Activation of B-Raf and regulation of the mitogen-activated protein kinase pathway by the G(o) alpha chain - PubMed (original) (raw)

Activation of B-Raf and regulation of the mitogen-activated protein kinase pathway by the G(o) alpha chain

V Antonelli et al. Mol Biol Cell. 2000 Apr.

Free PMC article

Abstract

Many receptors coupled to the pertussis toxin-sensitive G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the alpha chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Galpha(o) to regulate MAPK activity by transient expression of the activated mutant Galpha(o)-Q205L in Chinese hamster ovary cells. Galpha(o)-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Galpha(o)-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Galpha(o) stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Galpha(o). Moreover, Galpha(o)-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Galpha(o) can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of Galpha(o) caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Galpha(o) induces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway.

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Figures

Figure 1

Effect of activated Gαo on MAPK activity. CHO cells transfected in 6-cm dishes were placed for 18 h in 0.5% FCS medium and incubated for 5 min in the absence or presence of 100 ng/ml EGF. MAPK activity was determined as described in MATERIALS AND METHODS. (A) Endogenous p42 MAPK activity in cells transfected with 1.8 μg of Gαo-wild-type (WT) or Gαo-Q205L and, where indicated, 1.8 μg of EGF receptor (EGFR). The data represent means ± SE of two to four independent experiments performed in triplicate. (B) HA-p44 MAPK activity in cells transfected with 1.4 μg of HA-p44 MAPK, 1.4 μg of Gαo-WT or Gαo-Q205L and, where indicated, 1.4 μg of EGFR. The data are means ± SE of two independent experiments performed in duplicate. (C) Endogenous p42 MAPK activity in cells transfected with 1.8 μg of wild-type (WT) or activated (QL and RC) Gαi2, Gαi3, and Gαq subunits, 1.4 μg of Gβ1 and Gγ2 chains, and, where indicated, 1.8 μg of EGFR. Means ± SE from two independent experiments performed in duplicate are given. When nontransfected CHO cells were incubated with 100 ng/ml EGF for 5 min, the values of endogenous p42 MAPK activity in the absence and presence of the growth factor were 4097 ± 429 and 4278 ± 620 cpm, respectively (n = 4). Immunoblots show the expression of wild-type or activated Gαo (A) and HA-p44 MAPK (B) in cells transfected with different cDNAs. Similar results were obtained in two additional experiments.

Figure 2

MAPK stimulation by activated Gαo involves PKC and PI3K. (A–C and E) CHO cells in 6-cm dishes were transfected with 1.8 μg of EGFR and 1.8 μg of either empty vector or Gαo-Q205L and assayed for endogenous p42 MAPK activity as described in the legend to Figure 1. (A) Effect of PMA pretreatment on MAPK activity. Cells were incubated for 18 h in 0.5% FCS medium containing 1 μM PMA before stimulation with 100 ng/ml EGF or 1 μM PMA for 5 min. The data represent means ± SE of two independent experiments performed in duplicate. (B, C, and E) Effects of PKC or PI3K inhibitors on MAPK activity. Cells were serum starved for 18 h and incubated for 15 min with 1 μM GF 109203X (B), the indicated concentrations of wortmannin (C), or 20 μM LY 294002 (E) before stimulation with 100 ng/ml EGF for 5 min. The data are means ± SE of four to nine observations. Additional assays showed that GF 109203X completely abolished the increase in p42 MAPK activity induced by short exposure to PMA. (D) Effect of wortmannin on Akt phosphorylation. Nontransfected CHO cells grown in 6-cm dishes were serum starved for 18 h, incubated with the indicated concentrations of wortmannin for 15 min, and stimulated with 20 ng/ml PDGF for 10 min. Phosphorylated Akt was detected by immunoblot analysis of cell lysates with specific antibodies as described in MATERIALS AND METHODS. The molecular mass (kilodaltons) of a marker protein is shown to the left, and the position of phosphorylated Akt is indicated by an arrow. Data are representative of two similar experiments.

Figure 3

Effect of activated Gαo on EGF receptor tyrosine phosphorylation. CHO cells in 10-cm dishes were transfected with 5 μg of EGFR and 5 μg of empty vector, Gαo-WT, or Gαo-Q205L, serum starved for 18 h, and stimulated with 100 ng/ml EGF for 5 min. Immunoprecipitation (IP) with anti-EGFR antibodies and immunoblot analysis of the immunoprecipitates with anti-phosphotyrosine or anti-EGFR antibodies were performed as described in MATERIALS AND METHODS. The molecular masses (kilodaltons) of marker proteins are shown to the left, and the position of the EGFR is indicated by arrows. Data are representative of three similar experiments.

Figure 4

Effect of activated Gαo on Ras-stimulated MAPK activity. CHO cells transfected in 6-cm dishes were serum starved for 18 h and assayed for MAPK activity as described in the legend to Figure 1. (A) Endogenous p42 MAPK activity in cells transfected with Gαo-WT or Gαo-Q205L (1.8 μg) and either empty vector or Ras-Q61L (2.2 μg). (B) HA-p44 MAPK activity in cells transfected with HA-p44 MAPK (1.4 μg), Gαo-WT or Gαo-Q205L (1.4 μg), and either empty vector or Ras-Q61L (1.4 μg). Data represent means ± SE of two separate experiments performed in duplicate. Expression of Ras-Q61L in the absence of exogenous Gαo gave results similar to those obtained in cells transfected with the wild-type subunit.

Figure 5

Activated Gαo stimulates B-Raf activity. (A) Expression of Raf proteins in CHO cells. Raf-1, A-Raf, and B-Raf were immunoprecipitated from lysates of nontransfected cells and detected by immunoblotting with specific antibodies as described in MATERIALS AND METHODS. The molecular masses (kilodaltons) of marker proteins are shown to the left, and the positions of Raf-1, A-Raf, and B-Raf migration are indicated by arrows. (B–D) CHO cells transfected in 10-cm dishes were serum starved for 18 h and assayed for endogenous Raf-1, A-Raf, and B-Raf activities as described in MATERIALS AND METHODS. (B) Effect of EGFR stimulation on Raf activity. Cells were transfected with 5 μg of EGFR and incubated for 5 min in the absence or presence of 100 ng/ml EGF. Data represent means ± SE of two to three independent experiments performed in triplicate. *, Significantly higher than basal (p < 0.01 by unpaired t test). In similar experiments carried out with nontransfected CHO cells, basal B-Raf activity (106,370 ± 7071 cpm) was not significantly increased by EGF (102,468 ± 5748 cpm) (n = 4). (C) Effect of activated Gαo on B-Raf activity. Cells transfected with 5 μg of empty vector, Gαo-WT or Gαo-Q205L and, where indicated, 5 μg of EGFR were incubated for 5 min in the absence or presence of 100 ng/ml EGF. Values are means ± SE of three independent experiments performed in triplicate. EGF induced a significant increase in B-Raf activity in all transfected cells (p < 0.01 by unpaired t test). *, Significantly higher than values from cells transfected with vector or Gαo-WT (p < 0.01 by unpaired t test). (D) Effects of N17-Ras expression, PMA pretreatment, and PKC or PI3K inhibitors on Gαo-mediated B-Raf activation. Cells were transfected with 4 μg of empty vector or Gαo-Q205L, and, where indicated, 8 μg of N17-Ras. PMA (1 μM), wortmannin (25 nM), GF 109203X (1 μM), and LY 294002 (20 μM) were used as described in the legend to Figure 2. Results are expressed as percentages of inhibition of the increase induced by Gαo-Q205L and represent the mean ± SE of two experiments in duplicate determinations. Expression of N17-Ras, PMA pretreatment, and the various inhibitors did not modify B-Raf activity in cells transfected with vector. Additional assays showed that both PMA pretreatment and GF 109203X completely abolished the increase in B-Raf activity induced by 5-min stimulation with 1 μM PMA (∼50%).

Figure 6

Activated Gαo does not affect MAPK stimulation by FGF receptors. (A) Effect of FGF on Raf activity in nontransfected CHO cells grown in 10-cm dishes. (B) Effect of Gαo on FGF-stimulated MAPK activity in CHO cells transfected in 6-cm dishes with 1.4 μg of HA-p44 MAPK and 1.4 μg of empty vector, Gαo-WT, or Gαo-Q205L. Cells were serum starved for 18 h before stimulation with 40 ng/ml FGF for 5 min. Raf-1, A-Raf, B-Raf, and HA-p44 MAPK activities were determined as described in the legends to Figures 1 and 5. Data are means ± SE for duplicate samples from two independent experiments.

Figure 7

B-Raf activation by dopaminergic D2 receptors. CHO cells transfected in 6-cm dishes were serum starved for 18 h and either left untreated or stimulated with 100 nM dopamine (DA) for 5 or 20 min. B-Raf activity was assayed as described in the legend to Figure 5. Data shown represent means ± SE of two independent experiments performed in duplicate. (A) Cells were transfected with 1.8 μg of dopaminergic D2 receptor (D2R), 1.8 μg of wild-type or activated Gαi2 and Gαi3 subunits, or 1.4 μg of Gβ1 and Gγ2 chains. *, Significantly higher than basal (p < 0.05 by unpaired t test). (B) Cells were transfected with 1.8 μg of D2R and, where indicated, 1.8 μg of Gαo-WT. *, Significantly higher than values obtained in the absence of Gαo-WT (p < 0.01 by unpaired t test). (C) Cells were transfected with 1.4 μg of D2R and, where indicated, 2.8 μg of N17-Ras. PMA pretreatment and incubation with wortmannin were as described in the legend to Figure 5. Results are expressed as percentages of the increase induced by DA in control cells.

Figure 8

Effects of N17-Ras expression and PMA pretreatment on dopamine-stimulated MAPK activity. CHO cells transfected in 6-cm dishes were serum starved for 18 h and stimulated with 100 nM DA for 5 or 20 min. (A) Cells were transfected with 1.4 μg of D2R and 2.8 μg of either empty vector or N17-Ras. (B) Cells were transfected with 1.4 μg of D2R and pretreated with PMA as described in the legend to Figure 5. Endogenous p42 MAPK activity was measured as described in the legend to Figure 1. Data are means ± SE of triplicate determinations. Similar results were obtained in at least one additional experiment.

Figure 9

Model of signaling pathways regulating Raf and MAPK in CHO cells. The EGF receptor (EGFR) activates Ras and B-Raf, whereas the FGF receptor (FGFR) activates Ras, Raf-1, and A-Raf. Activated Gαo potentiates MAPK activation by Ras and EGFR but not FGFR. The G protein subunit stimulates the kinase activity of B-Raf by a Ras-independent and PKC- and PI3K-dependent mechanism, which is insufficient to promote MAPK activation. It is proposed that this mechanism can efficiently stimulate the MAPK pathway in the presence of a B-Raf-activating signal from Ras or the EGFR. The dopaminergic D2 receptor (D2R), which couples to both Gi and Go, appears to stimulate MAPK through Ras activation of Raf-1. This effect is presumably mediated by G protein βγ complexes and, possibly, Gαi subunits. Upon stimulation of this receptor, the Ras-independent and PKC- and PI3K-dependent activation of B-Raf induced by Gαo does not produce a MAPK response.

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Activation of B-Raf and regulation of the mitogen-activated protein kinase pathway by the G(o) alpha chain - PubMed (original) (raw)