Nek2 phosphorylates and stabilizes β-catenin at mitotic centrosomes downstream of Plk1

Abstract

β-Catenin is a multifunctional protein with critical roles in cell-cell adhesion, Wnt signaling, and the centrosome cycle. Whereas the regulation of β-catenin in cell-cell adhesion and Wnt signaling are well understood, how β-catenin is regulated at the centrosome is not. NIMA-related protein kinase 2 (Nek2), which regulates centrosome disjunction/splitting, binds to and phosphorylates β-catenin. Using in vitro and cell-based assays, we show that Nek2 phosphorylates the same regulatory sites in the N-terminus of β-catenin as glycogen synthase kinase 3β (GSK3β), which are recognized by a specific phospho-S33/S37/T41 antibody, as well as additional sites. Nek2 binding to β-catenin appears to inhibit binding of the E3 ligase β-TrCP and prevents β-catenin ubiquitination and degradation. Thus β-catenin phosphorylated by Nek2 is stabilized and accumulates at centrosomes in mitosis. We further show that polo-like kinase 1 (Plk1) regulates Nek2 phosphorylation and stabilization of β-catenin. Taken together, these results identify a novel mechanism for regulating β-catenin stability that is independent of GSK3β and provide new insight into a pathway involving Plk1, Nek2, and β-catenin that regulates the centrosome cycle.

FIGURE 1:

Nek2 phosphorylates β-catenin at known GSK3β S33/S37/T41 sites and additional residues. (A) Schematic representation of β-catenin, with the armadillo repeats highlighted in blue (not to scale). The phosphorylated amino acids identified by nano-LC/MS/MS are shown only for the sample incubated with ATP. As a control, an identical sample without ATP was also analyzed. Known GSK3β phosphorylation sites are highlighted in red. The bracketed residues indicate a region of the peptide in which specific phospho site(s) could not be distinguished. (B, C) In vitro phosphorylation assay of GST protein and GST-N-terminal β-catenin (amino acids 1–133) purified from bacteria and incubated with recombinant kinases as indicated for 60 min in the presence of ATP, separated by SDS–PAGE, and Western blotted with an antibody to GST (B) or an antibody to the phospho-S33/S37/T41 epitope (C); phosphorylated β-catenin is marked by an asterisk, degradation products by a square, and GST by a triangle. (D) HEK293 cells cotransfected as indicated and treated for 4 h with GSK3 inhibitor (20 μM SB21673), extracted with 1% SDS, and immunoblotted with antibodies to detect phospho-S33/S37/T41 β-catenin and Nek2. (E) Quantitation of phospho–β-catenin band intensity normalized for total GFP–β-catenin (AU, arbitrary units); error bars, SEM of three independent experiments.

FIGURE 2:

Nek2 stabilizes β-catenin. (A) HEK293 cells were cotransfected as indicated and extracted with NP40, and proteins were immunoprecipitated with polyclonal GFP antibody and immunoblotted with a monoclonal antibody to HA, a polyclonal antibody to GFP, or a monoclonal antibody to Nek2. (B) Quantitation of HA-ubiquitin band intensity (AU, arbitrary units); error bars, SEM of three independent experiments. (C) HEK293 cells were cotransfected as indicated and extracted with NP40, and proteins were immunoprecipitated with a Myc antibody and immunoblotted with antibodies specific for phospho-S33/S37/T41, β-catenin, GFP, and Myc. Whole-cell lysates were immunoblotted for GFP. (D) HEK293 cells cotransfected as indicated and treated for 4 h with proteasome inhibitor (25 μM MG132) and GSK3 inhibitor (20 μM SB21673) as indicated. Cell lysates were extracted with NP40, immunoprecipitated with a β-catenin antibody, and then immunoblotted for β-catenin and Myc. Whole-cell lysates were immunoblotted for Myc.

FIGURE 3:

Phospho–β-catenin is located at mitotic centrosomes. (A) HCT116 18^−/ΔS45 cells were synchronized in mitosis. Centrosomes were enriched in a sucrose gradient, and fractions were immunoblotted for phospho-S33/S37/T41, Aurora A, β-catenin, and γ-tubulin. (B) Centrosomes from an asynchronous population of HCT116 18^−/ΔS45 cells were enriched and immunoblotted as described in A. (C, D) Indicated protein bands from blots in A and B were quantified (square in top blots indicate HMW protein(s), and triangle in top mitotic blot indicates phospho-S33/S37/T41 β-catenin; double bands of phospho-S33/S37/T41 β-catenin and Aurora A were combined for quantitation). Distributions are presented on the same graph, but levels (AU) of proteins are not comparable, as different antibodies were used for each protein. (E) Centrosome-positive fractions (*13/14) and centrosome-negative fractions (21/22) from mitotic and asynchronous centrosome preparations, respectively, were immunoprecipitated with phospho-S33/S37/T41 antibody (p-S33/S37/T41 IP) and immunoblotted (IB) with antibodies to phospho-S33/37/T41 (p-S33/S37/T41) and β-catenin. Levels of immunoglobulin heavy chain (IgG) in the immunoprecipitations are shown as a control (IB: IgG in E). Part of the original fractions of the sucrose gradients shown in A and B that were used for immunoprecipitations were combined into one immunoblot for comparison of total β-catenin levels in these fractions (total fractions in E, immunoblotted for β-catenin).

FIGURE 4:

GSK3β activity is not required for phospho-S33/S37/T41 reactivity at spindle poles. (A) Spindles derived from three HCT116 lines expressing wild-type β-catenin and ΔS45 β-catenin (Par^WT/ΔS45), only ΔS45 β-catenin (18^−/ΔS45), or only wild-type β-catenin (85^WT/−) were stained for DNA with DAPI and immunostained with antibodies as indicated. Images were taken at identical exposure times and identically contrast enhanced for each stain. Scale bar, 5 μm. (B) Quantitation of phospho-S33/S37/T41 reactivity and (C) total β-catenin levels at spindle poles of cell lines in A. Error bars, SEM of ≥20 spindle poles; ***p < 0.001; **p < 0.01; *p < 0.05. Original unmodified images taken at identical exposure times were measured for the three cell lines (AU, arbitrary units). HCT116 18^{−/ΔS45 cells} had significantly more β-catenin but less phospho-S33/S37/T41 reactivity at spindle poles than parental Par^WT/ΔS45 and 85^WT/− cells (*p < 0.05; **p < 0.01; ***p < 0.001). (D) HCT116 Par^WT/ΔS45 cells were treated with 2% DMSO as a control or with different GSK3 inhibitors (20 μM SB21673, 5 μM GSK3 Inhibitor IX, or 20 mM LiCl) for 4 h and then processed for immunofluorescence of mitotic spindles with antibodies as indicated and costained with DAPI for DNA (blue in merge). For presentation of control spindles and different treatments, images were taken at identical exposure times and identically contrast enhanced for each stain. Scale bar, 5 μm. (E) Phospho-S33/S37/T41 reactivity at spindle poles in different HCT116 lines described in A, untreated (same as in graph 4B), or treated as described in D (AU, arbitrary units). Error bars, SEM of ≥18 spindle poles; ***p < 0.001 and **p < 0.01. Original unmodified images taken at identical exposure times were measured for controls and different treatments. The data are representative of two independent experiments done with all cell lines under identical conditions.

FIGURE 5:

Nek2 activity is required for phospho-S33/S37/T41 reactivity at spindle poles (A, C) U2OS cells were synchronized in mitosis by double-thymidine block and release, transfected with HA-KD Nek2 or HA-WT Nek2, and immunostained with antibodies as indicated. For presentation of control spindles and different treatments, images were taken at identical exposure times and identically contrast enhanced for each stain. Scale bar, 10 μm. (B, D) Levels of β-catenin (B) and phospho-S33/S37/T41 immunofluorescence (D; AU, arbitrary units) at spindle poles of untreated U2OS cells or cells treated as described in A and C. Error bars, SEM of ≥15 spindle poles for B and SEM of ≥15 spindle poles for D per experiment from two experiments; ***p < 0.001. Original unmodified images taken at identical exposure times were measured for controls and transfected cells.

FIGURE 6:

Plk1 activity regulates phospho-S33/S37/T41 reactivity at spindle poles. (A) Pathways for regulation of centrosome separation by Plk1 at the onset of mitosis. (B, D) U2OS cells synchronized in mitosis by double-thymidine block and release were treated with control (2% DMSO), Plk1 inhibitor (100 nM BI2536), Eg5 inhibitor (100 μM monastrol), or GSK3 inhibitor (20 μM SB21673) for the last 9 h of the second release, stained for DNA with DAPI, and immunostained with antibodies as indicated. For presentation of control spindles and different treatments, images were taken at identical exposure times and identically contrast enhanced for each stain. Scale bar, 10 μm. (C, E) Graphs show levels of β-catenin (C) and phospho-S33/S37/T41 immunofluorescence (E; AU, arbitrary units) at spindle poles of untreated U2OS cells or cells treated as described in B and D. Error bars, SEM of ≥16 spindle poles for C and SEM of ≥10 spindle poles for E; ***p < 0.001. Original unmodified images taken at identical exposure times were measured for controls and different treatments. The data are representative of two independent experiments done with all cell lines under identical conditions.

FIGURE 7:

Nek2 rescues Plk1 inhibition of phospho-S33/S37/T41 reactivity at spindle poles. (A) HCT116 18^−/ΔS45 cells were synchronized in mitosis by double-thymidine block and release and were transfected as indicated. Cells were treated with control (2% DMSO) or Plk1 inhibitor (100 nM BI2536) for 6 h, processed for immunofluorescence with antibodies as indicated, and stained for DNA with DAPI. For presentation of control spindles and different treatments, images were taken at identical exposure times and identically contrast enhanced for each stain. Scale bar, 10 μm. (B) Quantitation of phospho-S33/S37/T41 reactivity at spindle poles (AU, arbitrary units); error bars, SEM of ≥15 spindle poles (***p <0.001; **p < 0.01) per experiment from two experiments. Original unmodified images taken at identical exposure times were measured for controls and different treatments. (C) HCT116 18^−/ΔS45 cells treated as described in A were processed for immunofluorescence with antibodies as indicated. Images were taken and prepared as described for A. Scale bar, 10 μm. (D) Quantitation of C-Nap1 levels at spindle poles; error bars, SEM of ≥15 spindle poles per experiment from two experiments (***p < 0.001). Original unmodified images taken at identical exposure times were measured for controls and different treatments.

FIGURE 8:

Plk1 activity regulates phospho-S33/S37/T41 β-catenin levels. (A) HCT116 18^−/ΔS45 cells were synchronized in mitosis and treated with control (2% DMSO) or Plk1 inhibitor (100 nM BI2536). Whole-cell lysates were immunoblotted for glyceraldehyde-3-phosphate dehydrogenase, β-catenin, phospho-S33/S37/T41 β-catenin, and cyclin B1. Cell lysates were immunoprecipitated with the phospho-S33/37/T41 antibody and immunoprecipitates immunoblotted for β-catenin and phospho-S33/S37/T41 reactivity. Phospho-S33/S37/T41 β-catenin is detectable only after concentrating it by immunoprecipitation (30 μl of total lysate/lane vs. immunoprecipitate from 500 μl of total lysate/lane was loaded). (B) Quantitation of phospho–β-catenin band intensities as measured in the immunoblots of β-catenin coimmunoprecipitated with the phospho-S33/S37/T41 antibody (AU, arbitrary units); error bars, SEM of three independent experiments (**p < 0.008). (C) Model for regulation of Nek2 by Plk1 at the onset of mitosis, which results in removal of centrosomal linker proteins C-Nap1 and rootletin and activation of phospho-S33/S37/T41 β-catenin. Localization of β-catenin to interphase centrosomes is mediated by the linker proteins C-Nap1 and rootletin. At the onset of mitosis, activation of Plk1 and Nek2 at centrosomes leads to phosphorylation of the linker proteins and β-catenin. The linker proteins are removed from centrosomes, whereas phospho–β-catenin (p-β-cat) remains associated with the spindle poles. In monopolar spindles caused by inhibition of Plk1 the linker proteins are not removed and continue to provide binding sites for nonphosphorylated β-catenin at the spindle pole.