Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate β-catenin

doi:10.1016/j.cellsig.2006.08.011

Cellular Signalling

Volume 19, Issue 3, March 2007, Pages 610-616

https://doi.org/10.1016/j.cellsig.2006.08.011 Get rights and content

Abstract

The current view of canonical Wnt signalling is that following Wnt binding to its receptors (Frizzled-Lrp5/6), dishevelled (Dvl) becomes hyperphosphorylated, and the signal is transduced to the APC-GSK3β-axin-β-catenin multiprotein complex, which subsequently dissociates. As a result β-catenin is not phosphorylated, escapes proteosomal degradation and activates its target genes after translocation to the nucleus. Here, we analyzed the importance of the Wnt-3a-induced phosphorylation and shift in electrophoretic migration of Dvl (PS-Dvl) for the activation of β-catenin. Analysis of Wnt-3a time- and dose-responses in a dopaminergic cell line showed that β-catenin is activated rapidly (within minutes) and at a low dose of Wnt-3a (1 ng/ml). Surprisingly, PS-Dvl appeared only after 30 min and at greater doses (≥ 20 ng/ml) of Wnt-3a. Moreover, we found that a casein kinase 1 inhibitor (D4476) or siRNA for casein kinase 1 δ/ε (CK1δ/ε) blocked the Wnt-3a-induced PS-Dvl. Interestingly, CK1 inhibition or siRNA for CK1δ/ε did not ablate the activation of β-catenin by Wnt-3a, indicating that there is a PS-Dvl-independent path to activate β-catenin. The increase in β-catenin activation by Wnt-3a (PS-Dvl-dependent or -independent) were blocked by Dickkopf1 (Dkk1), suggesting that the effect of Wnt-3a is in both cases mediated by Lrp5/6 receptors. Thus, our results show that Wnt-3a rapidly induce a partial activation of β-catenin in the absence of PS-Dvl at low doses, while at high doses induce a full activation of β-catenin in a PS-Dvl-dependent manner.

Introduction

The Wnt signaling pathway is one of the most evolutionary conserved biochemical signalling pathways and is involved in a vast array of processes including embryonic development, adult tissue homeostasis and cancer (for review see [1], [2], [3]). The Wnts are secreted glyco-lipoproteins that bind to their membrane receptors (Frizzleds) and their co-receptors (Lrp5/6). The primary signalling components that transduce the Wnt signal are cytoplasmic proteins such as Dishevelled (Dvl), and the degradation complex, formed by Axin, APC, and GSK3β. Upon Wnt stimulation, Dvl is phosphorylated and the degradation complex is inhibited leading to the stabilization of β-catenin. β-catenin can then act in the cytosol, or in the nucleus, by binding to TCF/LEF and regulating transcription (for additional information see Wnt homepage [4]).

The current view of Wnt signalling is that canonical Wnt binds to Frizzled-Lrp5/6 containing receptor complexes, Dvl becomes hyperphosphorylated, and then the signal is transduced to the GSK3β-axin-β-catenin complex, which dissociates and consequently β-catenin is not phosphorylated and becomes activated. Intriguingly, some crucial details of the molecular pathways induced by Wnts in mammalian cells are still not fully understood. One of the most elusive parts of the pathway is the link between phosphorylated Dvl and GSK3β inhibition resulting in the activation and stabilization of β-catenin. It was shown previously in mammalian cells that canonical Wnts are able to induce the phosphorylation of Dvl and the activation of β-catenin [5]. However, the mutual link between these two signalling events induced by canonical Wnts was not investigated in detail.

We previously found that dopaminergic neurons respond to Wnt signalling [6], [7] and we now take advantage of the SN4741 dopaminergic neuronal cell line [8] as a model system. SN4741 cells, as midbrain dopaminergic neurons [9], [10], express a complex battery of Wnt signaling components (N. Rawal and G. Schulte, unpublished) and constitute a good model to study molecular mechanisms of the Wnt signalling [9], [10]. We hereby report that Wnt-3a induces an unexpectedly rapid activation of β-catenin that requires Lrp5/6 and low doses of Wnt-3a, but does not require the casein kinase-1-mediated Dvl phosphorylation. However, at late time points and at high doses, Wnt-3a potentiated the initial activation of β-catenin by a Dvl phosphorylation-dependent mechanism. Thus our findings suggest that β-catenin can be activated by two subsequent mechanisms that are independent or dependent of casein kinase 1 activity and PS-Dvl.

Section snippets

Cell culture and treatments

SN4741 cells were generously provided by Dr. J. H. Son [8] and grown in DMEM, 10% FCS, l-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 U/ml), glucose (0.6%) (all from Gibco Life Sciences). For analysis of intracellular signaling, 40,000 cells/well were seeded in 24 well plates, grown overnight in the absence of serum and in the same medium stimulated with recombinant mouse Wnt-3a (R&D Systems) for 2 h according to the figure legends. Control stimulations were done with equivalent

Wnt-3a induces activation of β-catenin and phosphorylation of Dvl

In order to characterize the pathways activated by Wnt-3a in dopaminergic cells we treated SN4741 cells with Wnt-3a (100 ng/ml) for 2 h. To monitor the activation of canonical Wnt-signaling we have used antibodies recognizing different phosphorylation states of β-catenin: a) active β-catenin (ABC) antibody [11] specific to forms of β-catenin dephosphorylated on GSK3β target sites (Ser37 or Thr41); and b) phospho-β-catenin (p-β-catenin) antibody specific to the forms of β-catenin targeted for

Discussion

Canonical Wnts (e.g. Wnt-3a) transduce their signal into the cell by inducing the phosphorylation of Dvl, which in turn results in inhibition of GSK3β preventing thus the phosphorylation of β-catenin. Although the precise mechanism connecting Dvl phosphorylation with GSK3β inhibition is not known (for review see e.g. [25], [26]), these events are considered to be sequential. Here we show using siRNA and pharmacological inhibitors that Wnt-3a-induced PS-Dvl is dependent on CK1δ/ε. Using these

Acknowledgments

Financial support was obtained from the Swedish Foundation for Strategic Research, Swedish Royal Academy of Sciences, Knut and Alice Wallenberg Foundation, European Commission, Swedish MRC, Karolinska Institutet and Lars Hiertas Minnesfond. G.S. was supported by a post-doctoral fellowship from the Swedish Society for Medical Research (SSMF).

We would like to thank Claudia Tello, Johny Söderlund and Annika Käller for technical and secretarial assistance and the members of Arenas lab for

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¹: both authors contributed equally to this work

²: present address: Karolinska Institutet, Dept. Physiology and Pharmacology, Receptor Biology and Signaling, S-17177 Stockholm, Sweden.

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Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate β-catenin

Abstract

Introduction

Section snippets

Cell culture and treatments

Wnt-3a induces activation of β-catenin and phosphorylation of Dvl

Discussion

Acknowledgments

Curr. Opin. Genet. Dev.

Curr. Biol.

Curr. Opin. Neurobiol.

J. Biol. Chem.

J. Biol. Chem.

Dev. Biol.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

Mol. Cell

Dev. Cell

J. Biol. Chem.

J. Biol. Chem.

Curr. Biol.

Mol. Cell Biol.

Proc. Natl. Acad. Sci. U. S. A.