Threonine 393 of beta-catenin regulates interaction with Axin

Abstract

CK2 is a regulatory kinase implicated in embryonic development and in cancer. Among the CK2 substrates is beta-catenin, a protein with dual function in Wnt signaling and cell adhesion. Previously, we reported that CK2 activity is required for beta-catenin stability and we identified threonine (T) 393 as a major CK2 phosphorylation site in beta-catenin. However, it is not known whether phosphorylation at T393 increases beta-catenin stability and if so, what is the mechanism. In this study we investigate the molecular mechanism of beta-catenin stabilization through phosphorylation at T393. We found that pseudophosphorylation of beta-catenin at T393 resulted in a stable activated form of beta-catenin with decreased affinity for Axin in vitro. This phosphomimetic mutant also displayed decreased regulation by Axin in vivo in a bioassay in Xenopus laevis embryos. In contrast, the binding of T393 pseudophosphorylated beta-catenin to E-cadherin was unaffected. Further analysis showed that pseudophosphorylation at T393 did not prevent beta-catenin phosphorylation by GSK3beta. Interestingly, we found that in the presence of pseudophophorylated beta-catenin and another activated form of beta-catenin, the recruitment of GSK3beta to Axin is enhanced. These findings indicate that phosphorylation of T393 by CK2 may affect the stability of beta-catenin through decreased binding to Axin. In addition, the increased recruitment of GSK3beta to the destruction complex in the presence of activated beta-catenin mutants could be a feedback mechanism to suppress overactive Wnt signaling.

Fig. 1

393D β-catenin is more stable than 393A β-catenin. A: C57MG cells were lysed 24 h after transfection with 393A, 393D and wildtype myc-β-catenin plasmids and processed for immunobloting with the indicated antibodies. One representative experiment of four is shown. B: Histogram representing normalized mean ± SD from four independent experiments as in (A). The myc-β-catenin band intensity was corrected for loading (β-tubulin) and transfection efficiency (GFP). 393D was normalized to 1. C: C57MG cells were transfected with 393A, 393D myc-β-catenin plasmids, and empty vector (pCS2). Twenty-four hours after transfection cells were incubated in media containing 0.1% FBS and harvested after 0 or 2.5 h. One representative experiment out of three is shown. D: Half-life analysis: 24 h after transfection with 393A, 393D or wildtype (not shown) myc-β-catenin, C57MG cells were treated with 50 µg/ml CHX and lysed at indicated times (in hours), lysates were subjected to immunoblot. E: Logarithmic plot of myc-β-catenin degradation over time. The myc-β-catenin band intensity from two independent experiments was corrected for loading (β-tubulin) and transfection efficiency (GFP), and normalized to the zero time point. The half-life was calculated according to the slope of the lines. Apparent protein molecular weights are shown in italics.

Fig. 2

CK2 pseudophosphorylated β-catenin has increased half-life in L cells and HEK293T. A: Twenty-four hours after transfection with 393D or 393A myc-β-catenin, L cells were trypsinized, resuspended in DMEM and cultured in suspension in 50 ml Falcon tubes (BD) and incubated in 37°C water bath. Cells were treated with 50 µg/ml CHX. One ml of cell suspension was taken at different time points, lysed directly in Laemmli buffer, and separated onto SDS–PAGE followed by immunoblot analysis with anti-c-myc and anti-tubulin antibody. B: Twenty-four hours after transfection with 393D or 393A myc-β-catenin, HEK293T cells were trypsinized and split into a 6-well plate. Fifty µg/ml CHX was added to the cells after replating. Cells were lysed at different time points for immunoblot analysis with anti-c-myc antibody to detect myc-β-catenin. Position and weight of the molecular markers utilized is indicated on the left of the immunoblots.

Fig. 3

T393 pseudophosphorylated β-catenin is transcriptionally more active than 393A. A: mRNA was isolated from pCS2 (control plasmid), WT, 393A or 393D myc-β-catenin transfected C57MG cells and subjected to RT-qPCR. Results are expressed as mRNA levels normalized to the control β-glucuronidase (GUS). Data represent mean ± SD is from four independent experiments. Asterisk (*) indicates P < 0.05. B: Endogenous β-catenin levels are not affected by mutant β-catenin transfection. C57MG cells were lysed 24 h after transfection with 393D or 393A myc-β-catenin or pCS2. Cell lysates were subjected to immunoblotting with the indicated antibodies. Black arrowhead represents myc-β-catenin, and white arrowhead points to endogenous β-catenin. Position and weight of the molecular markers utilized is indicated on the left of the immunoblots.

Fig. 4

T393 pseudophosphorylation of β-catenin affects Axin binding but not E-cadherin binding. A: HEK293T cells were transfected with 393A and 393D myc-β-catenin either alone or with E-cadherin. Twenty-four hours after transfection, cells were starved in medium containing 0.5% FBS for 4 h. Anti-c-myc (upper panel) and anti-E-cadherin (lower panel) were used for immunoprecipitation from the cell extracts from the transfected cells. Anti-HA antibody was used for mock IP. B: Histogram representing quantitation from three experiments shows that E-cadherin bind to 393D and 393A myc-β-catenin with equal affinity. C: HEK293T cells were transfected with 393A or 393D myc-β-catenin either alone or with HA-XAxin. Twenty-four hours after transfection, cells were starved in medium containing 0.5% FBS. Anti-HA (upper panel) and anti-c-myc (middle panel) antibodies were used for immunoprecipitations from the cell extracts; anti-FLAG antibody was used for mock IP. This was followed by immunoblot with indicated antibodies. Expression levels of exogenous proteins are shown in the lower panel (input). D: Histograms representing quantitation from three independent experiments: (left) relative myc-β-catenin coimmunoprecipitated by HA-XAxin (the ratio between 393A myc-β-catenin and HA-XAxin was normalized to 1), and (right) relative HA-XAxin coimmunoprecipitated by myc-β-catenin (the ratio between HA-XAxin and 393A myc-β-catenin was normalized to 1). Apparent protein molecular weights are shown in italics.

Fig. 5

Reduced regulation of T393 pseudophosphorylated β-catenin by Axin in vivo: Ectopic axis induction by 393D β-catenin is not affected by Axin. Two nanograms of mRNAs for 393A or 393D myc-β-catenin were ventrally injected in Xenopus laevis embryos with or without 1.2 ng of HA-XAxin coinjection. A: Vegetal view of stage 10.5 embryos showing endogenous (white arrowhead) and ectopic (black arrowhead) blastopore lips. The endogenous blastopore lip, dorsally located (D), is to the right; and ectopic blastopore lip, ventrally located (V), is to the left in all frames. B: Lateral view of embryos shown at stage 38. Note that both 393A and 393D induce an ectopic axis (indicated with lines). Top panels, HA-XAxin alone does not induce an ectopic axis. Middle panels, HA-XAxin prevents induction of an ectopic axis by 393A. Lower panels, HA-XAxin does not affect the formation of an ectopic axis induced by 393D. Some embryos injected with HA-XAxin were ventralized (top and middle panels on the right column), as observed by other researchers [Fukui et al., 2000] and in rescue experiments of other Wnt components [Zeng et al., 1997]. C: Histogram showing pooled data from four independent experiments. Embryos were categorized and scored as described in Materials and Methods. LacZ mRNA-injected and uninjected controls (ctrl) are included. Numbers on the X-axis reflect number of embryos in each treatment. Asterisk (*) indicates P < 0.05.

Fig. 6

Reduced phosphorylation of 393D β-catenin by CKI/GSK3β. A: C57MG cells were lysed 24 h after transfection with 393D or 393A myc-β-catenin or pCS2 (not shown). Cell lysates were subjected to immunoblotting with the indicated antibodies. B: Histogram representing quantitation from three independent experiments showing the relative N-terminal phosphorylation of myc-β-catenin mutants relative to their expression. Asterisk (*) indicates P < 0.05. C: Higher affinity of endogenous GSK3β for Axin in HEK293T cells expressing 393D β-catenin. The experimental protocol was the same as in Figure 4C. Expression levels of exogenous and endogenous proteins are shown in the lower panel (input). Endogenous GSK3β levels are not affected by β-catenin transfection. Representative immunoblots from four independent experiments. Indicated on the left of the immunoblots are the apparent molecular weights of the proteins (numbers in italics) or the position and weight of the molecular markers utilized (numbers in roman).

Fig. 7

Endogenous GSK3β co-immunoprecipitates more with Axin in HEK293T cells expressing a myc-tagged N-terminal deleted β-catenin. The experimental protocol was the same as in Figure 4C. Cell lysates were subjected to IP with anti-c-myc antibodies. GSK3β levels are not affected by β-catenin transfection (lower panel). Representative immunoblots from three independent experiments. Position and weight of the molecular markers utilized is indicated on the left of the immunoblots.