Late Activation of Stress-activated Protein Kinases/c-Jun N-terminal Kinases Triggered by Cisplatin-induced DNA Damage in Repair-defective Cells

Bernd Kaina,Zeljka Fiket,Gerhard Fritz,Maja Osmak,Julia Damrot,Lars Helbig,Anamaria Brozovic,Johannes Hülsenbeck,Beate Köberle

doi:10.1074/jbc.m110.190645

Abstract

Although stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK) are rapidly activated by genotoxins, the role of DNA damage in this response is not well defined. Here we show that the SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK (Thr-183/Tyr-185) correlates with the level of cisplatin-DNA adducts at late times (16-24 h) after drug treatment in both human and mouse cells. Transfection of platinated plasmid DNA also caused SAPK/JNK activation. A defect in transcription-coupled nucleotide excision repair resting on a mutation in Cockayne syndrome group B protein promoted the late SAPK/JNK activation following cisplatin exposure. Signaling to SAPK/JNK was accompanied by activation of Ataxia telangiectasia mutated- and Rad3-related kinase, replication protein A, and checkpoint kinases as well as by the formation of DNA double strand breaks (DSBs). Ionizing radiation-induced DSBs did not provoke SAPK/JNK activation, and inhibition of transcription also failed to provoke this response. Late activation of SAPK/JNK stimulated by cisplatin-induced DNA lesions was reduced in the absence of specific DNA repair proteins, such as xeroderma pigmentosum protein C, pointing to an essential function of individual repair factors in DNA damage signaling to SAPK/JNK. Collectively, the data indicate that late SAPK/JNK activation is triggered by non-repaired cisplatin adducts in transcribed genes and involves replication-associated events, DSBs, tyrosine kinases, Rho GTPases, and specific repair factors.

Highlights

1541/5-3. □S The on-line version of this article contains supplemental Figs
As a first step to substantiate the role of DNA damage in triggering this response, we investigated whether the level of SAPK/JNK phosphorylation coincides with cisplatininduced DNA adduct formation
To substantiate the data in a second experimental model system, Mouse embryonic fibroblasts (MEFs) derived from BALB/c mice were treated with increasing doses of cisplatin, and dual phosphorylation of SAPK/JNK and DNA platination were measured 24 h later

Summary

Introduction

1541/5-3. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. Evidence that DNA damage-dependent functions might impact genotoxin-induced signaling to SAPK/JNK rests on the analysis of cell lines compromised in particular DNA repair functions, such as nucleotide excision repair (NER) or mismatch repair (19 –21). UV-C light, the monofunctional alkylating agent methyl methanesulfonate (MMS), and the anticancer drug cisplatin are powerful and prototypical activators of SAPK/JNK (26 –28). These agents induce different types of DNA adducts. Not each cellular response to genotoxins can be classified as a DNA damage-induced response Bearing this in mind, the aim of the present study was to ascertain whether DNA damage is involved in the late activation of SAPK/JNK following cisplatin treatment and to characterize the molecular mechanisms involved

Objectives

Results

Conclusion