Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex - PubMed (original) (raw)

Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex

K Willert et al. Genes Dev. 1999.

Abstract

The stabilization of beta-catenin is a key regulatory step during cell fate changes and transformations to tumor cells. Several interacting proteins, including Axin, APC, and the protein kinase GSK-3beta are implicated in regulating beta-catenin phosphorylation and its subsequent degradation. Wnt signaling stabilizes beta-catenin, but it was not clear whether and how Wnt signaling regulates the beta-catenin complex. Here we show that Axin is dephosphorylated in response to Wnt signaling. The dephosphorylated Axin binds beta-catenin less efficiently than the phosphorylated form. Thus, Wnt signaling lowers Axin's affinity for beta-catenin, thereby disengaging beta-catenin from the degradation machinery.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Wnt signaling modifies Axin. (A) Western analysis of Axin and β-catenin protein from C57MG cells treated with either Wnt-1 or Wnt-3A. (B) Axin and β-catenin protein from L cells treated with Wnt-3A. C57MG and L cells were stimulated with control (−) or Wnt-3A (+)-conditioned medium for 2 hr prior to lysis. C57MG cells containing a tet-repressible Wnt-1 transgene were incubated in tet-containing medium to repress Wnt-1 expression (−) or in tet-free medium for 12 hr to induce Wnt-1 expression (+). (C) Time course of Axin protein modification in response to Wnt-3A. C57MG cells were stimulated with control (−) or Wnt-3A (+)-conditioned medium for the indicated times and immunoblotted with anti-Axin antibody.

Figure 2

Figure 2

The Axin protein is phosphorylated. (A) PP2A, but not PP1, dephosphorylates the Axin protein in vitro. Axin protein was immunoprecipitated from cell extracts of C57MG cells treated with control (−) or Wnt-3A (+)-conditioned medium. The immune complexes were treated with phosphatase buffer only, PP2A, or PP1 and immunoblotted with anti-Axin antibody. (B) The phosphatase inhibitor okadaic acid inhibits the dephosphorylation of Axin. Cell lysates were prepared from C57MG cells treated with control (−) or Wnt-3A (+)-conditioned medium and with or without 400 n

m

okadaic acid and immunoblotted with anti-Axin antibody. (C) Lithium, an inhibitor of GSK-3β, promotes the dephosphorylation of Axin. Cell lysates were prepared from C57MG cells first treated with or without 25 m

m

LiCl for 4 hr and treated with control (−) or Wnt3A (+)-conditioned medium for an additional 2 hr.

Figure 3

Figure 3

In the absence of a Wnt signal, Axin binds β-catenin with higher affinity. (A) β-Catenin coimmunoprecipitates with Axin. Axin protein was immunoprecipitated from L cells treated with control (−) or Wnt-3A (+)-conditioned medium and immunoblotted with either anti-Axin or anti-β-catenin antibodies. (B) Axin from Wnt-3A-stimulated cells binds β-catenin less efficiently. β-Catenin-coated beads were used to retrieve the Axin protein from cell extracts prepared from C57MG cells treated with control (−) or Wnt-3A (+)-conditioned medium in the absence (−) or presence (+) of 25 m

m

LiCl. The precipitating Axin protein was then visualized by immunoblotting with anti-Axin antibodies. The bracket indicates the position of phosphorylated Axin (Axin-P); the arrowhead indicates the position of un- or under-phosphorylated Axin (Axin).

Figure 4

Figure 4

Phosphorylation of Axin with GSK-3β increases Axin’s affinity for β-catenin. (A) Phosphorylation of GST–Axin with GSK-3β and subsequent dephosphorylation with PP2A. GST–Axin immobilized on glutathione–Sepharose beads was treated with (+) or without (−) purified GSK-3β. Phosphorylated or unphosphorylated GST–Axin subsequently was washed to remove the ATP and incubated in the absence (−) or presence (+) of purified PP2A. The GST–Axin protein was visualized by immunoblotting with anti-Axin antibody. (B) GSK-3β phosphorylated Axin binds β-catenin significantly better than unphosphorylated or dephosphorylated GST–Axin. The GSK-3β and PP2A-modified GST–Axin proteins were used to precipitate β-catenin from lysates of L cells treated with Wnt-3A. Phosphorylated and unphosphorylated GST–Axin was also used to precipitate bacterially produced β-catenin protein. Axin-binding proteins were immunoblotted with anti-β-catenin antibody.

Figure 5

Figure 5

A model for the role of Axin in Wnt signal transduction. In an unstimulated cell, GSK-3β is active and phosphorylates Axin, which in turn, recruits β-catenin into the Axin/GSK-3β complex. By virtue of its proximity to GSK-3β, β-catenin is then phosphorylated. Phosphorylated β-catenin is then targeted for degradation. Upon transduction of the Wnt signal through the Frizzled (Fz) receptors to Dishevelled (Dvl), GSK-3β kinase activity is inhibited so that PP2A dephosphorylates Axin. Unphosphorylated Axin, in turn, no longer recruits β-catenin to the complex. Failure of β-catenin to associate with the Axin/GSK-3β complex prevents its phosphorylation by GSK-3β so that it can accumulate to high levels in the cytoplasm and nucleus and activate transcription in concert with the Tcf/Lef-1 family of transcription factors. GSK-3β also phosphorylates APC, which may facilitate β-catenin recruitment into the complex; however, this event has not been shown to be regulated by Wnt signaling.

Similar articles

Cited by

References

    1. Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. β-catenin is a target for the ubiquitin-proteasome pathway. EMBO J. 1997;16:3797–3804. - PMC - PubMed
    1. Behrens J, Jerchow BA, Wurtele M, Grimm J, Asbrand C, Wirtz R, Kuhl M, Wedlich D, Birchmeier W. Functional interaction of an axin homolog, conductin, with β-catenin, APC, and GSK3β. Science. 1998;280:596–599. - PubMed
    1. Brown JD, Moon RT. Wnt signaling: Why is everything so negative? Curr Opin Cell Biol. 1998;10:182–187. - PubMed
    1. Cadigan KM, Nusse R. Wnt signaling: A common theme in animal development. Genes & Dev. 1997;11:3286–3305. - PubMed
    1. Cook D, Fry MJ, Hughes K, Sumathipala R, Woodgett JR, Dale TC. Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase c. EMBO J. 1996;15:4526–4536. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources