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First published online 2 August 2005
doi: 10.1242/jcs.02491

Journal of Cell Science 118, 3717-3726 (2005)
Published by The Company of Biologists 2005
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Abl tyrosine kinase regulates a Rac/JNK and a Rac/Nox pathway for DNA synthesis and Myc expression induced by growth factors

Anthony Boureux, Olivia Furstoss*, Valérie Simon and Serge Roche{ddagger}

CRBM, CNRS FRE2593, 1919 route de Mende, 34293 Montpellier Cedex 05, France



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  		Fig. 1. Abl regulates PDGF-induced Rac activation. (A) Activation of PDGFRß in Abl/Arg+/+3T3 and Abl/Arg–/–3T3 cells. In vitro kinase assay of the immunoprecipitated PDGFRß from indicated cell lysates as described in Materials and Methods. Autophosphorylated PDGFR (32P-PDGFR) is shown. The level of the immunoprecipitated receptor is also shown and was assessed by western blotting with the {alpha}PRC antibody. (B) PDGF-induced Rac activation is impaired in Abl/Arg–/–3T3 cells. (C) Abl-NLS– expression restored PDGF-induced Rac activation in Abl/Arg–/–3T3 cells (left panel). Cells were stimulated with PDGF for 0-240 minutes. GTP-bound Ras, Rho Rac and Cdc42 (shown as GTPase-GTP) were precipitated as described in Materials and Methods and detected with specific antibody. Total GTPase level is also shown. (C, right panel) Western blotting of immunoprecipitated Abl from indicated cell-lysates using Ab-3 antibody. The level of Abl-NLS– expressed in Abl/Arg–/–3T3 was comparable with the endogenous level of Abl in Abl/Arg+/+3T3 cells.

 


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  		Fig. 2. RacV12 alleviates the G1 block induced by Abl-PP-K–. (A) RacV12 rescues the G1 block induced by Abl-PP-K–. Quiescent NIH3T3 transfected with the indicated constructs were stimulated or not with PDGF or 10% fetal calf serum as indicated for 18 hours in the presence of BrdU. (B) RacV12 does not rescue the G1 block induced by dominant interfering mutants of Shc and Stat3. Quiescent NIH3T3 transfected with the indicated constructs were stimulated or not with PDGF and proceeded as in A. Cells were fixed and stained for BrdU incorporation and ectopic protein expression as described in Materials and Methods. The mean±s.d. of the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions is shown.

 


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  		Fig. 3. RacV12 also restores mitogenesis in cells with loss of Abl kinase activity. (A) RacV12 rescues the mitogenic defect in Abl/Arg–/–3T3 cells. Quiescent cells that were infected with wild-type (mock) or retrovirus as shown, were stimulated or not with indicated mitogen for 16 hours in the presence of BrdU. (Top panels) The PDGF responses; (bottom panels) the serum (10% fetal calf serum) responses. (B) RacV12 rescues the mitogenic inhibition induced by the Abl kinase inhibitor STI 571. NIH3T3 quiescent cells that were infected with wild-type (mock) or RacV12-expressing retrovirus were treated or not with various concentrations of STI 571 1 hour before stimulating cells with serum for 18 hours in the presence of BrdU as indicated. The mean±s.d. of the percentage of BrdU-positive cells under the specified conditions is shown.

 


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  		Fig. 4. The Rac mitogenic rescue implicates both a JNK and a Nox activity. (A) Structure-function analysis of the Rac mitogenic rescue. (B) The Rac mitogenic rescue is sensitive to inhibitors of JNK and Nox activities. (C) The biological defect of RacL61/N52L is compensated by activators of the JNK pathway. (D) The biological defect of the RacL61/D38N is compensated by an activator of the Nox pathway. Quiescent NIH3T3 transfected with Abl-PP-K– together or not with indicated constructs were stimulated or not with mitogen (20 ng/ml PDGF or 10% serum as shown) in the presence of BrdU and proceeded as in Fig. 2. In panel B, cells were treated with 0.5 µM SP600125, 20 µM SB203580, 0.5 µM DPI or 10,000 U catalase as indicated before stimulation. The mean±s.d. of the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions is shown. (E) Activity of various RacL61 mutants used in this study. NIH3T3 infected with control or indicated RacL61 retroviruses were incubated overnight with 0.5% serum and assayed for Rac activity as described in Fig. 1. Total Rac level and Rac activity (Rac-GTP) is shown. (F) In vivo activity of DA-MKK4 and 7 used in this study. Cells were transiently transfected with indicated DA-MKK and Flag-tagged JNK1 constructs as indicated. Immunoprecipitated Flag-JNK was assayed for in vitro activity as described in Materials and Methods. The level of phosphorylated Jun (32P-Gst-Jun) is shown.

 


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  		Fig. 5. Co-activation of a JNK and a Nox pathway mimic the Rac mitogenic rescue. Active DA-MKK4, DA-MKK7 (A) and p47SD (B) partially rescue the Abl-PP-K– G1 block. Quiescent NIH3T3 transfected with Abl-PP-K– together or not with the indicated constructs were stimulated or not with PDGF in the presence of BrdU and proceeded as in Fig. 2. The mean±s.d. of the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions is shown.

 


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  		Fig. 6. Abl regulates PDGF-induced JNK and Nox activation. (A) Abl regulates PDGF-induced JNK activation. (Top panels) The level of active MAPK (pMAPK) and JNK (pJNK) from cells stimulated with PDGF (0-30 minutes). The level of MAPKs and JNK1 is also shown. (Bottom panels) The level of Jun and pS63-Jun (pJun) from indicated cell-lysates. (B,C) Abl regulates PDGF-induced Noxo2 (p47phox) membrane translocation. (B) Immunofluorescence localization of p47phox-GFP, actin structure and nuclei from indicated cells stimulated or not with PDGF as shown. (C) Statistic analysis of p47 membrane recruitment upon PDGF stimulation. Indicated quiescent cells transfected with p47phox-GFP were stimulated with PDGF for 5 minutes and the percentage of cells with p47 membrane localization was counted. The mean±s.d. (n=5) of the percentage of cells that exhibited p47-GFP at the membrane is shown.

 


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  		Fig. 7. JNK and Nox signal to Myc for the induction of DNA synthesis. (A) RacV12 rescues the Myc induction defect in Abl/Arg–/– cells. Indicated quiescent cells infected with wild-type (mock) or indicated retroviruses were stimulated or not with PDGF for 1 hour. The level of Myc mRNA was quantified by real-time quantitative RT-PCR as described in Materials and Methods. The ratio of Myc mRNA in stimulated and non-stimulated cells is shown (Myc induction). (B) Myc induction is sensitive to inhibitors of JNK and Nox activities. Quiescent NIH3T3 treated with DMSO (control) or indicated inhibitors (10 µM or 10 mM for NAC) were stimulated or not for 1 hour with PDGF. Myc mRNA was assessed by northern blotting as described in Materials and Methods. % Myc mRNA level=[Myc mRNA level]/[Myc RNA level of control quiescent cells]. Myc rescues the G1 block induced by DN-MKK4 (C) and p67V204A (D). Quiescent NIH3T3 transfected with indicated constructs were stimulated or not with PDGF in the presence of BrdU and proceeded as in Fig. 1B. The mean±s.d. of the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions is shown.

 


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  		Fig. 8. A model of how Abl mediates mitogenesis in response to growth factors. Growth factor (GF)-induced SFK activation allows phosphorylation of Abl in the cytoplasm for increased catalytic activity. Abl then operates on a Rac/JNK and Rac/Nox pathway for Myc expression, a transcription factor required for induction of DNA synthesis. JNK and Nox may participate in Myc induction through mRNA stabilization and/or gene transcription. Note that Abl substrates for Rac activation are currently unknown. SFK-induced Myc implicates additional substrates, including Stat3 and Shc. The Ras signaling cascade may participate in mitogenesis through Myc protein stabilization and expression of other genes.

 

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© The Company of Biologists Ltd 2005