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First published online 27 March 2007
doi: 10.1242/jcs.03430

Journal of Cell Science 120, 1436-1446 (2007)
Published by The Company of Biologists 2007
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Bcr-Abl induces abnormal cytoskeleton remodeling, beta1 integrin clustering and increased cell adhesion to fibronectin through the Abl interactor 1 pathway

Yingzhu Li1, Nancy Clough1, Xiaolin Sun1, Weidong Yu1, Brian L. Abbott1, Christopher J. Hogan1,2 and Zonghan Dai1,2,3,*,{dagger}

1 Department of Medicine, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA
2 University of Colorado Cancer Center, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA
3 Cell and Developmental Biology, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA


Figure 1
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  		Fig. 1. Abi1 is a downstream substrate of Bcr-Abl tyrosine kinase. (A) Ba/F3 and 32D cells were transduced with empty retroviral vector (control) or retroviral vector expressing p185Bcr-Abl (p185wt). Total lysates from 1x106 cells were analyzed by western blot (WB) using indicated antibodies. (B) Tyrosine phosphorylation of Abi1 in Bcr-Abl-transformed hematopoietic cells. Ba/F3 cells and the Ba/F3 expressing either p185wt or p185K671R were treated with or without 1 µM imatinib mesylate, as indicated. The lysates from 2x107 cells were immunoprecipitated (IP) with anti-Abi1 antibody, followed by western blot analysis using anti-phosphotyrosine ({alpha}-pTyr) antibody (upper panel). The blots were then stripped and reprobed with anti-Abi1 antibody (lower panel). The relative molecular mass (Mr) is indicated and presented as kilodaltons (kd).

 

Figure 2
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  		Fig. 2. Complex formation and membrane translocation of Abi1/WAVE2 in Ba/F3 cells transformed by p185wt. (A) Expression of WAVE2 in Ba/F3 and p185wt-transformed Ba/F3 cells. Total lysates from 2x105 cells were analyzed by western blot using indicated antibodies. (B) Complex formation of Abi1 and WAVE2. The lysates from Ba/F3 cells transduced with either an empty retroviral vector (Ba/F3) or the retroviral vector expressing p185wt were immunoprecipitated with anti-Abi1 antibody, anti-WAVE2 antibody or pre-immune rabbit serum (pre), as indicated. The immunoprecipitates were analyzed by western blot using indicated antibodies. (C) Bcr-Abl-induced membrane translocation of Abi1/WAVE2. Retroviral vectors expressing GFP-Abi1 (a-c) and GFP-WAVE2 (d-f) were introduced into Ba/F3 cells or Ba/F3 cells expressing wild type and the mutant form of p185Bcr-Abl, as indicated. The subcellular localization of GFP-Abi1 and GFP-WAVE2 was analyzed by two-photon confocal microscopy. Nuclei were stained by DAPI (blue). Arrowheads indicate the membrane localization of Abi1 and WAVE2. Bar, 10 µm. (D) Subcellular distributions of Abi1 and WAVE2 in Ba/F3 and the Ba/F3 transformed by p185wt. The Ba/F3 cells and the Ba/F3 cells transformed by p185wt were lysed and fractionated to separate the cytosol (C) and plasma membrane (M). The equal amounts of total lysate (I, input), cytosol, and membrane fractions were separated on SDS-PAGE and analyzed by western blot using antibodies specific to Abi1 and WAVE2, as indicated. To monitor the quality of the fractionation, the blot was also probed with the antibodies to the beta-subunit of IL-3 receptor and {alpha}-tubulin, the proteins known to be in membrane and cytosol, respectively.

 

Figure 3
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  		Fig. 3. Colocalization of Abi1, Bcr-Abl and WAVE2 with an abnormal F-actin-rich structure. Ba/F3 cells expressing p185wt were probed with anti-Abi1 (a-c) and anti-Abl (d-f) antibodies, respectively. This was followed by staining with FITC-conjugated secondary antibody. Cells were then counterstained with TRITC-conjugated phalloidin and DAPI to visualize F-actin and nuclei, respectively. In g-i, the Ba/F3 cells expressing p185wt were transduced with retrovirus expressing GFP-WAVE2 and counterstained by TRITC-conjugated phalloidin and DAPI. Subcellular distribution of Abi1 (a, green), Bcr-Abl (d, green), GFP-WAVE2 (g, green) and F-actin structures (b,e,h, red) were visualized by two-photon confocal microscopy, as indicated by arrowheads. The colocalization of these proteins with abnormal F-actin structure is shown by merged images (c,f,i). Bar, 10 µm.

 

Figure 4
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  		Fig. 4. The expression of p185Bcr-Abl induced integrin clustering. (A) beta1-integrin clustering in p185wt-transformed Ba/F3 cells. The Ba/F3 cells transduced with indicated retroviruses were starved, fixed and stained with FITC-conjugated monoclonal antibody against beta1-integrin. Cells were then stained with DAPI to visualize nuclei. Images were captured by two-photon confocal microscopy and the beta1-integrin clustering is indicated by arrowheads. Bar, 10 µm. (B) Colocalization of beta1-integrin, paxillin and vinculin with abnormal actin-enriched structure. The Ba/F3 cells expressing p185wt were starved, fixed, permeabilized and probed with FITC-conjugated antibodies against beta1-integrin, paxillin, tubulin and vinculin (green), as indicated. The cells were then counterstained with TRITC-conjugated phalloidin (red) and DAPI (blue) to visualize F-actin and nuclei, respectively. Images were captured by two-photon confocal microscopy and the colocalization of integrin, paxillin and vinculin with abnormal actin-enriched structure is shown in merged images, as indicated by arrowheads. Bar, 10 µm.

 

Figure 5
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  		Fig. 5. The PI3K pathway is not required for Bcr-Abl-induced membrane translocation of Abi1. (A) Abi1 does not form a complex with the p85 subunit of PI3K in Ba/F3 cells and the Ba/F3 cells transformed by p185wt. The lysates from 2x107 Ba/F3 cells and the Ba/F3 cells transformed by p185wt were immunoprecipitated by anti-Abi1 (lanes 3 and 4, left panel) and anti-p85 (lanes 5 and 6, right panel) antibodies, respectively. The immunoprecipitates were analyzed by western blotting using the indicated antibodies. A portion of total cell lysates (TCL) equivalent to 2x105 cells (p85 and WAVE2) or 1x106 cells (Abi1) was also analyzed by western blotting to show the expression level of Abi1, p85 and WAVE2 (lanes 1 and 2, left panel). (B) The mutation at tyrosine 407 does not affect the Bcr-Abl-induced membrane translocation of Abi1Y407F. Left panel: lysates from p185wt-transformed Ba/F3 cells (lane 1) and the p185wt-transformed Ba/F3 cells expressing either GFP-Abi1 (lane 2) or GFP-Abi1Y407F (lane 3) were immunoprecipitated by anti-Abi1 antibody and analyzed by western blotting using the antibodies indicated. Right panel: Ba/F3 cells and Ba/F3 cells transformed by p185wt, as indicated, were transduced with retroviruses expressing either GFP-Abi1 or GFP-Abi1Y407F. The subcellular distribution of GFP-fusion proteins was visualized by two-photon confocal microscopy. (C) LY294002 failed to inhibit Bcr-Abl-induced membrane translocation of GFP-Abi1. The p185wt-transformed Ba/F3 cells were transduced with the retrovirus expressing GFP-Abi1. The cells were then left untreated or treated with either 10 µM imatinib or 50 µM LY294002, as indicated, for 8 hours. The cells were fixed and counterstained with TRITC-conjugated phalloidin and DAPI to visualize the F-actin and nuclei, respectively. Images were captured by two-photon confocal microscopy. A color picture is presented in supplementary material Fig. S2B.

 

Figure 6
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  		Fig. 6. The p185{Delta}SH3{Delta}C failed to induce the membrane translocation of Abi1/WAVE2, integrin clustering and abnormal actin remodeling. (A) Ba/F3 cells expressing either p185wt (a,c) or p185{Delta}SH3{Delta}C (b,d) were transduced with the retroviral vectors expressing either GFP-Abi1 (a,b) or GFP-WAVE2 (c,d). The cells were fixed and GFP-fusion proteins were visualized by two-photon confocal microscopy. (B) Ba/F3 cells transduced with either p185wt (e,g) or p185{Delta}SH3{Delta}C (f,h) were fixed and incubated with FITC-conjugated monoclonal antibody specific for beta1-integrin (e,f). The cells were also stained with TRITC-conjugated phalloidin to visualize F-actin (g,h). The nuclei of the cells were stained by DAPI (blue) and the arrowheads indicate the subcellular localization of GFP-Abi1 (a), WAVE2 (c), clustering beta1-integrin (e), as well as abnormal actin-enriched structures (g) in p185wt-transformed cells. Bar, 10 µm.

 

Figure 7
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  		Fig. 7. Expression of Abi1PPLL inhibited Bcr-Abl-induced abnormal actin cytoskeleton remodeling. (A) Schematic diagram of Abi1 and Abi1PPLL. (B) Abi1PPLL is defective in binding to Bcr-Abl. Ba/F3 cells (lane 1) and the Ba/F3 cells expressing p185wt alone (lane 2), p185wt plus HA-tagged Abi1 (lane 3) and p185wt plus HA-tagged Abi1PPLL (lane 4) were lysed and immunoprecipitated with anti-Abl antibody. The immunoprecipitates (middle and lower panels) and 1/50 of total cell lysates used for immunoprecipitation (IP) (Input, upper panel) were analyzed by western blot using indicated antibodies. The HA-tagged Abi1 and Abi1PPLL are indicated by arrowheads, whereas a non-specific band crossreacted with anti-HA antibody is indicated by the arrow (upper panel). The anti-Abl antibody recognized both Bcr-Abl and endogenous c-Abl, as indicated by arrowhead and arrow, respectively (middle panel). (C) Abi1PPLL failed to be tyrosine-phosphorylated by Bcr-Abl. Ba/F3 cells (lane 2) and Ba/F3 cells expressing p185wt alone (lane 1) or p185wt plus HA-tagged Abi1PPLL (lane 3) were lysed and immunoprecipitated with indicated antibodies. The immunoprecipitates were analyzed by western blot using indicated antibodies. (D) Inhibition of Bcr-Abl-induced abnormal actin remodeling by Abi1PPLL. Ba/F3 cells transformed by p185wt were transduced with retroviruses expressing either GFP-Abi1 (a-c) or GFP-Abi1PPLL (d-f). Cells were fixed and stained with TRITC-conjugated phalloidin and DAPI to visualize F-actin (red) and nuclei (blue), respectively. Subcellular localization of GFP-fusion proteins (b,e, green) and F-actin structure (a,d, red) were analyzed by two-photon confocal microscopy. The merged images were also shown (c,f). The arrowheads in panel c indicate abnormal actin-enriched structures that colocalize with GFP-Abi1, whereas arrows in panel f indicate the p185wt-transformed cells that express GFP-Abi1PPLL. An open arrowhead in panel f indicates abnormal actin-enriched structure in a cell that did not express Abi1PPLL. Bar, 10 µm.

 

Figure 8
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  		Fig. 8. Blockade of Abi1 pathway or beta1-integrin function impaired the ability of Bcr-Abl to stimulate cell adhesion to fibronectin. (A) The Abi1 pathway is required for Bcr-Abl-stimulated cell adhesion to fibronectin-coated surfaces. Left panel: Ba/F3 cells and the Ba/F3 cells expressing either p185wt or p185{Delta}SH3{Delta}C were grown in fibroncetin-coated six-well plates (2.5x105/well) for 16 hours. The total cells and the cells that were adherent to fibronectin-coated surfaces were counted and the percentage of adherent cells calculated. The vertical axis shows the percentage of the adherent cells and is expressed as the mean ± s.d. of duplicate wells. Data are representative of two independent experiments. Right panel: the Ba/F3 cells expressing p185wt alone or p185wt plus Abi1PPLL were grown in fibroncetin-coated plates (2.5x105/well) for 16 hours. The total cells and the cells that were adherent to fibronectin-coated surfaces were counted and the percentage of adherent cells calculated. The data represent the mean ± s.d. of triplicate wells. (B) The F-actin-rich structures are enriched in adherent p185wt-transformed Ba/F3 cells. The p185wt-transformed Ba/F3 cells were grown in fibronectin-coated plates with or without fibronectin supplemented in medium (5 µg/ml) for 16 hours. Adherent and non-adherent cells were harvested separately and stained by TRITC-conjugated phalloidin and DAPI to visualize F-actin and nuclei, respectively, by fluorescence microscopy (upper panel). The percentage of the cells containing the F-actin-rich structures (spots) from three randomly selected fields in adherent cells as well as non-adherent cells was statistically calculated and expressed as the mean ± s.d. (lower panel). The data are representative of three independent experiments. (C) Bcr-Abl-stimulated cell adhesion to fibronectin is beta1-integrin dependent. The p185wt-transformed Ba/F3 cells were treated with either Ha2/5 or a control hamster IgM (4 µg/2.5x105 cells) and plated in fibroncetin-coated plates (2.5x105 cells/well) for 16 hours. The total cells and the cells that were adherent to fibronectin-coated surfaces were counted and the percentage of adherent cells calculated. The data represent the mean ± s.d. of triplicate wells and are representative of two independent experiments.

 

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© The Company of Biologists Ltd 2007