Biochemical and Structural Characterization of β-Catenin Interactions with Nonphosphorylated and CK2-Phosphorylated Lef-1

In the Wnt/beta-catenin signaling pathway, beta-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 beta-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 beta-catenin. However, a second salt bridge in other TCF beta-catenin structures is absent in the structure of beta-catenin Lef-1 complex, indicating that this feature is not obligatory for beta-atenin binding. Casein kinase 11 (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 beta-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 beta-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 beta-catenin. These data indicate that the effect of CK2 on the Wnt/beta-catenin pathway does not appear to be at the level of the Lef-1-beta-catenin interaction. (C) 2010 Elsevier Ltd. All rights reserved.