Wnt signals are transmitted through N-terminally dephosphorylated beta-catenin

Abstract

beta-catenin mediates Wnt signaling by acting as the essential co-activator for TCF transcription factors. Wnt signaling increases the half-life and therefore the absolute level of beta-catenin in responding cells. The current model states that these changes in beta-catenin stability set the threshold for Wnt signaling. However, we find that pharmacological inhibition of proteasome activity by ALLN leads to accumulation of cytosolic beta-catenin but not to increased TCF-mediated transcription. In addition, in temperature-sensitive ubiquitylation mutant CHO cells inhibition of ubiquitylation increases beta-catenin levels, but does not induce transcriptional activation of TCF reporter genes. Using an antibody specific for beta-catenin dephosphorylated at residues Ser37 and Thr41, we show that Wnt signals specifically increase the levels of dephosphorylated beta-catenin, whereas ALLN does not. We conclude that changes in the phosphorylation status of the N-terminus of beta-catenin that occur upon Wnt signaling independently affect the signaling properties and half-life of beta-catenin. Hence, Wnt signals are transduced via N-terminally dephosphorylated beta-catenin.

Fig. 1. Lack of transactivation by ALLN-induced increase in β-catenin levels. 293T cells were treated with the proteasome inhibitor ALLN for 6 h and analysed for β-catenin levels by western blotting (A). Equal amounts of protein were loaded in each lane. (B) In parallel, 293T were transfected with TOP and FOP reporter plasmids, Renilla-luciferase control vectors and S33 β-catenin. ALLN treatment does not induce activation of the reporter constructs, despite accumulation of β-catenin. Co-transfection of S33 β-catenin can still induce TCF-dependent transcription under ALLN treatment, indicating that the cells are signaling-competent.

Fig. 2. Increased β-catenin half-life by inhibition of ubiquitylation does not lead to increased TCF-dependent transcription (A). ts20 cells were incubated at the restrictive temperature (40°C) for 6 h. Subsequently the cells were lysed and analyzed on western blot using the TL α-β-catenin antibody (B). ts20 cells were transfected with reporter constructs with or without S33 β-catenin and immediately shifted to restricted temperature (40°C) for 6 h. While β-catenin levels increased (Figure 2A), TOP/FOP ratios remained the same. In addition, under conditions where ubiquitylation is inhibited, the cells are still capable of activating TCF-dependent transcription when transfected with β-catenin.

Fig. 3. Levels of non-phosphorylated β-catenin correlate with the transcriptional activity. (A) 293T cells were transfected with S33 β-catenin, Wnt1 or treated with ALLN and LiCl. Cells were analyzed by western blot using a pan-anti-β-catenin antibody directed against the C-terminus. Equal amounts of protein, as confirmed by Ponceau S staining, were loaded onto each lane. (B) The same samples were analyzed by western blot with an antibody directed against N-terminally dephosphorylated β-catenin. Protein amounts (intensity of specific bands) were measured by densitometry. Wnt, 2-fold increase over untreated cells; lithium and S33 β-catenin, 10-fold; ALLN, no change. (C) Parallel samples of the conditions under (A) and (B) were transfected with optimal (TOP) or mutated (FOP) luciferase reporter plasmids and analyzed for TCF-dependent transcription by measuring luciferase activity. The ratio of the specific versus control signals (corrected for transfection efficiencies), termed TOP/FOP, is shown.

Fig. 4. ts20 CHO cells fail to show increased levels of dephosphorylated β-catenin. The ts20 CHO cells were kept at the restrictive temperature for 6 h, before they were analysed on a western blot for their levels of dephosphorylated β-catenin and for total β-catenin levels. Whereas total β-catenin levels increase, dephosphorylated levels remain low.

Fig. 5. Strongly increased levels of nuclear dephosphorylated β-catenin after Wnt signaling. 293T cells were treated with LiCl or transfected with Wnt, cytospins were prepared and slides were stained with an antibody specific for total β-catenin (top) or dephosphorylated β-catenin (bottom), followed by an FITC-labeled secondary antibody. The levels of β-catenin increase in all cases compared to mock-treated cells, but a dramatic increase in dephosphorylated β-catenin is seen with LiCl and to a lesser extend with Wnt1.