Quantitative phosphoproteomics to unravel the cellular response to chemical stressors with different modes of action

Bharath Sampadi,Bob Van De Water,Branislav Mišovic,Alex Pines,Stephanie Munk,Harry Vrieling,Leon H F Mullenders,Jesper V Olsen,Anton J De Groot

doi:10.1007/s00204-020-02712-7

Abstract

Damage to cellular macromolecules and organelles by chemical exposure evokes activation of various stress response pathways. To what extent different chemical stressors activate common and stressor-specific pathways is largely unknown. Here, we used quantitative phosphoproteomics to compare the signaling events induced by four stressors with different modes of action: the DNA damaging agent: cisplatin (CDDP), the topoisomerase II inhibitor: etoposide (ETO), the pro-oxidant: diethyl maleate (DEM) and the immunosuppressant: cyclosporine A (CsA) administered at an equitoxic dose to mouse embryonic stem cells. We observed major differences between the stressors in the number and identity of responsive phosphosites and the amplitude of phosphorylation. Kinase motif and pathway analyses indicated that the DNA damage response (DDR) activation by CDDP occurs predominantly through the replication-stress-related Atr kinase, whereas ETO triggers the DDR through Atr as well as the DNA double-strand-break-associated Atm kinase. CsA shares with ETO activation of CK2 kinase. Congruent with their known modes of action, CsA-mediated signaling is related to down-regulation of pathways that control hematopoietic differentiation and immunity, whereas oxidative stress is the most prominent initiator of DEM-modulated stress signaling. This study shows that even at equitoxic doses, different stressors induce distinctive and complex phosphorylation signaling cascades.

Highlights

Cells are equipped with versatile physiological stress responses to prevent hazardous consequences resulting from exposure to chemical insults of endogenous and exogenous origin
We focused on four stressors with distinctive properties and modes of action that do not require bioactivation, i.e. cisplatin (CDDP), etoposide (ETO), diethyl maleate (DEM), and cyclosporine (CsA)
Sub-confluent cultures of mES cells were exposed to either CDDP (5 μM) (Accord, 1 mg/ml), cyclosporine A (CsA) (20 μM) (Sigma, Catalog No 30024-25 mg), DEM (150 μM) (Sigma, Catalog No D97703-100G) or ETO (0.5 μM) (Sigma, Catalog No E1383-25 mg) by adding the drug directly to the culture medium and cells were incubated for different periods after administration (2, 4, 6 and Protein sample collection, phosphopeptide enrichment, and cleanup were performed according to previously published procedures (Pines et al 2011)

Summary

Introduction

Cells are equipped with versatile physiological stress responses to prevent hazardous consequences resulting from exposure to chemical insults of endogenous and exogenous origin. In case these stress responses fall short in their. Activation of cellular stress responses entails signaling and effector stages with the induction of post-translational modifications (PTM) of specific proteins and directed transcriptional alterations, respectively, as main molecular mechanisms. Among the various types of PTMs that are involved in cellular stress responses, one of the most frequent modifications is the reversible and dynamic phosphorylation of proteins at specific serine (pS), threonine (pT) and tyrosine (pY) residues (Bennetzen et al 2010; Olsen et al 2010). It has been estimated that almost all proteins in mammalian cells are phosphorylated at

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