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ABSTRACT: Dishevelled (Dvl) is a key component in the canonical Wnt signaling pathway and becomes hyperphosphorylated upon Wnt stimulation. Dvl is required for LRP6 phosphorylation, which is essential for subsequent steps of signal transduction, such as Axin recruitment and cytosolic β-catenin stabilization. Here, we identify the HECT-containing Nedd4-like ubiquitin E3 ligase ITCH as a new Dvl-binding protein. ITCH ubiquitinates the phosphorylated form of Dvl and promotes its degradation via the proteasome pathway, thereby inhibiting canonical Wnt signaling. Knockdown of ITCH by RNA interference increased the stability of phosphorylated Dvl and upregulated Wnt reporter gene activity as well as endogenous Wnt target gene expression induced by Wnt stimulation. In addition, we found that both the PPXY motif and the DEP domain of Dvl are critical for its interaction with ITCH, as mutation in the PPXY motif (Dvl2-Y568F) or deletion of the DEP domain led to reduced affinity for ITCH. Consistently, overexpression of ITCH inhibited wild-type Dvl2-induced, but not Dvl2-Y568F mutant-induced, Wnt reporter activity. Moreover, the Y568F mutant, but not wild-type Dvl2, can reverse the ITCH-mediated inhibition of Wnt-induced reporter activity. Collectively, these results indicate that ITCH plays a negative regulatory role in modulating canonical Wnt signaling by targeting the phosphorylated form of Dvl.
Molecular and cellular biology 07/2012; 32(19):3903-12. · 6.06 Impact Factor
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ABSTRACT: The Ca(2+) signaling pathway appears to regulate the processes of the early development through its antagonism of canonical Wnt/β-catenin signaling pathway. However, the underlying mechanism is still poorly understood. Here, we show that nuclear factor of activated T cells (NFAT), a component of Ca(2+) signaling, interacts directly with Dishevelled (Dvl) in a Ca(2+)-dependent manner. A dominant negative form of NFAT rescued the inhibition of the Wnt/β-catenin pathway triggered by the Ca(2+) signal. NFAT functioned downstream of β-catenin without interfering with its stability, but influencing the interaction of β-catenin with Dvl by its competitively binding to Dvl. Furthermore, we demonstrate that NFAT is a regulator in the proliferation and differentiation of neural progenitor cells by modulating canonical Wnt/β-catenin signaling pathway in the neural tube of chick embryo. Our findings suggest that NFAT negatively regulates canonical Wnt/β-catenin signaling by binding to Dvl, thereby participating in vertebrate neurogenesis.
Journal of Biological Chemistry 08/2011; 286(43):37399-405. · 4.77 Impact Factor
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ABSTRACT: The Ca2+ signaling pathway appears to regulate the processes of the early development through its antagonism of canonical
Wnt/β-catenin signaling pathway. However, the underlying mechanism is still poorly understood. Here, we show that NFAT, a
component of Ca2+ signaling, directly interacts with Dvl in a Ca2+-dependent manner. A dominant-negative form of NFAT rescued
the inhibition of the Wnt/β-catenin pathway triggered by Ca2+ signal. NFAT functioned downstream of β-catenin without interfering
with its stability, but influencing the interaction of β-catenin with Dvl by its competitively binding to Dvl. Furthermore,
we demonstrate that NFAT is a regulator in the proliferation and differentiation of neural progenitor cells by modulating
canonical Wnt/β-catenin signaling pathway in the neural tube of chick embryo. Our findings suggest that NFAT negatively regulates
canonical Wnt/β-catenin signaling by binding to Dvl, thereby participating in vertebrate neurogenesis.
Journal of Biological Chemistry 08/2011; · 4.77 Impact Factor
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ABSTRACT: The TAK1-NLK cascade is a mitogen-activated protein kinase-related pathway that plays an inhibitory role in canonical Wnt/beta-catenin signaling through regulating the LEF1/TCF family transcriptional factors. TAB2 (TAK1-binding protein 2) is a putative TAK1 interacting protein that is involved in the regulation of TAK1. Here, we found that TAB2 could directly interact with NLK and function as a scaffold protein to facilitate the interaction between TAK1 and NLK. Knocking down TAB2 using small interfering RNA abolished the interaction of TAK1 with NLK in mammalian cells. The intermediate region (residues 292-417) of TAB2 was mapped for its binding to NLK. TAB2-DeltaM, a TAB2 mutant lacking this region, showed a lower affinity for NLK and became defective in its scaffolding function. In addition, TAB2, but not TAB2-DeltaM, mediated TAK1-dependent activation of NLK and LEF1 polyubiquitylation, resulting in the inhibition of canonical Wnt signaling. Moreover, Wnt3a stimulation led to an increase in the interaction of TAB2 with NLK and the formation of a TAK1.TAB2.NLK complex, suggesting that this TAK1-TAB2-NLK pathway may constitute a negative feedback mechanism for canonical Wnt signaling.
Journal of Biological Chemistry 03/2010; 285(18):13397-404. · 4.77 Impact Factor
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ABSTRACT: Axin, a key modulator of the Wnt/beta-catenin pathway, acts as a scaffold protein in phosphorylating and degrading cytoplasmic beta-catenin. Canonical Wnt proteins appear to stabilize beta-catenin by inducing the interaction of LRP5/6 with Axin. This interaction requires the phosphorylation of the Ser or Thr residues in the PPPP(S/T)PX(T/S) motifs at the intracellular domain of LRP5/6. In this work, we identified a novel Axin-interacting protein, zinc-finger BED domain-containing 3 (Zbed3), by yeast two-hybrid screening. The interaction was confirmed in co-immunoprecipitation experiment in mammalian cells and in vitro pulldown assays. Moreover, we found Zbed3 also contains a PPPPSPT motif, which is crucial to its binding to Axin. The Ser and Thr residues in the motif appear to be also phosphorylated by glycogen synthase kinase 3beta (GSK3beta) and the CKI family kinases, as GSK3beta and CKIepsilon could enhance the interaction of Zbed3 with Axin. Mutation of the Ser (SA) or Thr (TA) residue to Ala in the motif markedly impaired its ability to interact with Axin. Expressing Zbed3, but not these mutants, led to inhibition of GSK3beta-mediated beta-catenin phosphorylation, cytoplasmic beta-catenin accumulation, and activation of lymphoid enhancer binding factor-1-dependent reporter gene transcription. Furthermore, knockdown of Zbed3 with RNA interference attenuated Wnt-induced beta-catenin accumulation, lymphoid enhancer binding factor-1-dependent luciferase reporter activity, and the Wnt target gene expression. These results together indicate that Zbed3 is a novel Axin-binding protein that is involved in Wnt/beta-catenin signaling modulation.
Journal of Biological Chemistry 02/2009; 284(11):6683-9. · 4.77 Impact Factor
