Effect of Hypoxia on Activation of Epidermal Growth Factor Receptor (EGFR) Kinase and Src Kinase in the Cerebral Cortex of Newborn Piglets

Abstract

EGFR kinase and Src kinase activation is associated with cell differentiation and cell death. The present study tests the hypothesis that hypoxia results in increased activation of EGFR kinase as well as Src kinase in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx, n=5) and hypoxic (Hx, n=5) groups. Hypoxia was induced by decreasing FiO2 to 0.07 for 60 min. Cortical cell membranes were prepared and tyrosine phosphorylation of EGFR kinase and Src kinase was determined by Western blot and EGFR kinase and Src kinase activity was determined using immunopurified enzymes. Proteins were separated by 12% SDS-PAGE, probed with anti-phospho (pTyr 1173)-EGFR kinase and anti-phospho (pTyr418)-Src kinase. Activity of EGFR and Src kinase was expressed as pmols/mg protein/ hr. Expression of phosphorylated EGFR kinase was 51.85 ± 1.55 in Nx and 195.00 ± 11.41 in Hx (p< 0.05 vs Nx). The activity of EGFR kinase (pmoles/mg protein/hr) was 4603±155 in Nx and 8493 ± 427 in Hx (p< 0.05 vs Nx). Expression of phosphorylated Src kinase was 111.78± 21.13 in Nx and 234.52 ± 23.82 in Hx (p< 0.05 vs Nx). The activity of Src kinase was 2472.66 ± 75.56 in Nx and 4556.33 ± 358.50 in Hx (p< 0.05 vs Nx). The results demonstrate that cerebral hypoxia leads to increased phosphorylation and increased activity of EGFR kinase and Src kinase. We conclude that the hypoxia results in increased activation of EGFR kinase and Src kinase in the cerebral cortex of newborn piglets. We propose that the increased tyrosine phosphorylation and activation of EGFR kinase and Src kinase is due to NO-mediated inhibition of protein tyrosine phosphatases SH-PTP-1 and SH-PTP-2 during hypoxia. (Supported by NIH-HD-20337 and 38079)