Bcr-Abl stabilizes beta-catenin in chronic myeloid leukemia through its tyrosine phosphorylation

Abstract

Self-renewal of Bcr-Abl(+) chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine-(S/T) unphosphorylated beta-catenin. Although beta-catenin can be tyrosine (Y)-phosphorylated, the occurrence and biological relevance of this covalent modification in Bcr-Abl-associated leukemogenesis is unknown. Here we show that Bcr-Abl levels control the degree of beta-catenin protein stabilization by affecting its Y/S/T-phospho content in CML cells. Bcr-Abl physically interacts with beta-catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate beta-catenin at Y86 and Y654 residues. This Y-phospho beta-catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr-Abl antagonist, impairs the beta-catenin/TCF-related transcription causing a rapid cytosolic retention of Y-unphosphorylated beta-catenin, which presents an increased binding affinity for the Axin/GSK3beta complex. Although Bcr-Abl does not affect GSK3beta autophosphorylation, it prevents, through its effect on beta-catenin Y phosphorylation, Axin/GSK3beta binding to beta-catenin and its subsequent S/T phosphorylation. Silencing of beta-catenin by small interfering RNA inhibited proliferation and clonogenicity of Bcr-Abl(+) CML cells, in synergism with Imatinib. These findings indicate the Bcr-Abl triggered Y phosphorylation of beta-catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML.

Figure 1

SB-216763 promotes nuclear accumulation of Y-phospho β-catenin and BC-CML cell growth. (A) Proliferation of Ku812 and fresh BC cells treated with the indicated doses (μM) of SB-216763 for 24 h. Errors bars indicate s.d. of triplicate experiments. (B) GSK3β (IP: αGSK3β) was immunoprecipitated from Ku812 (a and b) and BC (c and d) cells treated with DMSO (−) or 5 μM SB-216763 (+) for 2 h and immunoblotted with an anti-GSK3β antibody or an antibody against its phospho-Y216 residue. β-Catenin was also shown. (C) Upon 8 h of treatment with 5 μM SB-216763, whole lysates from BC cells were analyzed for total β-catenin levels with a C-terminal antibody (C-Term). β-Catenin was also probed with two antibodies recognizing its specific S/T-unphospho- (nonp-S/T) or phosphorylated (p-S/T) form. Total β-actin levels were indicated as a loading control. (D) Nuclear (N) and cytosolic (C) extracts from Ku812 cells cultured with DMSO (−) or 5 μM SB-216763 (+) for 8 h were analyzed with antibodies against β-catenin, its specific S/T-nonphospho form (nonp-S/T), nuclear Lamin B and cytoplasmic IkBα. (E, F) Nuclear (N) and cytosolic (C) extracts of Ku812 cells treated as described above were immunoprecipitated with an anti-β-catenin (IP: α β-cat, E) or an anti-phosphotyrosine (IP: α p-Y, F) antibody and then immunoblotted as indicated (HC=heavy chain of IgG antibody used for immunoprecipitation).

Figure 2

β-Catenin is coupled to Bcr-Abl and accumulates in a Y-phosphoform in CML. (A) A representative sample of normal (donor) or CML-BMMC isolated from a CP-CML (CP) or BC-CML (BC) patient was compared to Ku812 cells. Total cell lysates (±1 μM Imatinib for 2 h) were immunoblotted with the indicated antibodies. (B) Ls174T (a), Ku812 (b) and fresh BC-CML (c–h) cells were immunoprecipitated with an anti-β-catenin antibody (IP: α β-cat). Immunocomplexes were analyzed by SDS–PAGE for Bcr-Abl and β-catenin. The same blot was stripped and analyzed for Y-phospho β-catenin (p-Y β-cat). (C) Anti-Abl immunoprecipitates (IP: α Abl) from Ku812 (a and b) and BC-CML (c and d) cells treated with DMSO (−) or 1 μM Imatinib (+) for 2 h were analyzed for Bcr-Abl and β-catenin levels. The same blot was reprobed by using an anti-phosphotyrosine antibody indicating two proteins of 210 kDa (p-Y Bcr-Abl) and 97 kDa (p-Y β-cat) and with a nonphospho-S/T β-catenin antibody (nonp-S/T β-cat). (D) Ku812 (a and b) and BC-CML (c and d) lysates were immunoprecipitated with an anti-β-catenin antibody (IP: α β-cat) and probed with the indicated antibodies.

Figure 3

Tyrosine kinase activity of Bcr-Abl is required to trigger β-catenin Y phosphorylation in BC-CML cells. (A) Ku812 cells were treated with DMSO (a), SKI-606 (b), Imatinib (c) or SU6656 (d) for 2 h and then immunoprecipitated with an anti-Abl antibody (IP: α Abl). Protein levels and Y phosphorylation of Bcr-Abl (p-Y Bcr-Abl) were analyzed by immunoblotting. Activation of c-Src was also probed by using a specific anti-phospho-Y418 antibody (p-Y Src). (B) Anti-β-catenin immunoprecipitates (IP: α β-cat) from Ku812 cells treated with DMSO (a), SKI-606 (b), Imatinib (c) or SU6656 (d) for 2 h were immunoblotted with the indicated antibodies. (C) Ku812 cells were transiently transfected with siRNAs for c-Src (c), a control siRNAs pool (b) or oligofectine alone (a). After 48 h, cells were immunoprecipitated with an anti-Abl antibody (IP: α Abl) and analyzed for c-Src content and Y-phospho-Bcr-Abl (p-Y Bcr-Abl). Levels of Bcr-Abl were shown as loading control for the immunoprecipitation. (D) Lysates obtained from Ku812 described were immunoprecipitated with an anti-β-catenin antibody (IP: α β-cat) and probed with the indicated antibodies.

Figure 4

Bcr-Abl promotes β-catenin Y86-Y654 phosphorylation and stability. (A) HEK293T cells were transiently transfected with Bcr-Abl (b and h) or an empty vector (g). A histidine (His)-tagged plasmid encoding for WT β-catenin (A) and its tyrosine-to-phenylalanine (Y-to-F) mutants Y86F (c), Y142F (d), Y654F (e) and Y86F-Y654F (f) were coexpressed with Bcr-Abl (c–f). After 48 h, transfected cells were immunoprecipitated with an anti-His antibody (IP: α His) and blotted for Bcr-Abl, Axin and β-catenin. The same blot was stripped and assessed for Y-phospho β-catenin (p-Y-β-cat). Levels of phospho-S/T β-catenin (p-S/T β-cat) are also shown. (B) GST-purified WT β-catenin (A,B) and the indicated Y-to-F fusion proteins Y86F (C), Y654F (D) and Y86F-Y654F (E) were phosphorylated by recombinant Abl kinase in vitro. Samples were analyzed by immunoblotting with the indicated antibodies. (C) Total lysates from cells transfected as described in (A) were blotted as indicated. (D) Ku812 cells were labeled with [³⁵S]methionine for 1 h and then chased with nonradioactive medium without or with Imatinib for the indicated time points. Cells were then lysed and immunoprecipitated for β-catenin (IP: α β-cat). The results were analyzed by densitometry and expressed as a percentage of the intensity value at time 0.

Figure 5

Tyrosine-phosphorylated β-catenin does not bind Axin. (A) BC cells (5 × 10⁶) treated with DMSO (b) or 1 μM Imatinib (c) for 2 h were immunoprecipitated for β-catenin (IP: α β-cat). A lysate from 5 × 10⁶ BC cells not immunoprecipitated was also prepared (a). The protein lysates were immunoblotted with the indicated antibodies. (B) BC cells (5 × 10⁶) cultured with DMSO (−) or 1 μM Imatinib (+) for 2 h were immunoprecipitated with an anti-Axin antibody (IP: α Axin) and immunoblotted with the indicated antibodies. (C, D) Ku812 cells incubated with DMSO (−) or 1 μM Imatinib (+) for 2 h were immunoprecipitated for β-catenin (IP: α β-cat, C) or Axin (IP: α Axin, D) and immunoblotted with the indicated antibodies. (E) Ku812 cells cultured with DMSO (−) or 1 μM Imatinib (+) for 2 h were immunoprecipitated with an anti-β-catenin (IP: α β-cat) (a and b) or an anti-Axin antibody (IP: α Axin) (c and d). After removal of Axin immunocomplexes from cell lysates, Y phosphorylated proteins were immunoprecipitated by anti-phosphotyrosine antibody (IP: α sup-PY) and analyzed in Western blot for Axin and β-catenin. The same blot was stripped and assessed for Y phosphorylation of β-catenin (p-Y β-cat) with an antiphosphotyrosine antibody.

Figure 6

Imatinib reduces nuclear levels of Y-phospho β-catenin by impairing its TCF4-related transcription. (A) Ku812 cells were treated with DMSO (a) or with 1 μM Imatinib for 2 h (b) or 16 h (c), respectively. Cells were also incubated with 20 μM caspase-3 inhibitor Z-DEVD-fmk alone (d) or with 1 μM Imatinib (e) for 16 h. Total cell lysates were then immunoblotted as indicated. (B) TOP and FOP-flash plasmids were transfected into Ku812 cells. After 24 h of transfection, cells were treated for an additional 24 h and processed for determining the luciferase gene-reporter activity. Data indicate the mean+s.d. of three independent experiments. (C) Ku812 cells were treated with DMSO (a and d) or Imatinib for 2 h (b and e) or 16 h (c and f). Nuclear (N) and cytosolic (C) cell extracts were then prepared and probed as indicated. (D) Nuclear (N) and cytosolic (C) fractions described in (C) were also immunoprecipitated for β-catenin (IP: α β-cat) and then subjected to immunoblotting with the indicated antibodies.

Figure 7

β-Catenin downregulation cooperates with Imatinib in reducing Bcr-Abl⁺ BC-CML cell growth and clonogenicity. (A) Ku812 cells were treated with DMSO (−) or 1 μM Imatinib (+) for 16 h. TCF4 and β-catenin were immunoprecipitated using an anti-TCF4 antibody (IP: α TCF4). β-Catenin and Bcr-Abl protein levels were analyzed by immunoblotting. The blot was reprobed to assess TCF4 protein content. (B) Ku812 cells were transfected with 50 (c), 150 (d) or 250 nM (e) of siRNA for β-catenin, 250 nM of a control siRNA (b) or carrier alone (a). Total lysates prepared 48 h after transfection were analyzed for β-catenin and cyclin D1 expression. The amounts of β-actin were reported as loading control. (C) Proliferation assay of Ku812 cells transfected with a control siRNA (siCTR) or β-catenin siRNA (siβ-cat) for 24 h and then cultured in the presence of decreasing concentrations of Imatinib for an additional 24 h. A labeling with [³H]thymidine was carried out for the last 8 h. (D) Quantitative analysis of apoptosis in Ku812 cells transfected with siCTR or siβ-cat for 24 h and then cultured in the absence or presence of 1 μM Imatinib for additional 24 h was performed by flow cytometry. Values represent the mean±s.d. of three independent experiments (^*P<0.003 for siβcat and <0.05 for dn-TCF4). (E) Ku812 cells were cotransfected with a pEGFP plasmid and siCTR, siβ-cat or a dominant-negative TCF4 construct (dn-TCF4). Total 1000 GFP⁺ sorted cells by FACS analysis were transferred to 0.5% soft agar in the absence or presence of 1 μM Imatinib. Colonies were scored on an inverted Leica microscope on day 15. (F) Ku812 cells were transfected with either siCTR, siβ-cat or dn-TCF4. After 24 h, cells were cotransfected with TOP-flash or FOP-flash plasmid and cultured in the absence or presence of Imatinib for an additional 24 h when luciferase reporter activity was determined. Data represent the mean±s.d. of three independent experiments.

Figure 8

A model of β-catenin/TCF4 transcriptional activation in Bcr-Abl⁺ CML cells. The figure summarizes the effects of Bcr-Abl, Imatinib and SB-216763 on β-catenin protein stabilization and nuclear signaling.