Bcr-Abl stabilizes β-catenin in chronic myeloid leukemia through its tyrosine phosphorylation
2007; Springer Nature; Volume: 26; Issue: 5 Linguagem: Inglês
10.1038/sj.emboj.7601485
ISSN1460-2075
AutoresAddolorata Maria Luce Coluccia, Angelo Vacca, Mireia Duñach, Luca Mologni, Sara Redaelli, Victor Bustos, Daniela Benati, Lorenzo A. Pinna, Carlo Gambacorti‐Passerini,
Tópico(s)HER2/EGFR in Cancer Research
ResumoArticle22 February 2007free access Bcr-Abl stabilizes β-catenin in chronic myeloid leukemia through its tyrosine phosphorylation Addolorata Maria Luce Coluccia Corresponding Author Addolorata Maria Luce Coluccia Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Search for more papers by this author Angelo Vacca Angelo Vacca Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Search for more papers by this author Mireia Duñach Mireia Duñach Departament de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, Barcelona, Spain Search for more papers by this author Luca Mologni Luca Mologni Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy Search for more papers by this author Sara Redaelli Sara Redaelli Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy Search for more papers by this author Victor H Bustos Victor H Bustos Venetian Institute for Molecular Medicine (VIMM), Padova, Italy Search for more papers by this author Daniela Benati Daniela Benati Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy Search for more papers by this author Lorenzo A Pinna Lorenzo A Pinna Venetian Institute for Molecular Medicine (VIMM), Padova, Italy Department of Biological Chemistry, University of Padova, Padova, Italy Search for more papers by this author Carlo Gambacorti-Passerini Carlo Gambacorti-Passerini Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy Department of Oncology/JGH, McGill University, Montreal, Quebec, Canada Search for more papers by this author Addolorata Maria Luce Coluccia Corresponding Author Addolorata Maria Luce Coluccia Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Search for more papers by this author Angelo Vacca Angelo Vacca Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Search for more papers by this author Mireia Duñach Mireia Duñach Departament de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, Barcelona, Spain Search for more papers by this author Luca Mologni Luca Mologni Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy Search for more papers by this author Sara Redaelli Sara Redaelli Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy Search for more papers by this author Victor H Bustos Victor H Bustos Venetian Institute for Molecular Medicine (VIMM), Padova, Italy Search for more papers by this author Daniela Benati Daniela Benati Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy Search for more papers by this author Lorenzo A Pinna Lorenzo A Pinna Venetian Institute for Molecular Medicine (VIMM), Padova, Italy Department of Biological Chemistry, University of Padova, Padova, Italy Search for more papers by this author Carlo Gambacorti-Passerini Carlo Gambacorti-Passerini Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy Department of Oncology/JGH, McGill University, Montreal, Quebec, Canada Search for more papers by this author Author Information Addolorata Maria Luce Coluccia 1,2, Angelo Vacca2, Mireia Duñach3, Luca Mologni1, Sara Redaelli1, Victor H Bustos4, Daniela Benati1, Lorenzo A Pinna4,5 and Carlo Gambacorti-Passerini1,6 1Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy 2Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy 3Departament de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, Barcelona, Spain 4Venetian Institute for Molecular Medicine (VIMM), Padova, Italy 5Department of Biological Chemistry, University of Padova, Padova, Italy 6Department of Oncology/JGH, McGill University, Montreal, Quebec, Canada *Corresponding author. Department of Clinical Medicine, University of Milano-Bicocca, via Cadore 48, 20052 Monza, Milan, Italy. Tel.: +39 02 64488059; Fax: +39 02 64488363; E-mail: [email protected] The EMBO Journal (2007)26:1456-1466https://doi.org/10.1038/sj.emboj.7601485 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Self-renewal of Bcr-Abl+ chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine-(S/T) unphosphorylated β-catenin. Although β-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 β-catenin protein stabilization by affecting its Y/S/T-phospho content in CML cells. Bcr-Abl physically interacts with β-catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate β-catenin at Y86 and Y654 residues. This Y-phospho β-catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr-Abl antagonist, impairs the β-catenin/TCF-related transcription causing a rapid cytosolic retention of Y-unphosphorylated β-catenin, which presents an increased binding affinity for the Axin/GSK3β complex. Although Bcr-Abl does not affect GSK3β autophosphorylation, it prevents, through its effect on β-catenin Y phosphorylation, Axin/GSK3β binding to β-catenin and its subsequent S/T phosphorylation. Silencing of β-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 β-catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML. Introduction The WNT/β-catenin signalling promotes stem cell renewal by coordinating changes in gene expression and cell adhesion (Reya and Clevers, 2005). The key player in this network is β-catenin, which acts as a nuclear coactivator of the TCF/LEF (T-cell factor/lymphoid enhancer factor) transcription factors or as a structural adaptor protein at cell adherens junctions (Nelson and Nusse, 2004; Harris and Peifer, 2005). WNT factors are cysteine-rich lipid-modified proteins that bind to several Frizzled (FZD) receptors. Under physiological conditions, WNT proteins accumulate β-catenin by inhibiting its glycogen synthase kinase 3 (GSK3)-dependent serine/threonine (S/T) phosphorylation on specific N-terminal residues. As GSK3 targets β-catenin for ubiquitination and proteasome degradation (Klymkowsky, 2005), detection of a nuclear S/T-nonphospho β-catenin is a hallmark of its transcriptional activation. Expression of β-catenin/TCF-induced cell cycle regulators (such as c-Myc and cyclin D1) is crucial for maintaining cell homeostasis in normal proliferating tissues, such as colon and skin (Pinto and Clevers, 2005). The WNT/β-catenin cascade has also pivotal roles in the self-renewal of hematopoietic stem cells (HSC), as a forced expression of a nondegradable β-catenin (S33-mutant) is sufficient to perpetuate themselves in vitro and sustain bone marrow reconstitution in vivo (Reya et al, 2003). Whereas the loss of WNT responsiveness allows multilineage differentiation of HSC (Baba et al, 2005), this link appears uncoupled in several human malignancies as a result of an increased β-catenin expression and protein stabilization in committed myeloid and lymphoid progenitors (Staal and Clevers, 2005). Chronic myeloid leukemia (CML) begins as an indolent disease when an HSC expresses the oncogenic tyrosine kinase Bcr-Abl, which confers a proliferative advantage to its progeny. At this phase, Bcr-Abl does not interfere with HSC differentiation, and its levels decrease in committed progenitors (Daley, 2004; Huntly et al, 2004). An Abl kinase-selective inhibitor, imatinib mesylate (CGP57148B, STI571, and Gleevec/Glivec), represents the treatment of choice for CML inducing remissions in most CML patients in chronic phase (CP) (Goldman and Melo, 2003; Deininger et al, 2005). However, mutations in the catalytic site of Bcr-Abl or bcr-abl gene amplification (le Coutre et al, 2000; Krause and Van Etten, 2005) can select imatinib-resistant clones during CML progression. Blast crisis (BC) represents the terminal outcome of CML and was recently correlated with the expansion of committed granulocyte–macrophage precursors with persistence of high Bcr-Abl mRNA levels and accumulation of a nuclear S/T-nonphospho β-catenin (Jamieson et al, 2004). In this report, we investigated the molecular causes underlying β-catenin deregulation in CML and identified Bcr-Abl-mediated Y phosphorylation of β-catenin as a cause for its increased protein stability and transcriptional signalling activity. Results GSK3 inhibition promotes proliferation and nuclear accumulation of a Y-phospho β-catenin in Bcr-Abl+ BC-CML cells Nuclear accumulation of β-catenin is a hallmark of WNT signalling activation. We tested whether Bcr-Abl+ CML cells could contain an intact WNT/GSK3 pathway by using SB-216763, a GSK3 inhibitor, which promotes β-catenin stabilization. Proliferation of a Bcr-Abl+ BC cell line (Ku812) and of fresh BMMC isolated from a BC-CML patient was increased by SB-216763 (Figure 1A), correlating with an impaired Y216 autophosphorylation of GSK3β (Cole et al, 2004) (Figure 1B, α p-Y GSK3β, b,d versus a,c). These data indicated the integrity of an APC/Axin/GSK3 pathway, suggesting that the rate of stabilized β-catenin was suboptimal in these Bcr-Abl+ cells or still exogenously inducible. 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). Download figure Download PowerPoint To verify the effect of SB-216763 on GSK3-mediated phosphorylation, β-catenin was probed with an antibody against its N-terminal S/T-phospho residues (Ser33, Ser37 and Thr41) (α p-S/T). We also used an antibody specific for β-catenin that is not phosphorylated on Ser33 and Ser37 (α nonp-S/T) and a pan C-terminal antibody (α C-term) that does not distinguish between phosphorylated and dephosphorylated β-catenin (Figure 1C). SB-216763 enhanced β-catenin accumulation in fresh BC cells (α C-term, b compared to a), increasing its S/T-nonphospho levels (α nonp-S/T, d versus c). Consistent with GSK3 kinase inhibition, the S/T-phospho pool of β-catenin was reduced by SB-216763 (α p-S/T, f compared to e). Figure 1D shows that the proliferative effect of SB-216763 correlated with increased nuclear and cytoplasmic levels of β-catenin (α β-cat, b,d versus a,c), further identified as transcriptionally active (α nonp-S/T, b,d versus a,c). β-Catenin could also be immunoprecipitated in a Y-phospho form from enriched nuclear and cytosolic Ku812 extracts (Figure 1E) (α p-Y β-cat, a and c), and the SB-216763 released β-catenin was highly phosphorylated on tyrosine residue(s) (α p-Y β-cat, b,d versus a,c). Interestingly, Y-phospho β-catenin was associated with the nuclear transcription factor TCF4 (Figure 1E and F, α TCF4, b versus a) and, to a lesser extent, to Axin in the cytosol (Figure 1E, α Axin, d versus c). As Axin was never immunoprecipitated using an antiphosphotyrosine antibody (Figure 1F, α Axin), as instead observed for β-catenin or TCF4, the Y-phospho β-catenin could be considered a true Axin-uncomplexed fraction in Bcr-Abl+ CML cells either in the presence or in absence of a WNT signal. β-Catenin accumulates in CML as a Y-phosphoprotein coupled to Bcr-Abl Our initial evidence that β-catenin accumulation might correlate with Bcr-Abl protein levels derived from an analysis of Bcr-Abl and β-catenin expression in BMMC from four CML patients in CP and six in BC. Figure 2A presents the results obtained in a representative BC patient and in the CP patient with the highest Bcr-Abl expression, the other CP patients being negative for Bcr-Abl in total cell lysates. Equal numbers of cell (5 × 105) were analyzed. As an expected feature of CML progression (Barnes et al, 2005), higher expression of Bcr-Abl (α Bcr-Abl) in BC-CML (lanes e and f) and Ku812 (lanes g and h) cells compared to CP-CML (lanes c and d) was detected and correlated with higher levels of total (α β-cat) and transcriptionally active (α nonp-S/T β-cat) β-catenin. Whereas β-catenin accumulation in BC cells could be accounted for by restored mRNA transcription (Jamieson et al, 2004), we observed that imatinib reduced the active S/T-nonphospho pool of β-catenin, pointing to a Bcr-Abl-mediated β-catenin stabilization and transcriptional activation. 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. Download figure Download PowerPoint As β-catenin can interact directly with oncogenic tyrosine kinases such as c-MET (Hiscox and Jiang, 1999), RON (Danilkovitch-Miagkova et al, 2001) and c-erbB-2 (Kanai et al, 1995), we investigated a potential association of Bcr-Abl with β-catenin (Figure 2B) in Ku812 (b) and BC-CML (lanes c–h) cells. The CRC Ls174T cells (a), which contain high β-catenin levels, were included as negative controls for Bcr-Abl expression. Cells were immunoprecipitated with an anti-β-catenin antibody (IP: α β-cat). Bcr-Abl (α Bcr-Abl) co-precipitated with β-catenin (α β-cat), which was Y phosphorylated (α p-Y β-cat). As shown in Figure 2C, total lysates from Ku812 and fresh BC-CML (BC) cells treated with dimethyl sulfoxide (DMSO) (−) or imatinib (+) were also immunoprecipitated with an anti-Abl antibody (IP: α Abl). β-Catenin was detected in the anti-Abl immunoprecipitates and imatinib prevented both Bcr-Abl (α p-Y Bcr-Abl) and β-catenin Y activation (α p-Y β-cat), decreasing their physical interaction (α β-cat, a,c versus b,d). The finding that an S/T-nonphospho β-catenin is coupled to Bcr-Abl (α nonp-S/T, a and c) suggests that Bcr-Abl recruits a signalling competent pool of β-catenin. In Figure 2D, we performed reciprocal anti-β-catenin immunoprecipitates (IP: α β-cat). Whereas a comparable amount of β-catenin was detected in all samples (α β-cat), the co-precipitation of Bcr-Abl and β-catenin was impaired in cells treated with imatinib (α Bcr-Abl, b,d compared to a,c) as well as Y phosphorylation of β-catenin (α p-Y β-cat, b,d versus a,c) and its nonphospho-S/T levels (α nonp-S/T, b,d versus a,c). These data show a functional link between the increased expression of Bcr-Abl and the accumulation of a nuclear Y-phospho β-catenin in the BC phase of CML. Bcr-Abl kinase activity is required to trigger Y phosphorylation of β-catenin β-Catenin is a target for several members of the Src family tyrosine kinase (Piedra et al, 2001; Coluccia et al, 2006), which are known to contribute to Bcr-Abl+ leukemogenesis (Tipping et al, 2004). Therefore, β-catenin might be phosphorylated by either Bcr-Abl itself and/or its proximal Src effectors in human CML cells. A search for Src kinase inhibitors not active against Bcr-Abl identified SU6656 (IC50 of 20 nM for c-Src). In Figure 3A, Ku812 cells treated with DMSO (a), 1 μM SKI-606 (b), 1 μM imatinib (c) or 5 μM SU6656 (d) were immunoprecipitated with an anti-Abl antibody (IP: α Abl). Whereas SKI-606 and imatinib inhibited Bcr-Abl (α p-Y Bcr-Abl, b and c) and Src (α p-Y Src, b and c) Y phosphorylation, SU6656 selectively reduced the activation of Src kinases bound to Bcr-Abl (α p-Y Src, d) without affecting Bcr-Abl autoactivation (α p-Y Bcr-Abl, d). The ability of SKI-606, imatinib or SU6656 kinase inhibitors to prevent β-catenin Y phosphorylation was also analyzed by immunoprecipitating Ku812 cells with a C-terminal β-catenin antibody (Figure 3B). β-Catenin Y activation observed in the untreated control (α pY β-cat, a) was inhibited by SKI-606 (b), a dual Src/Abl inhibitor, or Imatinib (c), a specific Abl antagonist, whereas SU6656 (d), a Src family kinase inhibitor, had a minor effect. Interestingly, SU6656 was unable to disrupt the Bcr-Abl/β-catenin association, as instead observed for SKI-606 and Imatinib (α Bcr-Abl, d versus c and b). Imatinib reduced the levels of Y-phospho Src associated with Bcr-Abl to a greater extent than SU6656, suggesting a downstream effect of Bcr-Abl on c-Src activation. 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. Download figure Download PowerPoint To confirm these data further, we targeted Src expression by using a mixture of four selected double-stranded small interfering RNA (siRNA) directed against Src. As shown in Figure 3C, this procedure inhibited 80% of Src associated with Bcr-Abl (α Src, c versus b). Although the autoactivation of Bcr-Abl (α p-Y Bcr-Abl, c) appeared slightly decreased by silencing of Src, this did not inhibit the binding of Bcr-Abl to β-catenin and its Y phosphorylation (Figure 3D). These findings cannot exclude the contribution of other Src-related kinases, which can immunoprecipitate with the Bcr-Abl/β-catenin complex, but they indicate that an active Bcr-Abl tyrosine kinase is required to trigger Y phosphorylation of β-catenin in CML cells. They also indicate that Src phosphorylation in CML cells is dependent on Bcr-Abl kinase activity, but not vice versa. Bcr-Abl phosphorylates β-catenin at Y86 and Y654 and promotes its protein stabilization Tyrosine residues of β-catenin that can be phosphorylated by Src kinases were identified as Y86, Y142 and Y654 (Roura et al, 1999; Piedra et al, 2003). To validate the effects of Bcr-Abl on β-catenin Y phosphorylation and stability, human embryonic kidney (HEK293) T cells were transiently cotransfected with Bcr-Abl and histidine (His)-tagged plasmids encoding for wild-type (WT) β-catenin or its specific Y-to-F mutants Y86F, Y142F, Y654F and the double-mutant Y86F-Y654F (Figure 4A). Analysis of anti-His immunoprecipitates (IP: α His) confirmed the Bcr-Abl/β-catenin association also in these cells (α Bcr-Abl, b–f) and showed that β-catenin was differently modified on its Y/S/T residues in the presence of the oncogene. In fact, the exogenous WT β-catenin resulted phosphorylated on S/T (α p-S/T β-cat: a), but not on Y-residues (α p-Y β-cat: a) when transfected alone. The coexpression of the Bcr-Abl (α Bcr-Abl, b–f) prevented the S/T phosphorylation of β-catenin (α p-S/T β-cat: b) by triggering its Y-modification (α p-Y β-cat: b). Both Y86F (α p-Y β-cat: c) and Y654F (α pY β-cat: e) mutants were less Y phosphorylated (approximately 50%) in presence of Bcr-Abl than the Y142F (α p-Y β-cat: d) or WT β-catenin (α p-Y β-cat: b), whereas the Y phosphorylation of the double-mutant Y86F-Y654F (α p-Y β-cat: f) was completely inhibited. Interestingly, a lower degree of S/T phosphorylation of the Y654F mutant compared to Y86F (α p-S/T β-cat: e versus c) correlated with a decreased amount of Axin detectable in Y654F-immunoprecipitates (α Axin: e compared to c), indicating that the Bcr-Abl-mediated phosphorylation of β-catenin Y86 could be more efficient than Y654 in impairing its binding affinity to Axin. 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. Download figure Download PowerPoint The ability of recombinant Abl (rAbl) kinase to Y phosphorylate purified GST-β-catenin fusion proteins in vitro indicated that β-catenin is a direct substrate of Abl (Figure 4B). As shown in Figure 4C, expression of Bcr-Abl (α Bcr-Abl, b–f and lane h) in HEK293T cells increased the total protein levels of either endogenous (α β-cat: h versus g) or ectopically induced WT β-catenin (α β-cat, b versus a). Also, different total levels of Y-to-F β-catenin mutants (α β-cat, c–f) correlated proportionately to their degree of Y phosphorylation (α p-Y β-cat: c–f). These effects were not accompanied by changes in GSK3β Y216 phosphorylation, as also detected in empty vector (−)-transfected sample (α p-Y GSK3β, a–h). The effect of Bcr-Abl on β-catenin protein turnover was analyzed by performing a pulse-chase analysis of Bcr-Abl+ CML cells cultured with or without Imatinib. Autoradiography of anti-β-catenin immunoprecipitates prepared at different times during a chase showed that the estimated half-life of β-catenin was decreased from 3.1 to 1.5 h in the presence of Imatinib compared with untreated cells (Figure 4D). In conclusion, these data indicate that the delayed degradation of β-catenin correlated with its Bcr-Abl-mediated Y phosphorylation on Y86 and Y654. This evidence further supports a causal role for Bcr-Abl in promoting β-catenin stabilization without affecting GSK3β autophosphorylation. Tyrosine-phosphorylated β-catenin does not interact with the Axin/GSK3β complex Total β-catenin levels are tightly regulated by a regulatory multi-protein complex involving Axin, APC and GSK3 (Harris and Peifer, 2005; Klymkowsky, 2005). In Figure 5A, APC (α APC, b) and Axin (α Axin, b) were immunoprecipitated with β-catenin (IP: α β-cat) from BC-CML patient cells. Although Imanitib did not change the amount of APC coupled to β-catenin (α APC, c versus b), it significantly increased β-catenin/Axin association (α Axin, c compared to b) and binding of β-catenin to the Y-activated GSK3β kinase (α p-Y GSK3β, c versus b). By analyzing reciprocal anti-Axin immunoprecipitates obtained from the same BC-CML sample (Figure 5B, IP: α Axin), we observed that the amount of β-catenin captured by Axin was higher (α β-cat, b versus a) in the presence of Imatinib, justifying the increases on its S/T phosphorylation levels (α p-S/T β-cat, b versus a). A similar analysis was carried out in Ku812 cells (Figure 5C and D) obtaining comparable results. In addition, as Imatinib did not alter the Axin/GSK3β interaction (Figure 5C and D, α GSK3β, b versus a), these findings indicate that the Bcr-Abl-induced Y-phospho pool of β-catenin has a reduced binding affinity to Axin. In this view (Figure 5E), Ku812 cells were cultured in the absence (−) or presence (+) of Imatinib and then immunoprecipitated with either 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 total cell lysates, the supernatants were further immunoprecipitated by using an anti-phosphotyrosine antibody (IP: α supPY, e and f). The immunoprecipitation with an anti-Axin antibody showed that the β-catenin/Axin interaction was enhanced upon Imatinib treatment (α Axin: d versus c). Interestingly, the analysis of the Axin-coupled and Axin-uncoupled fractions for β-catenin (α β-cat, c–f) revealed that Y-phospho β-catenin could be immunoprecipitated only from the Axin-free cell lysate supernatants (α p-Y-β-cat: e versus c). 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. Download figure Download PowerPoint In conclusion, these data indicate that Bcr-Abl-induced Y phosphorylation of β-catenin could sterically modify the protein, preventing its recruitment by the Axin/GSK3β. Effect of Bcr-Abl kinase inhibition on β-catenin cellular distribution and nuclear signalling We tested if the β-catenin/TCF signalling could be impaired by inhibition of Bcr-Abl kinase activity (Figure 6A). In Ku812 cells treated for 2 h with DMSO or Imatinib, β-catenin protein levels were unchanged (α β-cat: a versus b). Cleavage products of β-catenin became detectable after 16 h of exposure to Imatinib (α Δβ-cat: c), whereas total levels of Bcr-Abl (α Bcr-Abl), Axin (α Axin) and TCF4 (α TCF4) were unaffected. Imatinib-induced β-catenin cleavage was associated with reduced levels of pro-caspase-3 (α pro-caspase-3) and blocked by the irreversible caspase-3 inhibitor Z-DEVD-fmk (α Δβ-cat: e compared to c). Figure 6.Imatinib reduces nuclear levels of Y-phospho β-catenin by impairing its TCF4-related transcr
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