c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis - PubMed (original) (raw)
c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis
Olivia Furstoss et al. EMBO J. 2002.
Abstract
The mechanism by which the ubiquitously expressed Src family kinases regulate mitogenesis is not well understood. Here we report that cytoplasmic tyrosine kinase c-Abl is an important effector of c-Src for PDGF- and serum-induced DNA synthesis. Inactivation of cytoplasmic c-Abl by the kinase-inactive Abl-PP-K(-) (AblP242E/P249E/K290M) or by microinjection of Abl neutralizing antibodies inhibited mitogenesis. The kinase-inactive SrcK295M induced a G(1) block that was overcome by the constitutively active Abl-PP (AblP242E/P249E). Conversely, the inhibitory effect of Abl-PP-K(-) was not compensated by Src. c-Src-induced c-Abl activation involves phosphorylation of Y245 and Y412, two residues required for c-Abl mitogenic function. Finally, we found that p53 inactivation and c-myc expression, two cell cycle events regulated by Src during mitogenesis, also implied c-Abl: c-Abl function was dispensable in cells deficient in active p53 and inhibition of c-Abl reduced mitogen-induced c-myc expression. These data identify a novel function of cytoplasmic c-Abl in the signalling pathways regulating growth factor-induced c-myc expression and we propose the existence of a tyrosine kinase signalling cascade (PDGFR/c-Src/c-Abl) important for mitogenesis.
Figures
Fig. 1. Subcellular localization of Abl and Abl-PP. (A) Schematic representation of Abl and Abl-PP. Abl-PP contains a point mutation in the linker region between the SH2 domain and the catalytic sequence, replacing two prolines with two glutamates (P242E/P249E), and resulting in constitutive activity of Abl. The myristoylation site (\/\/), SH2, SH3, linker, catalytic and C-terminal sequences are indicated. (B) Immunostaining of Abl and Abl-PP expressed in fibroblasts. NIH 3T3 cells were transiently transfected with Abl and Abl-PP, and fixed and stained for Abl expression by indirect immunofluorescence. Shown is a representative example of images that were obtained after digital restoration (Huygens software) and visualized using shadow projection (Imaris software) as described in Materials and methods. (C) Abl and Abl-PP levels in various subcellular fractions. Lysates of NIH 3T3 cells overexpressing Abl and Abl-PP were fractionated into cytoplasmic/membrane (membrane+cyto) and nuclear (Nuc) fractions as described in Materials and methods. Equal quantities of proteins were subjected to SDS–PAGE and the Abl level was assessed by western blotting using 24-21 monoclonal antibody. The position of Abl and the fraction used are indicated.
Fig. 2. Abl-PP-K–, but not Abl-PP, inhibits PDGF-induced DNA synthesis. NIH 3T3 cells plated onto coverslips were transiently transfected with the indicated Abl-PP or Abl-PP-NLS– mutants for 24 h. After serum starvation, cells were stimulated or not with PDGF in the presence of BrdU. Eighteen hours later, cells were fixed and stained for Abl expression and BrdU incorporation by double immunostaining, and analysed by microscopy. (A) An example of double immunostaining analysis. The white arrowheads mark the position of cells expressing the indicated ectopic protein. (B) Statistical analysis. Shown is the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions. For each coverslip, the percentage of BrdU incorporation was calculated according to the following formula: % of BrdU-positive cells = (number of BrdU-positive cells/number of cells) × 100. For each coverlip, approximately 150–200 cells were counted. The data from four independent experiments have been averaged, and the mean and standard deviation are shown.
Fig. 3. Like SrcK–, Abl-PP-K– G1 block is overcome when inhibiting p53 function. (A) Inactivation of p53 rescues the G1 block induced by kinase-inactive Src (SrcK–) in MEF cells. Cells were plated onto coverslips and transiently co-transfected with SrcK– and the indicated construct. After serum starvation, cells were stimulated or not with PDGF (top panel) or serum (bottom panel) in the presence of BrdU for 18 h. (B) Inactivation of p53 rescues the G1 block induced by kinase-inactive Abl (Abl-PP-K–) in MEF cells. Cells were plated onto coverslips and transiently co-transfected with Abl-PP-K– and the indicated construct, and treated as in (A). (C) Inactivation of p53 rescues the PDGF mitogenic response inhibited by SrcK– or Abl-PP-K– in NIH 3T3 cells. Cells were plated onto coverslips and transiently transfected either with SrcK– or Abl-PP-K– in the presence or absence of E1B, then treated as in (A). Cells were fixed, stained for kinase expression and BrdU incorporation by double immunostaining, and analysed by microscopy. Shown is the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions, as calculated in the legend to Figure 2. The data from three to five independent experiments have been averaged, and the mean and standard deviation are shown.
Fig. 3. Like SrcK–, Abl-PP-K– G1 block is overcome when inhibiting p53 function. (A) Inactivation of p53 rescues the G1 block induced by kinase-inactive Src (SrcK–) in MEF cells. Cells were plated onto coverslips and transiently co-transfected with SrcK– and the indicated construct. After serum starvation, cells were stimulated or not with PDGF (top panel) or serum (bottom panel) in the presence of BrdU for 18 h. (B) Inactivation of p53 rescues the G1 block induced by kinase-inactive Abl (Abl-PP-K–) in MEF cells. Cells were plated onto coverslips and transiently co-transfected with Abl-PP-K– and the indicated construct, and treated as in (A). (C) Inactivation of p53 rescues the PDGF mitogenic response inhibited by SrcK– or Abl-PP-K– in NIH 3T3 cells. Cells were plated onto coverslips and transiently transfected either with SrcK– or Abl-PP-K– in the presence or absence of E1B, then treated as in (A). Cells were fixed, stained for kinase expression and BrdU incorporation by double immunostaining, and analysed by microscopy. Shown is the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions, as calculated in the legend to Figure 2. The data from three to five independent experiments have been averaged, and the mean and standard deviation are shown.
Fig. 3. Like SrcK–, Abl-PP-K– G1 block is overcome when inhibiting p53 function. (A) Inactivation of p53 rescues the G1 block induced by kinase-inactive Src (SrcK–) in MEF cells. Cells were plated onto coverslips and transiently co-transfected with SrcK– and the indicated construct. After serum starvation, cells were stimulated or not with PDGF (top panel) or serum (bottom panel) in the presence of BrdU for 18 h. (B) Inactivation of p53 rescues the G1 block induced by kinase-inactive Abl (Abl-PP-K–) in MEF cells. Cells were plated onto coverslips and transiently co-transfected with Abl-PP-K– and the indicated construct, and treated as in (A). (C) Inactivation of p53 rescues the PDGF mitogenic response inhibited by SrcK– or Abl-PP-K– in NIH 3T3 cells. Cells were plated onto coverslips and transiently transfected either with SrcK– or Abl-PP-K– in the presence or absence of E1B, then treated as in (A). Cells were fixed, stained for kinase expression and BrdU incorporation by double immunostaining, and analysed by microscopy. Shown is the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions, as calculated in the legend to Figure 2. The data from three to five independent experiments have been averaged, and the mean and standard deviation are shown.
Fig. 4. Abl-PP largely restores the PDGF mitogenic response inhibited by SrcK–, while Src does not overcome the Abl-PP-K– inhibition. (A) Src does not rescue the G1 block induced by Abl-PP-K–. NIH 3T3 cells were plated onto coverslips and transiently co-transfected with Abl-PP-K– and the indicated Src construct. After serum starvation, cells were stimulated or not with PDGF in the presence of BrdU for 18 h. (B) Abl-PP largely rescues the G1 block induced by SrcK– and this compensatory effect requires both the SH2 and C-terminal domains of Abl. NIH 3T3 cells were plated onto coverslips and transiently co-transfected either with SrcK– or RasN17 and the indicated construct. After serum starvation, cells were stimulated or not with PDGF in the presence of BrdU for 18 h. Cells were then fixed and stained for Abl (A), Src (B) or Ras (B) expression and BrdU incorporation, as described in Materials and methods. The data are presented as the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions and was calculated as in Figure 2. The data from three to five independent experiments have been averaged, and the mean and standard deviation are shown.
Fig. 5. Cytoplasmic microinjection of antibodies specific for Abl inhibited PDGF mitogenic response in p53+/+, but not in p53–/– MEF cells. (A) Characterization of Abl antibodies. c-Abl that was in vitro translated in reticulocytes lysates in the presence of [35S]methionine was immunoprecipitated (ip) with the indicated antibody, followed by SDS–PAGE and autoradiography (top panel). Endogenous c-Abl was immunoprecipitated (ip) from a MEF cell lysate with the indicated antibody and revealed by western blotting (wb) (middle panel) using the Abl monoclonal 24-21 antibody. The position of Abl is shown. Purified Abl was incubated or not with the indicated antibody and subjected to an in vitro kinase assay using GST–Crk as a substrate (bottom panel). Indicated is the position of radiolabelled GST–Crk as well as the kinase activity obtained relative to the control (c-Abl activity in the absence of any antibody). This data represents one of two independent experiments. (B) Abl antibodies inhibit PDGF mitogenic response in p53+/+ MEF cells. p53+/+ MEF cells plated onto coverslips and made quiescent by serum starvation were microinjected into the cytoplasm with the indicated antibody and stimulated with PDGF in the presence of BrdU for 18 h. Cells were then fixed and stained for BrdU incorporation. The injected antibodies were visualized with a FITC-conjugated antibody. An example of a representative antibody microinjection experiment is shown in the top panel. White arrowheads mark the position of injected cells. The bottom panel represents the percentage of BrdU-positive cells present in injecting and non-injecting cells under the specified conditions, calculated as in Figure 2. The data were obtained from four or five independent experiments (with >80 injected cells for each experiment) that had been averaged, and the mean and standard deviation are shown. (C) Inhibition of endogenous cytoplasmic c-Abl fails to inhibit PDGF mitogenesis in MEF deficient in p53. p53–/– MEF cells plated onto coverslips and made quiescent by serum starvation were microinjected into the cytoplasm with the indicated antibody and stimulated with PDGF in the presence of BrdU for 18 h. Cells were fixed and stained for BrdU incorporation. The injected antibodies were visualized with a FITC-conjugated antibody. An example of a representative antibody microinjection experiment is shown on top panel. White arrows mark the position of injected cells. The bottom panel represents the percentage of BrdU-positive cells present in injecting and non-injecting cells under the specified conditions, as calculated in Figure 2. The data were obtained from four or five independent experiments (with >80 injected cells for each experiment) that had been averaged, and the mean and standard deviation are shown.
Fig. 5. Cytoplasmic microinjection of antibodies specific for Abl inhibited PDGF mitogenic response in p53+/+, but not in p53–/– MEF cells. (A) Characterization of Abl antibodies. c-Abl that was in vitro translated in reticulocytes lysates in the presence of [35S]methionine was immunoprecipitated (ip) with the indicated antibody, followed by SDS–PAGE and autoradiography (top panel). Endogenous c-Abl was immunoprecipitated (ip) from a MEF cell lysate with the indicated antibody and revealed by western blotting (wb) (middle panel) using the Abl monoclonal 24-21 antibody. The position of Abl is shown. Purified Abl was incubated or not with the indicated antibody and subjected to an in vitro kinase assay using GST–Crk as a substrate (bottom panel). Indicated is the position of radiolabelled GST–Crk as well as the kinase activity obtained relative to the control (c-Abl activity in the absence of any antibody). This data represents one of two independent experiments. (B) Abl antibodies inhibit PDGF mitogenic response in p53+/+ MEF cells. p53+/+ MEF cells plated onto coverslips and made quiescent by serum starvation were microinjected into the cytoplasm with the indicated antibody and stimulated with PDGF in the presence of BrdU for 18 h. Cells were then fixed and stained for BrdU incorporation. The injected antibodies were visualized with a FITC-conjugated antibody. An example of a representative antibody microinjection experiment is shown in the top panel. White arrowheads mark the position of injected cells. The bottom panel represents the percentage of BrdU-positive cells present in injecting and non-injecting cells under the specified conditions, calculated as in Figure 2. The data were obtained from four or five independent experiments (with >80 injected cells for each experiment) that had been averaged, and the mean and standard deviation are shown. (C) Inhibition of endogenous cytoplasmic c-Abl fails to inhibit PDGF mitogenesis in MEF deficient in p53. p53–/– MEF cells plated onto coverslips and made quiescent by serum starvation were microinjected into the cytoplasm with the indicated antibody and stimulated with PDGF in the presence of BrdU for 18 h. Cells were fixed and stained for BrdU incorporation. The injected antibodies were visualized with a FITC-conjugated antibody. An example of a representative antibody microinjection experiment is shown on top panel. White arrows mark the position of injected cells. The bottom panel represents the percentage of BrdU-positive cells present in injecting and non-injecting cells under the specified conditions, as calculated in Figure 2. The data were obtained from four or five independent experiments (with >80 injected cells for each experiment) that had been averaged, and the mean and standard deviation are shown.
Fig. 5. Cytoplasmic microinjection of antibodies specific for Abl inhibited PDGF mitogenic response in p53+/+, but not in p53–/– MEF cells. (A) Characterization of Abl antibodies. c-Abl that was in vitro translated in reticulocytes lysates in the presence of [35S]methionine was immunoprecipitated (ip) with the indicated antibody, followed by SDS–PAGE and autoradiography (top panel). Endogenous c-Abl was immunoprecipitated (ip) from a MEF cell lysate with the indicated antibody and revealed by western blotting (wb) (middle panel) using the Abl monoclonal 24-21 antibody. The position of Abl is shown. Purified Abl was incubated or not with the indicated antibody and subjected to an in vitro kinase assay using GST–Crk as a substrate (bottom panel). Indicated is the position of radiolabelled GST–Crk as well as the kinase activity obtained relative to the control (c-Abl activity in the absence of any antibody). This data represents one of two independent experiments. (B) Abl antibodies inhibit PDGF mitogenic response in p53+/+ MEF cells. p53+/+ MEF cells plated onto coverslips and made quiescent by serum starvation were microinjected into the cytoplasm with the indicated antibody and stimulated with PDGF in the presence of BrdU for 18 h. Cells were then fixed and stained for BrdU incorporation. The injected antibodies were visualized with a FITC-conjugated antibody. An example of a representative antibody microinjection experiment is shown in the top panel. White arrowheads mark the position of injected cells. The bottom panel represents the percentage of BrdU-positive cells present in injecting and non-injecting cells under the specified conditions, calculated as in Figure 2. The data were obtained from four or five independent experiments (with >80 injected cells for each experiment) that had been averaged, and the mean and standard deviation are shown. (C) Inhibition of endogenous cytoplasmic c-Abl fails to inhibit PDGF mitogenesis in MEF deficient in p53. p53–/– MEF cells plated onto coverslips and made quiescent by serum starvation were microinjected into the cytoplasm with the indicated antibody and stimulated with PDGF in the presence of BrdU for 18 h. Cells were fixed and stained for BrdU incorporation. The injected antibodies were visualized with a FITC-conjugated antibody. An example of a representative antibody microinjection experiment is shown on top panel. White arrows mark the position of injected cells. The bottom panel represents the percentage of BrdU-positive cells present in injecting and non-injecting cells under the specified conditions, as calculated in Figure 2. The data were obtained from four or five independent experiments (with >80 injected cells for each experiment) that had been averaged, and the mean and standard deviation are shown.
Fig. 6. Quiescent Abl/Arg–/–3T3 fibroblasts exhibit a 4–6 h delay in S phase entry when induced by PDGF or serum. (A) Cells deficient in Abl and Arg exhibit a 4–6 h delay in S phase entry induced by mitogens. Abl/Arg+/+ 3T3 (grey bars) and Abl/Arg–/– 3T3 (black bars) cells were grown on coverslips and made quiescent by serum starvation. Cells were stimulated or not with PDGF (top panel) or serum (bottom panel), as indicated in the presence of BrdU for indicated times, fixed, stained for BrdU incorporation by immunostaining and analysed by microscopy. Shown is the percentage of BrdU-positive cells under the specified conditions, calculated as in Figure 2. These data are representative of two independent experiments. (B) Abl-NLS– restores the actin reorganization induced by PDGF in Abl/Arg–/– 3T3 cells. Top panel: Abl/Arg–/– 3T3 and Abl/Arg–/– 3T3 expressing the indicated constructs grown onto coverslips were made quiescent by serum starvation, stimulated with PDGF for 5 min and fixed. Actin was visualized by staining with rhodamin–phalloidin. Pictures are representative of two independent experiments. Bottom panel: level of immunoprecipitated Abl from the cytoplasmic fraction of the indicated cells. (C) Abl-NLS– restores mitogenesis in Abl/Arg–/– 3T3 cells. Cells were stimulated or not with PDGF (20 ng/ml) or serum (10%), as indicated, in the presence of BrdU for 16 h, then fixed and stained for BrdU incorporation and analysed by microscopy. Shown is the percentage of BrdU-positive cells under the specified conditions, calculated as described in Figure 2. The data from three independent experiments have been averaged, and the mean and standard deviation are shown.
Fig. 6. Quiescent Abl/Arg–/–3T3 fibroblasts exhibit a 4–6 h delay in S phase entry when induced by PDGF or serum. (A) Cells deficient in Abl and Arg exhibit a 4–6 h delay in S phase entry induced by mitogens. Abl/Arg+/+ 3T3 (grey bars) and Abl/Arg–/– 3T3 (black bars) cells were grown on coverslips and made quiescent by serum starvation. Cells were stimulated or not with PDGF (top panel) or serum (bottom panel), as indicated in the presence of BrdU for indicated times, fixed, stained for BrdU incorporation by immunostaining and analysed by microscopy. Shown is the percentage of BrdU-positive cells under the specified conditions, calculated as in Figure 2. These data are representative of two independent experiments. (B) Abl-NLS– restores the actin reorganization induced by PDGF in Abl/Arg–/– 3T3 cells. Top panel: Abl/Arg–/– 3T3 and Abl/Arg–/– 3T3 expressing the indicated constructs grown onto coverslips were made quiescent by serum starvation, stimulated with PDGF for 5 min and fixed. Actin was visualized by staining with rhodamin–phalloidin. Pictures are representative of two independent experiments. Bottom panel: level of immunoprecipitated Abl from the cytoplasmic fraction of the indicated cells. (C) Abl-NLS– restores mitogenesis in Abl/Arg–/– 3T3 cells. Cells were stimulated or not with PDGF (20 ng/ml) or serum (10%), as indicated, in the presence of BrdU for 16 h, then fixed and stained for BrdU incorporation and analysed by microscopy. Shown is the percentage of BrdU-positive cells under the specified conditions, calculated as described in Figure 2. The data from three independent experiments have been averaged, and the mean and standard deviation are shown.
Fig. 7. Src-induced Abl activation involves phosphorylation of two residues required for c-Abl mitogenic function. (A and B) Src activates cytoplasmic Abl via phosphorylation of both Y412 and Y245. (A) HEK-293 cells were transiently transfected with the indicated Abl-NLS– construct together with SrcY527F or empty vector. (B) Quiescent Abl/Arg–/– 3T3 cells expressing Abl-NLS– or Abl-Y245F/Y412F-NLS–, as indicated, were pre-treated for 15 min with vanadate (100 µM) in the presence or absence of PP1 (1 µM), then stimulated or not with PDGF (25 ng/ml) for 10 min. Abl mutants were immunoprecipitated and analysed for Abl protein level and Abl tyrosine phosphorylation content by western blotting using 24-21 and 4G10 antibodies respectively, and for their in vitro kinase activity using GST–Crk as a substrate. The position of Abl, tyrosine phosphorylated Abl and radiolabelled GST–Crk are shown. (A, bottom panel) Src-induced Abl mutant activation obtained from three independent experiments that were averaged; the mean and standard deviation are shown. Kinase activation (fold control) was calculated as described in Materials and methods. Abl activation relative to the control (c-Abl activity from unstimulated cells) obtained in (B) is indicated, and is representative of two independent experiments. (C) Abl mitogenic function requires both Y412 and Y245. MEF cells plated onto coverslips were transiently transfected with the indicated Abl-NLS– construct. After serum starvation, cells were stimulated or not with PDGF in the presence of BrdU for 18 h. Cells were fixed and stained for Abl expression and BrdU incorporation by double immunostaining, and analysed by microscopy. Shown is the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions, calculated as in Figure 2. The data from four independent experiments have been averaged, and the mean and standard deviation are shown.
Fig. 7. Src-induced Abl activation involves phosphorylation of two residues required for c-Abl mitogenic function. (A and B) Src activates cytoplasmic Abl via phosphorylation of both Y412 and Y245. (A) HEK-293 cells were transiently transfected with the indicated Abl-NLS– construct together with SrcY527F or empty vector. (B) Quiescent Abl/Arg–/– 3T3 cells expressing Abl-NLS– or Abl-Y245F/Y412F-NLS–, as indicated, were pre-treated for 15 min with vanadate (100 µM) in the presence or absence of PP1 (1 µM), then stimulated or not with PDGF (25 ng/ml) for 10 min. Abl mutants were immunoprecipitated and analysed for Abl protein level and Abl tyrosine phosphorylation content by western blotting using 24-21 and 4G10 antibodies respectively, and for their in vitro kinase activity using GST–Crk as a substrate. The position of Abl, tyrosine phosphorylated Abl and radiolabelled GST–Crk are shown. (A, bottom panel) Src-induced Abl mutant activation obtained from three independent experiments that were averaged; the mean and standard deviation are shown. Kinase activation (fold control) was calculated as described in Materials and methods. Abl activation relative to the control (c-Abl activity from unstimulated cells) obtained in (B) is indicated, and is representative of two independent experiments. (C) Abl mitogenic function requires both Y412 and Y245. MEF cells plated onto coverslips were transiently transfected with the indicated Abl-NLS– construct. After serum starvation, cells were stimulated or not with PDGF in the presence of BrdU for 18 h. Cells were fixed and stained for Abl expression and BrdU incorporation by double immunostaining, and analysed by microscopy. Shown is the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions, calculated as in Figure 2. The data from four independent experiments have been averaged, and the mean and standard deviation are shown.
Fig. 8. c-Abl regulates PDGF- and serum-induced c-myc expression. (A) Functional interaction between Abl and Myc during PDGF mitogenesis. NIH 3T3 cells were plated onto coverslips and transiently co-transfected with Abl-PP-K– and Myc. After serum starvation, cells were stimulated or not with PDGF in the presence of BrdU for 18 h. Cells were fixed and stained for Abl expression and BrdU incorporation by indirect immunostaining, and analysed by microscopy. Shown is the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions, calculated as in Figure 2. The data from four independent experiments have been averaged, and the mean and standard deviation are shown. (B) c-myc mRNA expression during mitogenesis is reduced in 3T3 cells deficient in both Abl and Arg. After serum starvation, Abl/Arg+/+, Abl/Arg–/– or Abl/Arg–/– 3T3 cells expressing Abl-NLS– were stimulated or not with PDGF (top) or serum (bottom) for 1 h. Total RNA was isolated from all cell types and was blotted and probed with a probe specific for c-myc, or S26 as a control for RNA level. Shown is a nothern blot analysis representative of three independent experiments. The positions of c-myc and S26 mRNA are shown. (C and D) The small molecule inhibitor of Abl, STI 571, reduces serum-induced BrdU incorporation (C) and c-myc mRNA expression (D). NIH 3T3 cells were serum starved and treated with vehicle (DMSO), STI 571 (1–10 µM) or the small PDGF receptor inhibitor AG 1296 (10 µM) 2 h before serum stimulation. Cells were stimulated for 18 h in the presence of BrdU for DNA synthesis analysis (C) or for the indicated times for c-myc expression (D). (C) Shown is the percentage of BrdU incorporation of cells treated with the indicated inhibitor and analysed as described in Materials and methods. The data from four independent experiments have been averaged, and the mean and standard deviation are shown. (D) Top panel represents an example of a northern blot analysis from cells treated with the indicated drug and stimulated or not with 10% serum for 1 h. Bottom panel represents the statistical analysis obtained from cells treated with DMSO (open bars) or 10 µM STI 571 (grey bars). c-myc mRNA level was calculated as described in Materials and methods. The data from three independent experiments have been averaged, and the mean and standard deviation are shown.
Fig. 8. c-Abl regulates PDGF- and serum-induced c-myc expression. (A) Functional interaction between Abl and Myc during PDGF mitogenesis. NIH 3T3 cells were plated onto coverslips and transiently co-transfected with Abl-PP-K– and Myc. After serum starvation, cells were stimulated or not with PDGF in the presence of BrdU for 18 h. Cells were fixed and stained for Abl expression and BrdU incorporation by indirect immunostaining, and analysed by microscopy. Shown is the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions, calculated as in Figure 2. The data from four independent experiments have been averaged, and the mean and standard deviation are shown. (B) c-myc mRNA expression during mitogenesis is reduced in 3T3 cells deficient in both Abl and Arg. After serum starvation, Abl/Arg+/+, Abl/Arg–/– or Abl/Arg–/– 3T3 cells expressing Abl-NLS– were stimulated or not with PDGF (top) or serum (bottom) for 1 h. Total RNA was isolated from all cell types and was blotted and probed with a probe specific for c-myc, or S26 as a control for RNA level. Shown is a nothern blot analysis representative of three independent experiments. The positions of c-myc and S26 mRNA are shown. (C and D) The small molecule inhibitor of Abl, STI 571, reduces serum-induced BrdU incorporation (C) and c-myc mRNA expression (D). NIH 3T3 cells were serum starved and treated with vehicle (DMSO), STI 571 (1–10 µM) or the small PDGF receptor inhibitor AG 1296 (10 µM) 2 h before serum stimulation. Cells were stimulated for 18 h in the presence of BrdU for DNA synthesis analysis (C) or for the indicated times for c-myc expression (D). (C) Shown is the percentage of BrdU incorporation of cells treated with the indicated inhibitor and analysed as described in Materials and methods. The data from four independent experiments have been averaged, and the mean and standard deviation are shown. (D) Top panel represents an example of a northern blot analysis from cells treated with the indicated drug and stimulated or not with 10% serum for 1 h. Bottom panel represents the statistical analysis obtained from cells treated with DMSO (open bars) or 10 µM STI 571 (grey bars). c-myc mRNA level was calculated as described in Materials and methods. The data from three independent experiments have been averaged, and the mean and standard deviation are shown.
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