Bcr is a negative regulator of the Wnt signalling pathway

Abstract

The Wnt signalling pathway can activate transcription of genes such as c-myc through beta-catenin. Here, we describe the protein breakpoint cluster region, Bcr, as a negative regulator of this pathway. Bcr can form a complex with beta-catenin and negatively regulate expression of c-Myc. Knockdown of Bcr by short interfering RNA relieves the block and activates expression of c-Myc. Expression of Bcr in the human colon carcinoma cell line HCT116, which has a high level of endogenous beta-catenin, leads to reduced c-Myc expression. The negative effect is exerted by the amino terminus of Bcr, which does not harbour the kinase domain. Bcr-Abl, the oncogene protein expressed in chronic myelogenous leukaemia (CML), does not bind to beta-catenin. It phosphorylates Bcr in the first exon sequence on tyrosines, which abrogates the binding of Bcr to beta-catenin. The inhibitor of the Bcr-Abl tyrosine kinase, STI-571 or Gleevec, a drug against CML, reverses this effect. Our data contribute to the understanding of Bcr as a tumour suppressor in the Wnt signalling pathway, as well as in CML.

Figure 1

Interaction of Bcr with β-catenin. (A) Schematic diagrams of β-catenin, BcrWT, BcrNT, BcrΔNT and Bcr-Abl^p210 constructs used. Bcr is a serine/threonine (S/T) kinase with four tyrosine phosphorylation sites. GEF is a guanine nucleotide exchange factor and GAP is a GTPase-activating protein. Bcr–Abl is a tyrosine (Y) kinase and a fusion protein with various breakpoints of Bcr at 902 or 927. p210 indicates the molecular mass (kDa); SH2 and SH3 are Src-homology domains; PXXP is the proline-rich region; NIM is the nuclear import region; DB is a DNA-binding domain; FAB is an actin binding site; NEX is the nuclear export region. Horizontal bars mark binding regions of the proteins indicated. TA denotes transactivator. (B) T98G is a glioblastoma cell line. Immunoprecipitation (IP) and subsequent immunoblot (IB) were carried out with antibodies as indicated, and with an irrelevant antibody as a control. IgG heavy and light chains (H, L) and molecular mass in kDa are indicated as markers. (C) Same as (B) except using an A549 human lung carcinoma cell line. Protein expression levels were analysed in direct lysates (DL) by IB. (D) (Top) Co-IP of endogenous Bcr with β-catenin from cytoplasmic (cyt) and nuclear (nuc) extracts from human embryonic kidney (HEK)293 cells. IP, IB and DL were carried out as indicated. Cyclin D1 served as a nuclear marker and heatshock protein HSP 56 as a cytoplasmic marker. (E) The plasmids coding for BcrWT, BcrNT and BcrΔNT were transfected into HEK293 cells; pcDNA3 is an empty plasmid control. IPs were carried out with anti-β-catenin, followed by IB with anti-Bcr (N-20 and C-20 together). Protein expression controls are shown below. (F) A Myc-tagged β-catenin wild type and a point mutant of β-catenin (K435A) were expressed ectopically in HEK293 cells. Co-IP and IB were carried out as indicated (top). Protein expression controls are shown below.

Figure 2

Effect of Bcr on gene expression. (A) Human embryonic kidney (HEK)293 cells or the human colon carcinoma cell line HCT116 was transfected with TOPflash and its control FOPflash (0.5 μg each) together with plasmid coding for β-catenin (1 μg) and BcrWT, BcrNT and BcrΔNT (1, 3, 4 and 7 μg each). Luciferase activity was determined by spectrophotometer. The error bars were obtained from four independent experiments. (B) BcrNT effect on c-Myc expression. HCT116 cells were transfected with plasmid DNA for protein expression. For immunoblot of direct lysates (IB of DL), a mixture of two antibodies against Bcr (N-20 and C-20) was used. (C) The effect of Bcr on expression of the c-Myc was analysed by means of short interfering RNA (siRNA) oligonucleotides. HEK293 cells were transfected with 50 or 100 nM of Bcrspecific siRNA duplexes and an irrelevant interleukin-12 (IL-12) siRNA as a control, which downregulated IL-12 but not Bcr messenger RNA. IB with DL are shown as indicated. (D) Reporter assay, as in (A), with HEK293 cells stimulated by LiCl with or without siRNA against Bcr or the control, as in (C).

Figure 3

Tyrosine phosphorylation regulates Bcr and β-catenin complex formation. (A) Human embryonic kidney (HEK)293 cells were transfected with Bcr-Abl^p210-expressing construct and treated for co-immunoprecipitation (Co-IP), immunoblotting (IB) and direct lysates (DL), as indicated, using Bcr antibodies (N-20), which would also recognize Bcr–Abl. (B) Cells as shown in (A) were treated with STI-571 (2 μM, 1 h). IB was carried out with phosphotyrosinespecific antibodies (PY). For control, Bcr–Abl and Bcr proteins are shown after IB with a Bcr-specific antibody (N-20) in DL. (C) Complex formation regulated by Bcr–Abl kinase. Cells as in (A) were treated with STI-571, fractionated as in Fig 1D and then IP and IB were carried out using Bcr antibodies (C-20). Cell fractionation was controlled by antibodies against HSP 56 and cyclin D1. (D) Human chronic myelogenous leukaemia (CML) cell analysis. The human patient cell line MEG-01 expressing endogenous Bcr and Bcr-Abl^p210 was used to detect the phosphorylated forms of proteins by P-Y antibodies in the absence or presence of STI-571 (2 μM, 24 h). Bcr was detected by C-20 antibodies. (E) Co-IP of Bcr (C-20 antibodies) with β-catenin in MEG-01 cells in the presence or absence of STI-571.

Figure 4

Model on Bcr effects on β-catenin. Negative regulation is indicated by bars and positive regulation by arrows. Dotted lines indicate downregulation of c-Myc protein levels by Bcr (Mahon et al, 2003). The figure depicts an unstimulated cell (left), Bcr–Abl-expressing tumour cell (middle) and STI-571 effect (right). The thick arrows indicate strong activation of c-Myc. For more details, see text.