The N-terminal domain of the non-receptor tyrosine kinase ABL confers protein instability and suppresses tumorigenesis

Abstract

Chromosome translocation can lead to chimeric proteins that may become oncogenic drivers. A classic example is the fusion of the BCR activator of RhoGEF and GTPase and the ABL proto-oncogene nonreceptor tyrosine kinase, a result of a chromosome abnormality (Philadelphia chromosome) that causes leukemia. To unravel the mechanism underlying BCR-ABL-mediated tumorigenesis, here we compared the stability of ABL and the BCR-ABL fusion. Using protein degradation, cell proliferation, 5-ethynyl-2-deoxyuridine, and apoptosis assays, along with xenograft tumor analysis, we found that the N-terminal segment of ABL, which is lost in the BCR-ABL fusion, confers degradation capacity that is promoted by SMAD-specific E3 ubiquitin protein ligase 1. We further demonstrate that the N-terminal deletion renders ABL more stable and stimulates cell growth and tumorigenesis. The findings of our study suggest that altered protein stability may contribute to chromosome translocation-induced cancer development.

Keywords: ABL kinase; ABL proto-oncogene non-receptor tyrosine kinase; BCR activator of RhoGEF and GTPase; Philadelphia chromosome; SMAD-specific E3 ubiquitin protein ligase 1 (Smurf1); chromosome rearrangement; chromosome translocation; leukemia; oncogene; protein chimera; protein degradation; proteolysis; ubiquitin.

Figure 1.

ABL is preferentially degraded by the ubiquitin proteasome system. A, degradation kinetics of ectopically expressed ABL and BCR-ABL fusion, analyzed by a protein expression shut-off assay. HEK-293 cells transfected with FLAG-ABL or GST-tagged BCR-ABL were treated with 100 μg/ml cycloheximide to shut off protein synthesis. Samples collected at various time points were then processed for immunoblotting with FLAG or GST antibody (upper). Similar amounts of protein extracts were used, as ascertained by blotting with anti-GAPDH (lower). All experiments were performed at least three times. B, protein stability of endogenous ABL and BCR-ABL in TF-1 or K562 cells, respectively. Degradation kinetics of endogenous ABL or BCR-ABL were determined as in A except that ABL antibody was used. C and D, quantification of the data in A and B, respectively. The results are derived from more than three experiments. E and F, ABL turnover is mediated by the proteasome. Degradation kinetics of ectopically expressed (E) or endogenous (F) ABL were analyzed in the absence or presence of the proteasome inhibitor MG132. G and H, quantification of the data in E and F, respectively. I, ABL is modified by ubiquitin. HEK-293 cells bearing FLAG-ABL with or without MG132 treatment were lysed and immunoprecipitated with FLAG antibody. Immunoprecipitates were then subjected to Western blotting with anti-ubiquitin to detect ubiquitylated ABL species (upper). The amounts of ABL and GAPDH control in protein extracts were also assessed by Western blotting (lower).

Figure 2.

The degradation signal lies in the N-terminal domain of ABL. A, domain structures of ABL, BCR-ABL (p210), and ABL^△45. The first 45 amino acids of ABL are lost in the BCR-ABL fusion and deleted in ABL^△45. B, the N-terminal region is critical for ABL degradation. HEK-293 cells expressing FLAG-tagged ABL or ABL^△45 were processed as described in Fig. 1A, to determine the protein stability of ABL and ABL^△45. C, the N-terminal fragment of ABL confers protein instability. The degradation kinetics of GFP and ABL⁴⁵-GFP fusion were analyzed by the protein expression shut-off assay. D, the degradation induced by the N-terminal ABL fragment requires the proteasome. The stability of ABL⁴⁵-GFP was analyzed in the absence or presence of MG132. E–G, quantification of the data in B–D, respectively.

Figure 3.

Ubiquitin ligase Smurf1 promotes ABL turnover via its N-terminal domain. A–D, Smurf1-stimulated ABL degradation requires its N-terminal region. Cells expressing endogenous ABL (A) or FLAG-tagged ABL (B) or ABL^△45 (C) or GST-tagged BCR-ABL (D) were transfected with a vector plasmid or the plasmid bearing Myc-tagged Smurf1. Levels of ABL derivatives, Myc-Smurf1, and GAPDH were assessed by Western blotting with various antibodies. E, Smurf1 promotes the ubiquitylation of ABL but not the ABL^△45 mutant. Ubiquitylation profiles of ABL and ABL^△45 with or without ectopic expression of Myc-Smurf1 were determined as described in Fig. 1E. F, the association between Smurf1 and ABL requires its N-terminal region. Cells expressing Myc-Smurf1 and/or FLAG-tagged ABL derivatives were harvested and lysed. Protein extracts were incubated with IgG beads coated with FLAG antibody. The immunoprecipitates were resolved by SDS-PAGE and processed for Western blotting using Myc or FLAG antibody as indicated. The inputs are shown in the lower panels. G, Smurf1-promoted ABL degradation requires lysine residues in the N-terminal region of ABL. Cells expressing FLAG-tagged WT ABL or the indicated lysine mutants were transfected with the plasmid with or without Myc-Smurf1. The effects on the expression of ABL derivatives were determined. H, four lysine residues in the N-terminal region are important for ABL degradation. The degradation kinetics of the indicated ABL derivatives were analyzed by the protein expression shut-off assay. I, quantification of the data in H.

Figure 4.

Effects of ABL derivatives on cell growth and survival. A, ABL derivatives promote TF-1 cell proliferation in the presence of GM-CSF. Cell proliferation was determined by hemocytometer measurements on the indicated days. B, effects of ABL derivatives on TF-1 cell growth in the presence of GM-CSF. The EdU assay was employed to measure DNA synthesis after cells were incubated for 24 h. EdU-positive cells were indicated by red fluorescence. 4[prime],6-Diamidino-2-phenylindole (DAPI) stains for nuclei (blue). Bar, 50 μm. C, quantification of the data in B. The percentage of EdU-positive nuclei, relative to the total number of nuclei, is shown. p values determined by student's t test are indicated, **, p < 0.01. D, BCR-ABL and ABL^△45, but not ABL, promote TF-1 cell proliferation in the absence of GM-CSF. Cell proliferation was performed as described in A except that cells were cultured in GM-CSF-free medium. E, effects of ABL derivatives on TF-1 cell death in the absence of GM-CSF. TF-1 cells expressing ABL derivatives were cultured in GM-CSF-free medium for 48 h and then stained with annexin V and propidium iodide to determine the apoptosis rate. F, quantification of the data in E.

Figure 5.

Tumor formation in mouse xenografts expressing ABL derivatives. A, tumors dissected from xenograft mice. TF-1 cells expressing ABL derivatives indicated were subcutaneously implanted into nude mice (n = 6 for each group). Tumor size was monitored by Vernier caliper. Different termination times were indicated, to prevent the tumors from growing too large. B, tumor growth curve in xenograft mice expressing ABL derivatives. Beginning at 7 days postinjection, tumor size was calculated every other day. BCR-ABL-expressing mice were sacrificed earlier because tumors grew quickly.