Kinetic Responses of β-Catenin Specify the Sites of Wnt Control
Ana R. Hernández*,
Allon M. Klein*, and
Marc W. Kirschner
Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
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Fig. 1. Dynamics of β-catenin during the Wnt response. (A to D) Total, GSK3- and CK1α-phosphorylated β-catenin after Wnt stimulation. α-tubulin: loading control. (B) shows absolute concentration of total β-catenin. (E) Accumulation rate of β-catenin [the derivative of (C)]. In (C) to (E), lines show fits to the kinetic model described in Fig. 2. (F) Accumulation of β-catenin in Wnt-stimulated RKO cells treated with proteasome inhibitor MG132. (G and H) Response of phosphorylated β-catenin to Wnt stimulation after inhibition by LiCl or various shRNAs. (I) Ratio of GSK3-phosphorylated β-catenin to total β-catenin relative to initial levels [calculated from (C) and (D)]. Error bars, mean ± SD (N = 3 replicates).
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Fig. 2. Flux analysis of the points of Wnt action on β-catenin degradation. (A) The β-catenin life cycle. (B) The three possible qualitative behaviors of phosphorylated β-catenin in response to Wnt, as predicted by flux analysis. (C) Quantification of total, GSK3-, and CK1α-phosphorylated β-catenin in Wnt-stimulated RKO cells. (D and E) The abundance of ubiquitylated phospho-β-catenin during Wnt stimulation in human embryonic kidney HEK293T (D) and RKO (E) cells. (F and G) The rates of GSK3- and CK1α-mediated phosphorylation, calculated from the measurements in (C) (supplementary materials, section S-IV). Gray curves show bounds on the CK1α phosphorylation rate: If β-catenin dephosphorylation is rare, the rate is described by the lower bound; if dephosphorylation occurs at a higher rate than phosphorylation and ubiquitylation, the upper bound holds. The black curve shows the dynamics if dephosphorylation and phosphorylation occur at comparable rates. Error bars, mean ± SD (N = 3 replicates).
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Fig. 3. The size of the unphosphorylated fraction of β-catenin. (A) Abundance of nonphospho β-catenin during Wnt stimulation in RKO cells. (B) Dephosphorylation of RKO cell lysates with protein phosphatase from bacteriophage  increased the signal of unphosphorylated β-catenin by 5 ± 5%. (C) Immunodepletion of GSK3-phosphorylated β-catenin decreased the signal of total β-catenin by 20 ± 5%. (A) and (B) show qualitative immunoblots; fractions (mean ± SD; N = 3 replicates) were determined by quantitative immunoblots (see supplementary materials).
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