Contribution of S6K1/MAPK signaling pathways in the response to oxidative stress: activation of RSK and MSK by hydrogen peroxide.
Cells respond to different kind of stress through the coordinated activation of signaling pathways such as MAPK or p53. To find which molecular mechanisms are involved, we need to understand their cell adaptation. The ribosomal protein, S6 kinase 1 (S6K1), is a common downstream target of signaling by hormonal or nutritional stress. Here, we investigated the initial contribution of S6K1/MAPK signaling pathways in the cell response to oxidative stress produced by hydrogen peroxide (H2O2). To analyze S6K1 activation, we used the commercial anti-phospho-Thr389-S6K1 antibody most frequently mentioned in the bibliography. We found that this antibody detected an 80-90 kDa protein that was rapidly phosphorylated in response to H2O2 in several human cells. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI3K inhibitors, and knock-down experiments showed that this protein was not S6K1. RSK and MSK proteins were candidate targets of this phosphorylation. We demonstrated that H2O2 stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. This phosphorylation required the activity of either p38 or ERK MAP kinases. Kinase assays showed activation of RSK and MSK by H2O2. Experiments with mouse embryonic fibroblasts from p38 animals' knockout confirmed these observations. Altogether, these findings show that the S6K1 signaling pathway is not activated under these conditions, clarify previous observations probably misinterpreted by non-specific detection of proteins RSK and MSK by the anti-phospho-Thr389-S6K1 antibody, and demonstrate the specific activation of MAPK signaling pathways through ERK/p38/RSK/MSK by H2O2.
Departament de Ciències Fisiològiques II, Campus de Bellvitge, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain ; Laboratório de Neuroquímica e Psicofarmacologia, Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul, PUCRS, Porto Alegre, Rio Grande do Sul, Brazil.
Anna
A
Siebel
Monica
M
Cubillos-Rojas
Roberto Christ
RC
Santos
Taiane
T
Schneider
Carla Denise
CD
Bonan
Ramon
R
Bartrons
Francesc
F
Ventura
Jarbas
J
Rodrigues de Oliveira
Jose Luis
JL
Rosa
D000818:Animals; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000235:genetics; D051379:Mice; D008817:Mice, Mutant Strains; D016877:Oxidants / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics / Q000378:metabolism*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000235:genetics / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism
D016877:Oxidants; D047428:Protein Kinase Inhibitors; D006861:Hydrogen Peroxide; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C512964:Rps6ka1 protein, mouse; D058570:TOR Serine-Threonine Kinases; C448901:ribosomal protein S6 kinase, 70kD, polypeptide 1