Regulation of epidermal growth factor receptor signaling during oxidative stress

Abstract

This thesis described the effects of exposure of cells to oxidative stress,induced by H 2 O 2 ,on the functioning of proteins involved in signal transduction pathways.In addition, H 2 O 2 was chosen as oxidant in order to produce cellular screening assays to measure antioxidant efficacy in preventing the H 2 O 2 -induced modifications of protein functioning. Afamily of kinases that plays a key role in the transduction of extracellular signals into intracellular events is formed by the MAP kinases.1 Moreover,MAP kinases are rapidly activated in response to various extracellular signals,including different types of cellular stress.2-5 Therefore,the effect of H 2 O 2 on the phosphorylation of MAP kinase was investigated and this revealed that exposure of Rat-1 fibroblasts resulted in a transient phosphorylation of p44/p42 MAPK .Subsequently,the H 2 O 2 -induced phosphorylation of p44/p42 MAPK was used as a marker for oxidative stress and the availability of a phospho- specific p44/p42 MAPK antibody provided us the ability to develop a cellular enzyme-linked immunosorbent assay (Cell-ELISA)to measure the phosphorylation of p44/p42 MAPK (chapter 2 ).This assay was subsequently used for the screening of antioxidant efficacy in Rat-1 fibroblasts and in addition,the assay was applicable to test other stresses,such as menadione,cumene hydroperoxide,AMVN and hypoxanthine/xanthine oxidase. Asecond screening assay was developed to measure the internalization of the EGF receptor in 96-well plates (chapter 4 ).Internalization and subsequent degradation of activated EGF receptors,also referred to as receptor downregulation or receptor-mediated endocytosis,results in a reduction of the amount of EGF receptors expressed at the plasma membrane and therefore in a reduction of binding sites for EGF.Another cellular feedback mechanism to attenuate receptor signaling involves the activation of phosphatases.6 Dephosphorylation of the C-terminal Tyr residues of the EGF receptor,for instance, abrogates docking sites for downstream signaling proteins and furthermore,the enzymatic activity of several signaling proteins can be negatively regulated by dephosphorylation. Finally,a third mechanism to regulate EGF-induced signaling is receptor transmodulation, which results in lowered affinity of the receptor for its ligand and in addition,receptor Tyr kinase activity is reduced.7 We decided to investigate the effect of H 2 O 2 on receptor downregulation,because an inhibition of EGF receptor-mediated endocytosis had been described for different forms of cellular stress,8 suggesting that oxidative stress might interfere with this process as well.H 2 O 2 was found to inhibit the internalization of the EGF receptor in HER14 fibroblasts (chapter 3 )and this inhibition was subsequently considered as a marker for oxidative stress.T easily study ligand-induced internalization,a cellular screening assay in 96-well plates was developed,which was partly based on the cellular MAP kinase assay as described in chapter 2 .In this assay,internalization was studied using biotin-conjugated EGF and we showed that the results obtained with this internalization assay were comparable with results obtained with radioactive labeled EGF (chapter 4 ). In conclusion,the newly developed 96-well plate assays are nonradioactive, relatively fast and reliable methods for quantitative detection of changes in phosphorylation of MAP kinases or changes in ligand-induced internalization in 96-well plates.Furthermore, both assays are applicable for the screening of various stress conditions on these processes and for testing a variety of antioxidants in their capacity to prevent or reduce the H 2 O 2 - induced changes in these cellular responses.