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Characteristic ERK1/2 signaling dynamics distinguishes necroptosis from apoptosis.

iScience (2021-09-28)
François Sipieter, Benjamin Cappe, Aymeric Leray, Elke De Schutter, Jolien Bridelance, Paco Hulpiau, Guy Van Camp, Wim Declercq, Laurent Héliot, Pierre Vincent, Peter Vandenabeele, Franck B Riquet
ABSTRACT

ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular responses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensitizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and necroptosis. We also decrypted a temporally shifted amplitude- and frequency-modulated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
RSK Inhibitor II - CAS 501437-28-1 - Calbiochem, The RSK Inhibitor II, also referenced under CAS 501437-28-1, controls the biological activity of RSK. This small molecule/inhibitor is primarily used for Cell Signaling applications.
Sigma-Aldrich
Doxycycline hyclate
Sigma-Aldrich
Phorbol 12-myristate 13-acetate, ≥99% (TLC), film or powder
Sigma-Aldrich
U0126 monoethanolate, ≥98% (HPLC), powder
Sigma-Aldrich
Anti-MLKL Antibody, clone 3H1, clone 3H1, from rat
Sigma-Aldrich
Anti-Fas Antibody (human, activating), clone CH11, clone CH11, Upstate®, from mouse
Sigma-Aldrich
Anti-phospho-MLKL (Ser345) Antibody, clone 7C6.1, clone 7C6.1, from mouse
Sigma-Aldrich
Anti-MAP Kinase, Activated (Diphosphorylated ERK-1&2) antibody, Mouse monoclonal, clone MAPK-YT, purified from hybridoma cell culture
Sigma-Aldrich
PD184352, ≥98% (HPLC)
Sigma-Aldrich
AZ628, ≥98% (HPLC)