The
epidermal growth factor receptor (EGFR) is linked to the
phosphatidylinositol 3-kinase (PI3K)/
protein kinase B (AKT) and Raf/
mitogen-activated protein kinase (MAPK)/
extracellular signal-regulated kinase (ERK1/2) signaling pathways. During
brain ischemia/reperfusion, EGFR could be transactivated, which stimulates these intracellular signaling cascades that either protect cells or potentiate cell injury. In the present study, we investigated the activation of EGFR, PI3K/AKT, and Raf/MAPK/ERK1/2 during
ischemia or reperfusion of the brain using the
middle cerebral artery occlusion model. We found that EGFR was phosphorylated and transactivated during both
ischemia and reperfusion periods. During
ischemia, the activity of PI3K/AKT pathway was significantly increased, as judged from the strong phosphorylation of AKT; this activation was suppressed by the inhibitors of EGFR and Zn-dependent
metalloproteinase.
Ischemia, however, did not induce ERK1/2 phosphorylation, which was dependent on reperfusion. Coimmunoprecipitation of Son of sevenless 1 (SOS1) with EGFR showed increased association between the receptor and SOS1 in
ischemia, indicating the inhibitory node downstream of SOS1. The inhibitory phosphorylation site of Raf-1 at Ser259, but not its stimulatory phosphorylation site at Ser338, was phosphorylated during
ischemia. Furthermore,
ischemia prompted the interaction between Raf-1 and AKT, while both the inhibitors of PI3K and AKT not only abolished AKT phosphorylation but also restored ERK1/2 phosphorylation. All these findings suggest that Raf/MAPK/ERK1/2 signal pathway is inhibited by AKT via direct phosphorylation and inhibition at Raf-1 node during
ischemia. During reperfusion, we observed a significant increase of ERK1/2 phosphorylation but no change in AKT phosphorylation. Inhibitors of
reactive oxygen species and
phosphatase and
tensin homolog restored AKT phosphorylation but abolished ERK1/2 phosphorylation, suggesting that the
reactive oxygen species-dependent increase in
phosphatase and
tensin homolog activity in reperfusion period relieves ERK1/2 from inhibition of AKT.