STD and TRNOESY NMR Studies on the Conformation of the Oncogenic Protein β-Catenin Containing the Phosphorylated Motif DpSGXXpS Bound to the β-TrCP Protein

Abstract

␤-TrCP is the F-box protein component of an Skp1/ Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that ␤-TrCP targets the oncogenic protein ␤-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from ␤-catenin containing the phosphorylated motif DpSGXXpS (P-␤-Cat 17-48 ) and ␤-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the ␤-TrCP binding pocket. The amide and aromatic protons of the His 24 and Trp 25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter ␤-catenin fragment with the ␤-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated ␤-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.The ubiquitin-proteasome pathway of protein degradation is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction (1, 2). This work is based on the study of the oncogenic protein ␤-catenin (␤-Cat), 1 which plays an essential role in the Wingless/Wnt signaling pathway and is an important component of cadherin cell-adhesion complexes (Fig. 1A). The abundance of ␤-catenin in the cytoplasm is regulated by ubiquitindependent proteolysis (3), and Wnt signaling is regulated by the presence or absence of the intracellular protein ␤-catenin. When Wnt signal is absent, the signal transduction pathway is off because ␤-catenin is rapidly destroyed. A large multiprotein machine normally facilitates the addition of phosphate groups to ␤-catenin by glycogen synthase kinase-3␤ (GSK3␤). Phosphorylated ␤-catenin binds to a protein called ␤-TrCP and is then modified by the covalent addition of a small protein called ubiquitin. Proteins tagged with ubiquitin are degraded by the 26 S proteasome, the protein-recycling center of the cell. When cells are exposed to the Wnt signal, it binds to cell surface receptors. Receptor activation blocks ␤-catenin phosphorylation, and its subsequent ubiquitination by an unknown mechanism that requires the intervention of the Disheveled protein (Dsh). ␤-Catenin is thus diverted from the proteasome. It accumulates and enters the nucleus, where it finds a par...

Negative feedback loop in T cell activation through IκB kinase-induced phosphorylation and degradation of Bcl10

“…Interestingly, it has been observed in the case of ␤-catenin (see Fig. 2 A) that this aspartic acid, which is specifically involved in the interaction with ␤-TrCP (28,29), is subjected to mutations in a large number of cancers (30)(31)(32).…”

Post-translational modifications regulate distinct functions of CARMA1 and BCL10

“…Interestingly, mutations that affect the conserved aspartic acid (D) of the consensus sequence (D 80 S/TcXXS/ T) of BCL10 have been described in a case of diffuse large B cell lymphoma [55] and in a mesothelioma [33]. A corresponding aspartic acid present in b-catenin, which is specifically involved in the interaction with b-TrCP [56,57], is subject to mutations in a large number of cancers [58][59][60]. Nuclear BCL10 might promote tumor progression by at least two distinct, but not exclusive, mechanisms: BCL10 stabilization could increase NF-kB activation and the subsequent expression of pro-survival genes, and/or BCL10 stabilization and nuclear translocation could have a role in the transcriptional regulation of a particular subset of genes.…”

Mechanistic insights from structural studies of β-catenin and its binding partners

“…The 3D structures of ␤-catenin peptides containing the Nterminal GSK3 phosphorylation sites (S33 and S37) have also been determined (Megy et al, 2005a;Megy et al, 2005b;Wu et al, 2003) since this is the recognition site for the ␤-TrCP ubiquitin ligase (Hart et al, 1999;Kitagawa et al, 1999;Latres et al, 1999;Liu et al, 1999;Winston et al, 1999). These structures showed that the region covering ␤-catenin residues 20-31 has a tendency to form a helix, which facilitates the recognition of the DpS 33 GXpS 37 motif ( denotes a hydrophobic amino acid and pS are the serines phosphorylated by GSK3) by ␤-TrCP (Megy et al, 2005a;Megy et al, 2005b;Wu et al, 2003).…”

“…These structures showed that the region covering ␤-catenin residues 20-31 has a tendency to form a helix, which facilitates the recognition of the DpS 33 GXpS 37 motif ( denotes a hydrophobic amino acid and pS are the serines phosphorylated by GSK3) by ␤-TrCP (Megy et al, 2005a;Megy et al, 2005b;Wu et al, 2003).…”