Axel Knebel

Dephosphorylation of receptor tyrosine kinases as target of regulation by radiation, oxidants or alkylating agents.

Several non‐physiologic agents such as radiation, oxidants and alkylating agents induce ligand‐independent activation of numerous receptor tyrosine kinases (RTKs) and of protein tyrosine kinases at the inner side of the plasma membrane (e.g. Dévary et al., 1992; Sachsenmaier et al., 1994; Schieven et al., 1994; Coffer et al., 1995). Here we show additional evidence for the activation of epidermal growth factor receptor (EGFR), and we show activation of v‐ErbB, ErbB2 and platelet‐derived growth factor receptor. As a common principle of action the inducing agents such as UVC, UVB, UVA, hydrogen peroxide and iodoacetamide inhibit receptor tyrosine dephosphorylation in a thiol‐sensitive and, with the exception of the SH‐alkylating agent, reversible manner. EGFR dephosphorylation can also be modulated by these non‐physiologic agents in isolated plasma membranes in the presence of Triton X‐100. Further, substrate (EGFR) and phosphatase have been separated: a membrane preparation of cells that have been treated with epidermal growth factor (EGF) and whose dephosphorylating enzymes have been permanently destroyed by iodoacetamide can be mixed with a membrane preparation from untreated cells which re‐establishes EGFR dephosphorylation. This dephosphorylation can be modulated in vitro by UV and thiol agents. We conclude that RTKs exhibit significant spontaneous protein kinase activity; several adverse agents target (an) essential SH‐group(s) carried by (a) membrane‐bound protein tyrosine phosphatase(s).

UV-INDUCED SIGNAL TRANSDUCTION

Irradiation of cells with wavelength ultraviolet (UVA, B and C) induces the transcription of many genes. The program overlaps with that induced by oxidants and alkylating agents and has both protective and other functions. Genes transcribed in response to UV irradiation include genes encoding transcription factors, proteases and viral proteins. While the transcription factor encoding genes is initiated in minutes after UV irradiation (immediate response genes) and depends exclusively on performed proteins, the transcription of protease encoding occurs only many hours after UV irradiation. Transcription factors controlling the activity of immediate response genes are activated by protein kinases belonging to the group of proline directed protein kinases immediately after UV irradiation. Experimental evidence suggests that these kinases are activated in UV irradiated cells through pathways which are used by growth factors. In fact, the first cellular reaction detectable in UV irradiated cells is the phosphorylation of several growth factor receptors at tyrosine residues. This phosphorylation does not depend on UV induced DNA damage, but is due to an inhibition of the activity of tyrosine phosphatases. In contrast, for late cellular reactions to UV, an obligatory role of DNA damage in transcribed regions of the genome can be demonstrated. Thus, UV is absorbed by several target molecules relevant for cellular signaling, and it appears that numerous signal transduction pathways are stimulated. The combined action of these pathways establishes the genetic program that determines the fate of UV irradiated cells.

The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents

Monofunctional alkylating agents like methyl methanesulfonate (MMS) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) are potent inducers of cellular stress leading to chromosomal aberrations, point mutations, and cell killing. We show that these agents induce a specific cellular stress response program which includes the activation of Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), p38 mitogen-activated protein kinase, and the upstream kinase SEK1/MKK4 and which depends on the reaction mechanism of the alkylating agent in question. Similar to another inducer of cellular stress, UV irradiation, damage of nuclear DNA by alkylation is not involved in the MMS-induced response. However, in contrast to UV and other inducers of the JNK/SAPKs and p38 pathways, activation of growth factor and G-protein-coupled receptors does not play a role in the MMS response. We identified the intracellular glutathione (GSH) level as critical for JNK/SAPK activation by MMS: enhancing the GSH level by pretreatment of the cells with GSH or N-acetylcysteine inhibits, whereas depletion of the cellular GSH pool causes hyperinduction of JNK/SAPK activity by MMS. In light of the JNK/SAPK-dependent induction of c-jun and c-fos transcription, and the Jun/Fos-induced transcription of xenobiotic-metabolizing enzymes, these data provide a potential critical role of JNK/SAPK and p38 in the induction of a cellular defense program against cytotoxic xenobiotics such as MMS.

Synthesis and biological evaluation of aeroplysinin analogues: a new class of receptor tyrosine kinase inhibitors

Receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR), are critically involved in the transduction of mitogenic signals across the plasma membrane and therefore in the regulation of cell growth and proliferation. Enhanced RTK activity is associated with proliferative diseases such as cancer, psoriasis and atherosclerosis, while decreased function may be associated for instance with diabetes. EGFR and PDGFR are selectively inhibited by analogues of the marine natural product aeroplysinin. The synthetic inhibitors display IC50 values in the low micromolar range and in contrast to the natural product show pronounced inhibitory activity in cultured cells in vivo. The mechanism of inhibition is likely based on a covalent modification of the target enzymes by reaction of epoxy ketone 8 with various nucleophiles.

[24] Radiation-induced signal transduction

Publisher Summary Cells depend for almost all their life reactions, from survival to apoptosis, from circadian rhythm to changes in behavior and differentiation, on extracellular cues. Much information has accumulated on the mechanisms of cell-cell communication and on the network of signaling inside the cell. It is now known the way hormones act and the way cytokines address the cellular program of gene expression and some of the components of the signal transduction network are known from the cell membrane to the cytoskeleton and to the nucleus. There are still many gaps in our understanding. One research field that may contribute to a better understanding of such pathways concerns cellular responses to light, to other types of radiation, and to non-natural chemicals. This chapter addresses some of the methodology that has been employed to study radiation-induced signaling and that led to the recognition of radiation-induced pathways and of some of the primary target molecules.

An Oxidant Sensor at the Plasma Membrane

The expression of genes is predominantly determined by conditions of the microenvironment of cells. Prime examples of such regulation are found in embryonic development of all multicellular organisms and also in the adult when various cytokines and hormones exert highly inducer-specific influences on genes. The naturally occurring regulating agents interact with specific receptors: e.g., the retinoids, vitamin D3, thyroid hormones and the steroid hormones with appropriate nuclear receptors (RARs, RXRs, VDR and the specific steroid hormone receptors), or the members of the large TGFβ and FGF families with their respective cell surface receptors. While nuclear receptors act as transcription factors themselves and select their genes by receptor-specific recognition elements, the growth factors induce, through their cell surface receptors, a complex process of signal transduction to the nucleus (for reviews see Beato, 1989; Karin, 1994; Gilbert, 1994; Angel and Herrlich, 1994; McCormick, 1995; Howe and Weiss, 1995; Ullrich and Simon, 1995).