An EGF receptor/Ral-GTPase signaling cascade regulates c-Src activity and substrate specificity - PubMed (original) (raw)

An EGF receptor/Ral-GTPase signaling cascade regulates c-Src activity and substrate specificity

T Goi et al. EMBO J. 2000.

Abstract

c-Src is a membrane-associated tyrosine kinase that can be activated by many types of extracellular signals, and can regulate the function of a variety of cellular protein substrates. We demonstrate that epidermal growth factor (EGF) and beta-adrenergic receptors activate c-Src by different mechanisms leading to the phosphorylation of distinct sets of c-Src substrates. In particular, we found that EGF receptors, but not beta(2)-adrenergic receptors, activated c-Src by a Ral-GTPase-dependent mechanism. Also, c-Src activated by EGF treatment or expression of constitutively activated Ral-GTPase led to tyrosine phosphorylation of Stat3 and cortactin, but not Shc or subsequent Erk activation. In contrast, c-Src activated by isoproterenol led to tyrosine phosphorylation of Shc and subsequent Erk activation, but not tyrosine phosphorylation of cortactin or Stat3. These results identify a role for Ral-GTPases in the activation of c-Src by EGF receptors and the coupling of EGF to transcription through Stat3 and the actin cytoskeleton through cortactin. They also show that c-Src kinase activity can be used differently by individual extracellular stimuli, possibly contributing to their ability to generate unique cellular responses.

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Figures

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Fig. 1. Ral signaling modulates tyrosine phosphorylation of Stat3 in PC12 cells. PC12 cells were transiently transfected with either control vector (C), vector expressing the Ral-specific exchange factor Rgr, a constitutively activated Ral allele, RalA72L, or a dominant-negative Ral allele, Ral28N, along with HA-tagged Stat3. After 48 h, the cells were treated with either buffer or IL–6 (10 ng/ml) for 10 min. The tagged Stat3 was then immunoprecipitated and blotted with anti-phosphotyrosine-specific Stat3 antibodies (p–Stat3). Total HA-Stat3 expression (Stat3) in all of the experiments is shown at the bottom. The data represent the results of at least three independent experiments.

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Fig. 2. Ral activates c–Src kinase activity. (A) Top: 293 cells were transiently transfected with control vector (C), or vector encoding Rgr or RalA72L. After 24 h, the cells were serum starved (0.5%) for 12 h and then some cells were treated with EGF for 10 min. The cells were lysed and c–Src was immunoprecipitated. The immunoprecipitates were used in a kinase assay in vitro using denatured enolase as substrate. The labeled reaction was then run on SDS–gels, and exposed to a phosphoimager. The fold increase in enolase phosphorylation compared with control sample (C) is marked on top of the radioactive image. Bottom: data from at least three independent experiments were pooled and plotted with standard errors of the mean indicated. (B) 3Y1(EGFR) cells or 3Y1(EGFR) cells expressing RalA72L were serum starved for 12 h and then some cells were exposed to EGF for 10 min. c–Src was then immunoprecipitated and Src-like activity was assayed in an in vitro kinase assay as described above. Total c–Src levels in cell lysates are depicted at the bottom. The data are representative of two independent experiments. (C) Activated Ras (Ras61L) and Ras effector mutants that preferentially activate Ral-GEFs (Ras12V37G) or Raf (Ras12V35S) were transiently transfected into 293 cells and assayed for c–Src activity as described above. Total c–Src and Ras levels in cell lysates are shown at the bottom. The data are representative of two independent experiments.

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Fig. 2. Ral activates c–Src kinase activity. (A) Top: 293 cells were transiently transfected with control vector (C), or vector encoding Rgr or RalA72L. After 24 h, the cells were serum starved (0.5%) for 12 h and then some cells were treated with EGF for 10 min. The cells were lysed and c–Src was immunoprecipitated. The immunoprecipitates were used in a kinase assay in vitro using denatured enolase as substrate. The labeled reaction was then run on SDS–gels, and exposed to a phosphoimager. The fold increase in enolase phosphorylation compared with control sample (C) is marked on top of the radioactive image. Bottom: data from at least three independent experiments were pooled and plotted with standard errors of the mean indicated. (B) 3Y1(EGFR) cells or 3Y1(EGFR) cells expressing RalA72L were serum starved for 12 h and then some cells were exposed to EGF for 10 min. c–Src was then immunoprecipitated and Src-like activity was assayed in an in vitro kinase assay as described above. Total c–Src levels in cell lysates are depicted at the bottom. The data are representative of two independent experiments. (C) Activated Ras (Ras61L) and Ras effector mutants that preferentially activate Ral-GEFs (Ras12V37G) or Raf (Ras12V35S) were transiently transfected into 293 cells and assayed for c–Src activity as described above. Total c–Src and Ras levels in cell lysates are shown at the bottom. The data are representative of two independent experiments.

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Fig. 3. Inhibition of Ral-GEF or Ras-GEF activities suppresses EGF- but not isoproterenol-induced activation of c–Src. (A) 293 cells were transiently transfected with either control vector or vector encoding dominant-negative RalA28N. After 48 h, cells were serum starved and some cells were treated with buffer, EGF or isoproterenol for10 min. c–Src was immunoprecipitated and assayed in vitro as described in Figure 2. The fold increase in c–Src activity is indicated on top of the radioactive image. Total c–Src levels in cell lysates are shown at the bottom. The data are representative of at least three independent experiments. (B) 3Y1(EGFR) cells or 3Y1(EGFR) cells expressing RalA28N were serum starved and then some cells were treated with EGF for 10 min. c–Src was then immunoprecipitated and assayed in vitro as described in Figure 2. Total c–Src levels in cells lysates are shown below. Below that are immunoblots of these lysates using phospho-specific ERK antibodies. (C) The experiments were performed as in (A) except that Ras17N was substituted for Ral28N. The data are representative of at least three independent experiments.

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Fig. 3. Inhibition of Ral-GEF or Ras-GEF activities suppresses EGF- but not isoproterenol-induced activation of c–Src. (A) 293 cells were transiently transfected with either control vector or vector encoding dominant-negative RalA28N. After 48 h, cells were serum starved and some cells were treated with buffer, EGF or isoproterenol for10 min. c–Src was immunoprecipitated and assayed in vitro as described in Figure 2. The fold increase in c–Src activity is indicated on top of the radioactive image. Total c–Src levels in cell lysates are shown at the bottom. The data are representative of at least three independent experiments. (B) 3Y1(EGFR) cells or 3Y1(EGFR) cells expressing RalA28N were serum starved and then some cells were treated with EGF for 10 min. c–Src was then immunoprecipitated and assayed in vitro as described in Figure 2. Total c–Src levels in cells lysates are shown below. Below that are immunoblots of these lysates using phospho-specific ERK antibodies. (C) The experiments were performed as in (A) except that Ras17N was substituted for Ral28N. The data are representative of at least three independent experiments.

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Fig. 4. Ral activity regulates the c–Src-induced tyrosine phosphorylation of Stat3. (A) 293 cells were transiently transfected with vector control, or vector expressing Ral28N or Ral72L. After 24 h, cells were serum starved for 12 h and some cells were exposed to EGF for 10 min. c–Src was immunoprecipitated and the precipitates were immunoblotted with either anti-Stat3 antibodies (upper lanes) or anti-phosphotyrosine-specific Stat3 antibodies (lower lanes). The data are representative of two independent experiments. (B) 3Y1(EGFR) cells or 3Y1(EGFR) cells expressing either RalA72L or RalA28N were serum starved for 12 h and then some cells were exposed to EGF for 10 min. c–Src was then immunoprecipitated from cell lysates and immunoblotted with anti-phosphotyrosine-specific Stat3 antibodies.

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Fig. 5. Ral activity regulates the tyrosine phosphorylation of cortactin, but not that of Shc or the activation of Erk. (A) 293 cells were transiently transfected with either control vector, RalA72L or RalA28N and then serum starved for 12 h. Some cells were then exposed to EGF for 10 min. Cell lysates were immunoprecipitated with α–cortactin antibodies and then immunoblotted with α–phospho– tyrosine-specific antibodies. The data are representative of two independent experiments. Total cortactin levels in cell lysates are displayed below. (B) 293 cells were treated as in (A) except that cell lysates were immunoprecipitated with α–Shc antibodies and then immunoblotted with anti-phosphotyrosine antibodies. (C) Cells were treated as in (A) except that cell lystates were immunoblotted with α–phospho-specific Erk antibodies.

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Fig. 6. Isoproterenol stimulation leads to Erk activation but not to cortactin or Stat3 tyrosine phosphorylation. 293 cells were serum starved for 24 h and then exposed to either EGF or isoproterenol for 10 min. Top: cortactin was immunoprecipitated and then the immunoprecipitates were blotted with anti-phosphotyrosine-specific antibodies. Middle: c–Src was also immunoprecipitated from the lysates and then blotted with anti-Stat3 antibodies. Bottom: cell lysates were run directly on SDS–gels and then blotted with phospho-specific Erk antibodies. The results are representative of at least two independent experiments.

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Fig. 7. EGF and β–adrenergic receptors activate c–Src by different mechanisms that lead to the tyrosine phosphorylation of different sets of c–Src substrates. EGF receptors, but not β–adrenergic receptors (βAR), activate c–Src by a Ras- and Ral-dependent signaling pathway. Importantly, Ral-induced c–Src activation leads to Stat3 and cortactin tyrosine phosphorylation, but not Shc phosphorylation and Erk activation. In contrast, βAR activation of c–Src leads to Shc but not cortactin or Stat3 tyrosine phosphorylation. Ral can also be activated by Ras-independent pathways, such as through calcium, which would allow c–Src to activate downstream targets without activating Ras and its multiple effectors. Overall, these findings suggest how different receptors may generate specific cellular effects through a common kinase.

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