No abstract

Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H(2)O(2)) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the H(2)O(2)-induced inhibition of internalization, a screening assay was developed to measure the internalization in 96-well plates. In this assay, cells are exposed to biotin-conjugated EGF and the amount of internalized EGF is detected with horseradish peroxidase-conjugated streptavidin. We show that the results obtained by this new assay are comparable with those from internalization studies performed with radioactive labeled EGF. Therefore, the cellular internalization assay as presented here is a reliable method to measure EGF receptor internalization. Moreover, because elaborate processing of the cells is not required, the assay is a relatively fast and inexpensive method to study ligand-induced internalization in 96-well plates and thereby is suitable for large-scale screening of compounds or conditions interfering with this internalization.

Recently, we demonstrated that hydrogen peroxide (H2O2) inhibits the internalization of the epidermal growth factor (EGF) receptor and the EGF-induced mono-ubiquitination of EGF receptor pathway substrate clone #15 (Eps15) in fibroblasts. In addition, it was suggested that EGF receptor internalization might be inhibited by H2O2 by inhibition of ubiquitination of proteins involved in endocytosis. Here, we show that H2O2 also inhibits the poly-ubiquitination of the EGF receptor in fibroblasts. Furthermore, recovery of the cells resulted in re-establishment of ubiquitination of both the EGF receptor and Eps15 and coincided with restoration of internalization of those receptors that had bound EGF in the presence of H2O2. In addition, EGF receptor internalization was inhibited by the sulphydryl reagent N-ethylmaleimide (NEM), indicating that intact SH groups might be required for receptor-mediated endocytosis. Furthermore, H2O2 rapidly induced an increase in the cellular ratio of GSSG:GSH (oxidized glutathione:reduced glutathione) and removal of H2O2 resulted in a fast restoration of the ratio of GSSG:GSH. Therefore, these results suggest a relation between the inhibition of internalization ubiquitination and an increase in GSSG:GSH ratio, which strengthens the hypothesis that H2O2 inhibits EGF receptor internalization by an inhibition of ubiquitination of proteins involved in EGF receptor-mediated endocytosis.

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that are activated by phosphorylation and are involved in the cellular response to various physiologic stimuli and stress conditions. Because MAP kinases play an important role in cellular functioning, a screening assay to determine the phosphorylation of MAP kinase upon various conditions was desirable. Therefore, we have developed a cellular enzyme-linked immunosorbent assay (Cell-ELISA), in which the phosphorylated forms of p42MAPK and p44MAPK are detected. We show that in this Cell-ELISA, MAP kinase becomes phosphorylated in a dose- and time-dependent manner under proliferative or stress conditions. This dose- and time-dependent phosphorylation agrees with observations using classical gel-electrophoresis and Western blotting techniques. Furthermore, we show that our assay is applicable to different cell types and that serum-starvation is not required for detection of an increase in MAP kinase phosphorylation. From these experiments, it is concluded that the Cell-ELISA is a reliable and fast method for quantitative detection of the phosphorylation, and thus the activation, of MAP kinase. This assay is applicable for a large-scale screening of the effectivity of biological or chemical compounds that modulate the cellular response to physiologic stimuli or stress through phosphorylation and activation of MAP kinase.

The 85-kDa Ca2+-dependent cytosolic phospholipase A 2 (cPLA2) is of special interest since it selectively hydrolyzes glycerophospholipids at the sn-2 position, generating arachidonic acid, which plays an important rote in various cellular processes. In many cell types, cPLA 2 can be activated by various stimuli, such as cytokines and growth factors. Growth factors stimulate cPLA 2 activity by activating the mitogenactivated protein kinase (MAPK) cascade, resulting in a phosphorylation of cPLA 2 by MAPK on Ser-505. cPLA 2 is fully activated when it is translocated to membranes in a Ca2+-dependent manner. Localization studies of endogenous cPLA 2 revealed a punctate pattern randomly distributed throughout the cytoplasm. Recent evidence suggests that reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H202), function as intracellular second messengers. Furthermore, exogenously H202 could mimic growth factor-induced tyrosine phosphorylation in A431 cells. Recently we demonstrated that epidermal growth factor (EGF) induced a twofold cPLA 2 activation in Her t4 fibroblasts. This activation is mediated by the MAPK isoforms ERK-1 and ERK-2, which are phosphorylated and thus activated upon EGF stimulation. Because ROS are thought to function as intracellular second messengers, we investigated whether H202 is also able to activate MAPK and cPLA 2 in Her 14 fibroblasts. To this extent cells were stimulated for 30 min with 0.1, 0.5, 1, 2, or 5 mM H202, respectively, in phosphate-buffered saline containing 0.9 mM Ca 2+, 0.5 mM Mg 2+, and 5 mM glucose, whereafter the cells were scraped in buffer (50 mM HEPES pH 7.4; 0.25 M sucrose; 50 mM NaF; 250 ~tM Na3VO4; 1 mM EGTA; 10 laM leupeptin; 1 gM pepstatin; 1 mM phenyl methylsulfony! fluoride). The proteins were separated on SDS-polyacry-lamide gel electrophoresis and stained with a phospho-specific antibody for MAPK. H202 was found to phosphorylate ERK-1 and ERK-2 MAPK in a dose-dependent manner with a maximal effect at 5 raM. Under these conditions, no release of lactate dehydrogenase was detected, indicating that they are still viable. Subsequently, the time course of ERK-1 and ERK-2 phosphorylation induced by 5 mM H202 was examined, showing a rapid phosphorylation, with a maximal effect at 10 min. We then investigated the effect of H202 on cPLA 2 activity, cPLA 2 activity was measured by the release of radiolabeled arachidonic acid from the sn-2 position of lstearoyl-2-[ 1-14C]arachidonoyl-glycero-3-phosphocholine in vitro. H202 stimulated cPLA 2 activity in a dose-dependent way with a maximum at 1 raM. cPLA 2 activity was already maximal after 5 min and remained increased up to 40 min. In EGF-induced signal transduction, MAPK is activated by MAPK kinase (MEK) which in turn is activated by Raf. In order to establish whether in H202-stimulated cells MAPK as well as cPLA 2 is activated through this pathway, we preincubated the cells with the MEK inhibitor PD98059 which inhibits the phosphorylation of MEK by Raf. Preliminary results using this inhibitor in H2O...