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␤-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...

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Key events leading to terminal differentiation of preadipocytes into adipocytes have been identified in recent years. However, signaling pathways involved in the decision of stem cells to follow the adipogenic lineage have not yet been characterized. We have previously shown that differentiating mouse embryonic stem (mES) cells give rise to functional adipocytes upon an early treatment with retinoic acid (RA). The goal of this work was to identify regulators of RA-induced commitment of mES cells to the adipocyte lineage. First, we investigated the role of RA receptor (RAR) isotypes in the induction of mES cell adipogenesis. Using synthetic retinoids selective of RAR isotypes, we show that RARbeta activation is both sufficient and necessary to trigger commitment of mES cells to adipocytes. Then, we performed a small-scale drug screening to find signaling pathways involved in RARbeta-induced mES cell adipogenesis. We show that pharmacological inhibitors of glycogen synthase kinase (GSK) 3, completely inhibit RARbeta-induced adipogenesis in mES cells. This finding uncovers the requirement of active GSK3 in RARbeta-induced commitment of mES cells toward the adipocyte lineage. Finally, we investigated the role of the Wnt pathway, in which GSK3 is a critical negative regulator, in adipocyte commitment by analyzing Wnt pathway activity in RA- and RARbeta-induced mES cell adipogenesis. Our results suggest that although RARbeta and active GSK3 are required for RA-induced adipogenesis, they might be acting through a Wnt pathway-independent mechanism.