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. 2001 Sep 1;21(17):6459-66.
doi: 10.1523/JNEUROSCI.21-17-06459.2001.

Requirement of Ras for the activation of mitogen-activated protein kinase by calcium influx, cAMP, and neurotrophin in hippocampal neurons

N Iida  1 , K Namikawa, H Kiyama, H Ueno, S Nakamura, S Hattori
Affiliations

Requirement of Ras for the activation of mitogen-activated protein kinase by calcium influx, cAMP, and neurotrophin in hippocampal neurons

N Iida et al. J Neurosci. .

Abstract

Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term potentiation both in vitro and in living animals. MAP kinase is activated in response to a broad range of stimuli, including calcium influx through NMDA receptor and L-type calcium channel, cAMP, and neurotrophins. To investigate the role of Ras in the activation of MAP kinase and cAMP response element-binding protein (CREB) in hippocampal neurons, we inhibited Ras function by overexpressing a Ras GTPase-activating protein, Gap1(m), or dominant negative Ras by means of adenovirus vectors. Gap1(m) expression almost completely suppressed MAP kinase activation in response to NMDA, calcium ionophore, membrane depolarization, forskolin, and brain-derived neurotrophic factor (BDNF). Dominant negative Ras also showed similar effects. On the other hand, Rap1GAP did not significantly inhibit the forskolin-induced activation of MAP kinase. In contrast to MAP kinase activation, the inactivation of Ras activity did not inhibit significantly NMDA-induced CREB phosphorylation, whereas BDNF-induced CREB phosphorylation was inhibited almost completely. These results demonstrate that Ras transduces signals elicited by a broad range of stimuli to MAP kinase in hippocampal neurons and further suggest that CREB phosphorylation depends on multiple pathways.

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Figures

Fig. 1.
Fig. 1.
Inhibition of Ras/MAP kinase pathway by Adex-Gap1m (A) and pEFBOS-MycGap1m (B) in PC12 cells. A, PC12 cells were infected with Adex-LacZ or Adex-Gap1m at moi 100 and incubated for 2 d. Cells were stimulated with the indicated concentrations of NGF for 5 min. The relative amount of active Ras and phosphorylated MAP kinase (pMAPK) was analyzed as described in Materials and Methods. B, PC12 cells were transfected with pEFBOS-MycGap1m or pEFBOS-MycΔGap1m and incubated for 2 d. Cells were treated with 100 ng/ml NGF for another 2 d and immunostained for Myc-tagged proteins (myc) or neurofilaments (NF). Arrows indicate cells expressing Myc-tagged proteins.
Fig. 2.
Fig. 2.
Inhibition of Ras activation by Adex-Gap1m (A) and MAP kinase activation by pEFBOS-MycGap1m(B) in hippocampal neurons. A, Hippocampal neurons at DIV 6 were infected with Adex-LacZ (top panel) or with either Adex-LacZ or Adex-Gap1m (bottom panel) at moi 500. Two days later, the cells were stimulated with 10 μm NMDA or 100 ng/ml BDNF for 5 min, and the relative amount of active Ras (indicated by arrows) was determined as in Figure 1. B, Cells were transfected with pEFBOS-MycGap1m and incubated for 2 d. Cells were then stimulated with BDNF (100 ng/ml) or NMDA (10 μm) for 5 min and immunostained with anti-Myc (myc) or anti-phospho-MAP kinase (pMAPK) antibodies. Myc-positive cells and negative cells are indicated by arrows andarrowheads, respectively.
Fig. 3.
Fig. 3.
Inhibition of MAP kinase activation by NMDA, membrane depolarization, ionomycin, and BDNF by Adex-Gap1m and AdRasY57. Hippocampal neurons (DIV 6) were infected with Adex-LacZ (LacZ), Adex-Gap1m (Gap1m), or AdRasY57 (dnRas) at moi 500 and incubated for 2 d. The cells were then stimulated with NMDA (100 μm), KCl (60 mm), ionomycin (0.5 μg/ml), or BDNF (100 ng/ml) for 5 min. The cell lysates (corresponding to 3 × 104 cells) were subjected to Western blots using antibodies against the phosphorylated form of MAP kinase (pMAPK), MEK (pMEK), and CREB (pCREB), as well as the antibody recognizing both dephosphorylated and phosphorylated forms of MAP kinase (total MAP kinase). Representative data of three independent experiments are shown.
Fig. 4.
Fig. 4.
Dose dependency of NMDA and BDNF and the effect of Adex-Gap1m infection on the phosphorylation of MAP kinase and CREB. Neurons were infected with Adex-LacZ (gray bars) or Adex-Gap1m(black bars) at moi 500 and stimulated with various concentrations of BDNF or NMDA for 5 min as in Figure 3(n = 3). The relative amounts of phosphorylated MAP kinase and CREB are quantitated using NIH Image software and shown with SDs.
Fig. 5.
Fig. 5.
The effect of PD098059 and Adex-Gap1m on the activation of MAP kinase and CREB. Cells (DIV 6) were infected with Adex-LacZ (LacZ) or Adex-Gap1m (Gap1m) and incubated for 2 d. Cells pretreated with either 50 μm PD098059 or a control vehicle solution for 30 min were stimulated with 1, 3, and 10 μm NMDA for 5 min (n = 3). The phosphorylation of MAP kinase and CREB was examined as in Figure 3 (top panel) and quantitatively shown (bottom panel).
Fig. 6.
Fig. 6.
The effect of a MEK inhibitor, U0126, on NMDA- and BDNF-induced CREB phosphorylation. Cells (DIV 6) pretreated with 20 or 40 μm U0126 for 30 min were stimulated with 10 μm NMDA or 10 μg/ml BDNF for 5 min (n = 3). The phosphorylation of MAP kinase and CREB was examined as in Figure 5.
Fig. 7.
Fig. 7.
The effect of Adex-Gap1m on the phosphorylation of MAP kinase and CREB in NMDA-stimulated hippocampal neurons. Neuronal cells (DIV 6) were infected with Adex-LacZ (▪) or Adex-Gap1m (●) and stimulated with NMDA (10 μm) for the indicated periods of time. The cell lysates were subjected to Western blots as in Figure 3, and the relative amounts of phosphorylated MAP kinase and CREB were quantitated (n = 3).
Fig. 8.
Fig. 8.
Inhibition of Rap1 activation by Adex-Rap1GAP.A, COS7 cells grown on 60 mm dishes (1 × 106 cells per plate) were transfected with pCXN2-FLAG-Rap1 (Mochizuki et al., 1999) or pCXN2-FLAG-Rap1 plus pCAGGS-C3G (Gotoh et al., 1995) and infected with Adex-LacZ or Adex-Rap1GAP at moi 10. Two days later, the active Rap1 and total Rap1 were analyzed as described in Materials and Methods. B, Cortical neurons (2 × 107 cells per 90-mm-diameter dishes) at DIV 4 were infected with either Adex-LacZ or Adex-Rap1GAP (expressing Myc-tagged Rap1GAP) at 10 moi, and the cells were further cultured for 2 d. Cells were stimulated with a mixture of forskolin (100 μm) and TPA (1 μm) and then KCl depolarized (60 mm) for 15 min. Cell lysates from three dishes were combined, and the relative amounts of active Rap1 in the lysates were determined by pull-down assay. The expression of Myc-Rap1GAP, the activation of MAP kinase (pMAPK), and the amount of total MAP kinase were analyzed as in Figure 3. Representative data of two independent experiments are shown.
Fig. 9.
Fig. 9.
The effect of Adex-Gap1m or Adex-Rap1GAP on forskolin-induced activation of MAP kinase and CREB. Cells (DIV 6) were infected with the indicated adenoviruses at moi 500 and incubated for 2 d. Cells were stimulated with 100 μm forskolin for various times specified in the figure. At each point, activation of MAP kinase and CREB was examined as in Figure 3.
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