds to nonphosphorylated or CK2-phosphorylated Lef-1 with the same affinity, which is consistent with the absence of phospho-Ser40 interactions in the crystal structure of phosphorylated Lef-1 N-terminal domain bound to ?-catenin. These data indicate that the effect of CK2 on the Wnt/?-catenin pathway does not appear to be at the level of the Lef-1-?-catenin interaction.

SUBMITTER: Sun J

PROVIDER: S-EPMC3017643 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Biochemical and structural characterization of β-catenin interactions with nonphosphorylated and CK2-phosphorylated Lef-1.

Sun Jinglucy J Weis William I WI

Journal of molecular biology 20101112 2

In the Wnt/β-catenin signaling pathway, β-catenin activates target genes through its interactions with the T-cell factor/lymphoid enhancer-binding factor (TCF/Lef) family of transcription factors. The crystal structures of complexes between the β-catenin armadillo domain and the Lef-1 N-terminal domain show that the overall conformation and many of the interactions are similar to other published structures of TCFs bound to β-catenin. However, a second salt bridge in other TCF-β-catenin structure ...[more]

PMID: 21075118

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Project description:BackgroundThe Wnt/β-catenin signaling pathway is a prolific regulator of cell-to-cell communication and gene expression. Canonical Wnt/β-catenin signaling involves partnering of β-catenin with members of the TCF/LEF family of transcription factors (TCF1, TCF3, TCF4, LEF1) to regulate gene expression. IL-6 is a key cytokine involved in inflammation and is particularly a hallmark of inflammation in the brain. Astrocytes, specialized glial cells in the brain, secrete IL-6. How astrocytes regulate IL-6 expression is not entirely clear, although in other cells NFκB and C/EBP pathways play a role. We evaluated here the interface between β-catenin, TCFs/LEF and C/EBP and NF-κB in relation to IL-6 gene regulation in astrocytes.MethodsWe performed molecular loss and/or gain of function studies of β-catenin, TCF/LEF, NFκB, and C/EBP to assess IL-6 regulation in human astrocytes. Specifically, siRNA mediated target gene knockdown, cDNA over expression of target gene, and pharmacological agents for regulation of target proteins were used. IL-6 levels was evaluated by real time quantitative PCR and ELISA. We also cloned the IL-6 promoter under a firefly luciferase reporter and used bioinformatics, site directed mutagenesis, and chromatin immunoprecipitation to probe the interaction between β-catenin/TCFs/LEFs and IL-6 promoter activity.Resultsβ-catenin binds to TCF/LEF to inhibits IL-6 while TCFs/LEF induce IL-6 transcription through interaction with ATF-2/SMADs. β-catenin independent of TCFs/LEF positively regulates C/EBP and NF-κB, which in turn activate IL-6 expression. The IL-6 promoter has two putative regions for TCFs/LEF binding, a proximal site located at -91 nt and a distal site at -948 nt from the transcription start site, both required for TCF/LEF induction of IL-6 independent of β-catenin.ConclusionIL-6 regulation in human astrocytes engages a discordant interaction between β-catenin and TCF/LEF. These findings are intriguing given that no role for β-catenin nor TCFs/LEF to date is associated with IL-6 regulation and suggest that β-catenin expression in astrocytes is a critical regulator of anti-inflammatory responses and its disruption can potentially mediate persistent neuroinflammation. Video Abstract.

Dataset Information

Biochemical and structural characterization of ?-catenin interactions with nonphosphorylated and CK2-phosphorylated Lef-1.

Publications

Biochemical and structural characterization of β-catenin interactions with nonphosphorylated and CK2-phosphorylated Lef-1.

Similar Datasets

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