Chibby cooperates with 14-3-3 to regulate β-catenin subcellular distribution and signaling activity

β-Catenin functions in both cell–cell adhesion and as a transcriptional coactivator in the canonical Wnt pathway. Nuclear accumulation of β-catenin is the hallmark of active Wnt signaling and is frequently observed in human cancers. Although β-catenin shuttles in and out of the nucleus, the molecular mechanisms underlying its translocation remain poorly understood. Chibby (Cby) is an evolutionarily conserved molecule that inhibits β-catenin–mediated transcriptional activation. Here, we identified 14-3-3ε and 14-3-3ζ as Cby-binding partners using affinity purification/mass spectrometry. 14-3-3 proteins specifically recognize serine 20 within the 14-3-3–binding motif of Cby when phosphorylated by Akt kinase. Notably, 14-3-3 binding results in sequestration of Cby into the cytoplasm. Moreover, Cby and 14-3-3 form a stable tripartite complex with β-catenin, causing β-catenin to partition into the cytoplasm. Our results therefore suggest a novel paradigm through which Cby acts in concert with 14-3-3 proteins to facilitate nuclear export of β-catenin, thereby antagonizing β-catenin signaling

Chibby forms a homodimer through a heptad repeat of leucine residues in its C-terminal coiled-coil motif

BACKGROUND: The Wnt/β-catenin signaling pathway plays crucial roles in embryonic development and in maintenance of organs and tissues in adults. Chibby (Cby) is an evolutionarily conserved molecule that physically interacts with the key downstream coactivator β-catenin and represses its transcriptional activation potential. Although Cby harbors a predicted coiled-coil motif in the C-terminal region, its molecular nature and functional importance remain largely unexplored. RESULTS: Here we report that Cby forms a stable complex with itself. Alanine substitutions of two or more of four critical leucine residues within the C-terminal heptad repeats completely eliminate the Cby-Cby interaction. The Cby oligomer predominantly exists as a homodimer. Furthermore, we found that dimerization-deficient Cby mutants still retain the ability to bind to β-catenin and to repress β-catenin-dependent gene activation. More importantly, Cby homodimerization is required for its efficient interaction with the nuclear import receptor importin-α and subsequent nuclear translocation. CONCLUSION: Our comprehensive mutational analysis of the Cby coiled-coil domain reveals that the four heptad leucine residues play an essential role in mediating Cby homodimerization. Although monomeric Cby is sufficient to bind to β-catenin and block β-catenin-mediated transcriptional activation, homodimer formation of Cby is indispensable for its efficient nuclear import

Chibby Promotes Adipocyte Differentiation through Inhibition of β-Catenin Signaling▿

The canonical Wnt/β-catenin signaling pathway plays diverse roles in embryonic development and disease. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and in mice. Here, we report that the β-catenin antagonist Chibby (Cby) is required for adipocyte differentiation. Cby is expressed in adipose tissue in mice, and Cby protein levels increase during adipogenic differentiation of 3T3-L1 cells. Ectopic expression of Cby induces spontaneous differentiation of these cells into mature adipocytes to an extent similar to that of dominant-negative Tcf-4. In contrast, depletion of Cby by RNA interference potently blocks adipogenesis of 3T3-L1 and mouse embryonic stem cells. In support of this, embryonic fibroblasts obtained from Cby-deficient embryos display attenuated differentiation to the adipogenic lineage. Mechanistically, Cby promotes adipocyte differentiation, in part by inhibiting β-catenin, since gain or loss of function of Cby influences β-catenin signaling in 3T3-L1 cells. Our results therefore establish Cby as a novel proadipogenic factor required for adipocyte differentiation

Nuclear-Cytoplasmic Shuttling of Chibby Controls β-Catenin Signaling

Chibby (Cby) acts with 14-3-3 to regulate β-catenin localization in the canonical Wnt pathway. We show that Cby harbors functional NLS and NES motifs, and shuttles between the nucleus and cytoplasm. Cby distribution at steady state is controlled by an intricate cooperation between 14-3-3, CRM1 and importin-α, which impacts on β-catenin signaling