Large Scale Screening Assay for the Phosphorylation of Mitogen-Activated Protein Kinase in Cells

Abstract

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.

Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells

“…Also the oxidative stressinduced phosphorylation and activation of the Ser/Thr kinase Raf-1, a downstream effector molecule of p21 ras , has been described in several cell types as well (Kasid et al, 1996;Abe et al, 1998). Other studies have established that oxidative stress induces the phosphorylation and activation of MAPKs (Guyton et al, 1996;Lander et al, 1996;Peus et al, 1999a;Dérijard et al, 1994;Stevenson et al, 1994;Zhang et al, 2000;De Wit et al, 1998;Bapat et al, 2001).…”

“…Various studies have established that oxygen free radicals can cause the phosphorylation and activation of numerous signaling proteins (Finkel and Holbrook, 2000), including Receptor Tyrosine Kinases (RTK) (Gamou and Shimizu, 1995;González-Rubio et al, 1996), PKC (Gopalakrishna and Jaken, 2000), PLCg1 (Schieven et al, 1993), Src kinases (Aikawa et al, 1997;Hardwick and Sefton, 1997), MAPKs (Dérijard et al, 1994;Stevenson et al, 1994;De Wit et al, 1998;Bapat et al, 2001), protein kinase B , and transcription factors (Devary et al, 1991;Meyer et al, 1993). As an example the effects on the RTK and MAPK pathway will be discussed.…”

Peroxynitrite activates mitogen‐activated protein kinase (MAPK) via a MEK‐independent pathway: a role for protein kinase C

“…There is increasing evidence suggesting that reactive oxygen species activate or inactivate signaling pathways involving tyrosine and serine/threonine kinases and thereby, regulate phosphorylation of MAPKs. Indeed, MAPKs are reported to be activated in response to various forms of oxidative stress such as UV irradiation [1], hydrogen peroxide [2], nitric oxide [3], and peroxynitrite (ONOO − ) [4–7]. Of particular interest is the highly reactive ONOO − , which is involved in oxidative damage detected in several pathological conditions such as inflammation, ischemia/reperfusion injury, atherosclerosis and neurological disorders (reviewed in [8,9]).…”

Hydrogen peroxide reversibly inhibits epidermal growth factor (EGF) receptor internalization and coincident ubiquitination of the EGF receptor and Eps15

“…In the past few years, the involvement of oxygen free radicals in intrinsic signal transduction pathways leading to cell division has been studied in great detail. It has been demonstrated that extracellular addition of inducers of oxidative stress, such as hydrogen peroxide (H 2 O 2 ), can induce the phosphorylation and activation of several proteins that are involved in these signaling pathways (6)(7)(8)(9)(10)(11)(12)(13). Moreover, recent studies revealed that H 2 O 2 is produced upon stimulation of cells with epidermal growth factor (EGF) (14) or platelet-derived growth factor (PDGF) (15,16).…”