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Association of Bcr-Abl with the Proto-oncogene Vav Is Implicated in Activation of the Rac-1 Pathway

Bassermann, Florian, Jahn, Thomas, Miething, Cornelius, Seipel, Petra, Bai, Ren-Yuan, Coutinho, Sunita, Peschel, Christian, Duyster, Justus, Tybulewicz, Victor L.·2002·Journal of Biological Chemistry
DOI 10.1074/jbc.m112397200
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CC-BY · From the PDF
FIG. 1 A , a C-terminal Vav fragment binds to kinase-active Bcr-Abl in yeast. *, growth under tryptophan and leucine selection reflecting the transfection efficiency; † , growth under additional histidine selection reflecting the specificity of interaction. B , the Vav/F46 fragment binds directly to Bcr-Abl in vitro , Bcr-Abl was in vitro translated and S 35 -labeled. 50 𝛍 l of the translation mix was allowed to autophosphorylate and was then incubated with 5 𝛍 g of GST-Vav/F46 or GST alone as a control. Input ( right lane ) and bound fractions on GST-Vav/F46 ( middle lane ) and GST as control ( left lane ) were resolved by SDS-PAGE and visualized by autoradiography.
FIG. 1 A , a C-terminal Vav fragment binds to kinase-active Bcr-Abl in yeast. *, growth under tryptophan and leucine selection reflecting the transfection efficiency; † , growth under additional histidine selection reflecting the specificity of interaction. B , the Vav/F46 fragment binds directly to Bcr-Abl in vitro , Bcr-Abl was in vitro translated and S 35 -labeled. 50 𝛍 l of the translation mix was allowed to autophosphorylate and was then incubated with 5 𝛍 g of GST-Vav/F46 or GST alone as a control. Input ( right lane ) and bound fractions on GST-Vav/F46 ( middle lane ) and GST as control ( left lane ) were resolved by SDS-PAGE and visualized by autoradiography.CC-BY · From the PDF
FIG. 2 Vav interacts with Bcr-Abl and v-Abl in vivo. A , a temperature-sensitive mutant of Bcr-Abl ( TS Bcr-Abl ) was transiently transfected into 293 cells. TSBcr-Abl-transfected 293 cells were kept at 39 ° C(restrictive temperature) or shifted to 32 ° C(permissive temperature) for 4 h. Cell lysates were run on SDS-PAGE and immunoblotted with an anti-phosphotyrosine antibody (PY20) ( upper panel ) or an anti-Abl antibody (8E9) ( lower panel ). B , cell lysates from 1 × 10 7 TSBcr-Abl-transfected 293 cells kept at the restrictive (39 ° C) or permissive (32 ° C) temperature were immunoprecipitated with an anti-Vav antibody or a rabbit anti-mouse ( R 𝛂 M ) antibody as control. Protein complexes were washed thoroughly and resolved by SDS-PAGE followed by immunoblotting with anti-Vav antibody ( upper panel ), anti-phosphotyrosine antibody ( anti-ptyr , middle panel ), or anti-Abl antibody ( lower panel ). C , lysates of 5 × 10 6 K562 cells ( left panel ), Lama84 cells ( middle panel ), and MO7E overexpressing Bcr-Abl ( right panel ) were immunoprecipitated with anti-Vav antibody or rabbit anti-mouse antibody as control. Bound fractions were resolved by SDS-PAGE and immunoblotted with anti-Vav antibody. D , 293 cells were transiently cotransfected with v-Abl or Bcr-Abl together with Flag-tagged full-length Vav ( Vav ). Cell lysates were prepared and immunoprecipitated with rabbit anti-mouse antibody as control or anti-Flag antibody to precipitate Flag-tagged Vav. Precipitated proteins ( upper left and lower left panels ) and lysates ( right panel ) were run on SDS-PAGE and visualized with anti-Abl and anti-Flag antibodies as indicated.
FIG. 2 Vav interacts with Bcr-Abl and v-Abl in vivo. A , a temperature-sensitive mutant of Bcr-Abl ( TS Bcr-Abl ) was transiently transfected into 293 cells. TSBcr-Abl-transfected 293 cells were kept at 39 ° C(restrictive temperature) or shifted to 32 ° C(permissive temperature) for 4 h. Cell lysates were run on SDS-PAGE and immunoblotted with an anti-phosphotyrosine antibody (PY20) ( upper panel ) or an anti-Abl antibody (8E9) ( lower panel ). B , cell lysates from 1 × 10 7 TSBcr-Abl-transfected 293 cells kept at the restrictive (39 ° C) or permissive (32 ° C) temperature were immunoprecipitated with an anti-Vav antibody or a rabbit anti-mouse ( R 𝛂 M ) antibody as control. Protein complexes were washed thoroughly and resolved by SDS-PAGE followed by immunoblotting with anti-Vav antibody ( upper panel ), anti-phosphotyrosine antibody ( anti-ptyr , middle panel ), or anti-Abl antibody ( lower panel ). C , lysates of 5 × 10 6 K562 cells ( left panel ), Lama84 cells ( middle panel ), and MO7E overexpressing Bcr-Abl ( right panel ) were immunoprecipitated with anti-Vav antibody or rabbit anti-mouse antibody as control. Bound fractions were resolved by SDS-PAGE and immunoblotted with anti-Vav antibody. D , 293 cells were transiently cotransfected with v-Abl or Bcr-Abl together with Flag-tagged full-length Vav ( Vav ). Cell lysates were prepared and immunoprecipitated with rabbit anti-mouse antibody as control or anti-Flag antibody to precipitate Flag-tagged Vav. Precipitated proteins ( upper left and lower left panels ) and lysates ( right panel ) were run on SDS-PAGE and visualized with anti-Abl and anti-Flag antibodies as indicated.CC-BY · From the PDF
Image 4
CC-BY · From the PDF
FIG. 4 The Vav SH2 domain is required but not sufficient to mediate the complex with Bcr-Abl in K562 cells. Lysates of 5 × 10 6 K562 cells were incubated with GST-Vav/F46 and GST-Vav/ F46R696L (SH2-mutant) ( A ) or GST-Vav-SH2 ( B ). Input and bound fractions were resolved by SDS-PAGE. Proteins were visualized by immunoblotting with anti-Abl antibody and by Amido Black staining as indicated. C , 293 cells were transiently transfected with Xpress ® -tagged F46 lacking the C-terminal SH3 domain ( F46 𝚫 CSH3 ) or F46 with a proline-to-alanine substitution of 4 proline residues and a deletion of the C-terminal SH3 domain (F 46AA 𝚫 CSH3 ). Cell lysates were prepared and incubated with GST-F46CSH3. Input and bound fractions were resolved by SDS-PAGE. Proteins were visualized by immunoblotting with anti-Xpress ® antibody and by Amido Black staining as indicated.
FIG. 4 The Vav SH2 domain is required but not sufficient to mediate the complex with Bcr-Abl in K562 cells. Lysates of 5 × 10 6 K562 cells were incubated with GST-Vav/F46 and GST-Vav/ F46R696L (SH2-mutant) ( A ) or GST-Vav-SH2 ( B ). Input and bound fractions were resolved by SDS-PAGE. Proteins were visualized by immunoblotting with anti-Abl antibody and by Amido Black staining as indicated. C , 293 cells were transiently transfected with Xpress ® -tagged F46 lacking the C-terminal SH3 domain ( F46 𝚫 CSH3 ) or F46 with a proline-to-alanine substitution of 4 proline residues and a deletion of the C-terminal SH3 domain (F 46AA 𝚫 CSH3 ). Cell lysates were prepared and incubated with GST-F46CSH3. Input and bound fractions were resolved by SDS-PAGE. Proteins were visualized by immunoblotting with anti-Xpress ® antibody and by Amido Black staining as indicated.CC-BY · From the PDF
FIG. 5 The Vav SH2 and SH3 domain and a proline-rich region are required for the Vav/Bcr-Abl complex in vivo. 293 cells were transiently transfected with wild-type Bcr-Abl or kinase-defective BcrAbl ( KDBcr-Abl ) together with Flag-tagged full-length Vav ( Vav ), Flagtagged full-length Vav with a mutation in the SH2 domain ( VavR696L ), or Flag-tagged full-length Vav with a proline-to-alanine substitution of 4 proline residues and a deletion of the C-terminal SH3 domain ( VavAA 𝚫 CSH3 ). Cell lysates were prepared and immunoprecipitated ( IP ) with rabbit anti-mouse ( R 𝛂 M ) antibody as control ( upper panel ) or anti-Flag antibody to precipitate Flag-tagged Vav ( middle two panels ). Precipitated proteins and lysates ( lower panel ) were run on SDS-PAGE and visualized with anti-Abl and anti-Flag antibodies as indicated.
FIG. 5 The Vav SH2 and SH3 domain and a proline-rich region are required for the Vav/Bcr-Abl complex in vivo. 293 cells were transiently transfected with wild-type Bcr-Abl or kinase-defective BcrAbl ( KDBcr-Abl ) together with Flag-tagged full-length Vav ( Vav ), Flagtagged full-length Vav with a mutation in the SH2 domain ( VavR696L ), or Flag-tagged full-length Vav with a proline-to-alanine substitution of 4 proline residues and a deletion of the C-terminal SH3 domain ( VavAA 𝚫 CSH3 ). Cell lysates were prepared and immunoprecipitated ( IP ) with rabbit anti-mouse ( R 𝛂 M ) antibody as control ( upper panel ) or anti-Flag antibody to precipitate Flag-tagged Vav ( middle two panels ). Precipitated proteins and lysates ( lower panel ) were run on SDS-PAGE and visualized with anti-Abl and anti-Flag antibodies as indicated.CC-BY · From the PDF
FIG. 6 Bcr-Abl utilizes Vav to activate Rac-1. 293 cells were transiently transfected with Rac-1 and empty vectors ( ø ); Rac-1, WT-Bcr-Abl, or KD-Bcr-Abl and empty vector ( Bcr-Abl, KD-Bcr-Abl ); Rac-1, Bcr-Abl, and Vav/F46 as a dominant-negative; or Rac-1, Bcr-Abl, and Vav/ F46AA 𝚫 CSH3. The amount of DNA was equal in all transfections. 72 h after transfection, lysates were prepared from 1 × 10 7 starved cells. A GST pull-down assay with the GST-p21-binding domain of PAK, which specifically precipitates Rac and Cdc42 in their GTP-bound form (43), was done in duplicates. Bound fractions were resolved by SDS-PAGE and analyzed by immunoblotting with a monoclonal anti-Rac antibody, a monoclonal anti-Abl antibody, and an anti-tag antibody to detect Flag-tagged Vav mutants as indicated. The amount of GST-PAK was analyzed by Coomassie staining of the gel. A representative experiment is shown that was done independently three times with similar results.
FIG. 6 Bcr-Abl utilizes Vav to activate Rac-1. 293 cells were transiently transfected with Rac-1 and empty vectors ( ø ); Rac-1, WT-Bcr-Abl, or KD-Bcr-Abl and empty vector ( Bcr-Abl, KD-Bcr-Abl ); Rac-1, Bcr-Abl, and Vav/F46 as a dominant-negative; or Rac-1, Bcr-Abl, and Vav/ F46AA 𝚫 CSH3. The amount of DNA was equal in all transfections. 72 h after transfection, lysates were prepared from 1 × 10 7 starved cells. A GST pull-down assay with the GST-p21-binding domain of PAK, which specifically precipitates Rac and Cdc42 in their GTP-bound form (43), was done in duplicates. Bound fractions were resolved by SDS-PAGE and analyzed by immunoblotting with a monoclonal anti-Rac antibody, a monoclonal anti-Abl antibody, and an anti-tag antibody to detect Flag-tagged Vav mutants as indicated. The amount of GST-PAK was analyzed by Coomassie staining of the gel. A representative experiment is shown that was done independently three times with similar results.CC-BY · From the PDF
FIG. 7 Overexpression of Vav does not enhance the transformation potential of Bcr-Abl in NIH3T3 fibroblasts. A , immunoblot analysis showing the expression of Vav and Bcr-Abl proteins in stable NIH3T3 cell lines used in B . NIH3T3 cells were transfected with the pcDNA 3.1-Flag-Vav construct containing a Zeocin ® selection marker or the empty vector, respectively. Zeocin ® -resistant clones were picked, expanded, and retrovirally transduced with a Mig-Bcr-Abl construct. B , the indicated NIH3T3 cell lines were plated into soft agar, and colonies were photographed after 2 weeks.
FIG. 7 Overexpression of Vav does not enhance the transformation potential of Bcr-Abl in NIH3T3 fibroblasts. A , immunoblot analysis showing the expression of Vav and Bcr-Abl proteins in stable NIH3T3 cell lines used in B . NIH3T3 cells were transfected with the pcDNA 3.1-Flag-Vav construct containing a Zeocin ® selection marker or the empty vector, respectively. Zeocin ® -resistant clones were picked, expanded, and retrovirally transduced with a Mig-Bcr-Abl construct. B , the indicated NIH3T3 cell lines were plated into soft agar, and colonies were photographed after 2 weeks.CC-BY · From the PDF
Image 9
CC-BY · From the PDF
FIG. 8 Reduced transformation of Vav H11546 / H11546 compared with Vav ÷ / ÷ bone marrow by Bcr-Abl. 1 × 10 5 bone marrow cells from wild-type ( Vav + / + ) or Vav knock-out ( Vav -/ -) mice retrovirally transduced with empty vector ( Mig ) or Bcr-Abl ( p210 ) were plated in bone marrow medium as described under ' Materials and Methods. ' As a further control, growth of cells in the presence of 1 ng/ml IL-3 was measured. Bars represent the numbers of cells after 10 days of culture. Data represent means ± standard deviation of an experiment done in triplicate. Similar results were obtained in three independent experiments.
FIG. 8 Reduced transformation of Vav H11546 / H11546 compared with Vav ÷ / ÷ bone marrow by Bcr-Abl. 1 × 10 5 bone marrow cells from wild-type ( Vav + / + ) or Vav knock-out ( Vav -/ -) mice retrovirally transduced with empty vector ( Mig ) or Bcr-Abl ( p210 ) were plated in bone marrow medium as described under ' Materials and Methods. ' As a further control, growth of cells in the presence of 1 ng/ml IL-3 was measured. Bars represent the numbers of cells after 10 days of culture. Data represent means ± standard deviation of an experiment done in triplicate. Similar results were obtained in three independent experiments.CC-BY · From the PDF

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Cited by

Metadata

Authors
Bassermann, F., Jahn, T., Miething, C., Seipel, P., Bai, R.-Y., Coutinho, S., Peschel, C., Duyster, J., & Tybulewicz, V. L.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2002-04
Volume
277
Issue
14
Pages
12437-12445
ISSN
0021-9258
DOI
10.1074/jbc.m112397200
Cited by
47
References
0
Language
en
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