Visit our Expo - Redox and Inflammation signaling 2012

Session III : Protein kinase cascades as therapeutic targets Poster III, 46
Tyrosine nitration of MEK and ERK induces their autophosphorylation and activation
in vivo and in vitro
Elena Pinzar*, Maria Garrido°, Daniela Urrecheaga°*, Guillaume Bonnot*, Patrick
Levy* and Serge P. Bottari *#
*Laboratoire HP2, UFR de Médecine, Grenoble Universités, Domaine de la Merci,
38700 La Tronche, France, °Facultad de Farmacia, Universidad Central de Venezuela,
Caracas, Venezuela, #Département de Biologie Intégrée, CHU de Grenoble, 38043
Grenoble, France. E-mail: Serge.Bottari@ujf-grenoble.fr
Angiotensin II (Ang II) has been shown to activate MAPK pathways and to induce production
of reactive oxygen and nitrogen species. Nitric oxide (NO) and superoxide (O2-.) as well as
peroxinitrite (ONOO-) have been previously reported to stimulate MAPK in a few cell types.
We therefore investigated whether ERK could be nitrated in rat vascular smooth muscle cells
(VSMC) upon exposure to Ang II. Stimulation of cells with 10 nM Ang II induced a
prominant and sustained nitration of ERK1/2 as revealed by immunoprecipitation and
immunoblotting. ERK nitration was accompanied by activation of the enzyme and showed a
kinetic profile different than that of phosphorylation. Both Ang II-induced ERK nitration and
phosphorylation were mediated by the AT1 receptor. The ONOO- donor 3morpholinosydnonimine
hydrocloride (SIN-1) also stimulated nitration and activation of
recombinant unactive ERK and MEK. Nitration and phosphorylation of ERK1/2 by Ang II
was significantly inhibited by the NAD(P)Hoxidase inhibitors 4-(2-aminoethyl)
benzenesulfonyl fluoride (AEBSF) and diphenylene iodonium (DPI). Moreover, the selective
inducible nitric-oxide synthase (iNOS) inhibitor 1400W but not the endothelial NOS inhibitor
L-NAME, completely inhibited nitration and activation of ERK. The ONOO- and O2-.
scavengers, respectively ebselen and myricetin, drastically blunted Ang II-stimulated nitration
and activation of ERK. Conversely, the MEK inhibitor U0126, did not affect ERK nitration,
but completely blocked ERK phosphorylation and activation. Taken together these data
indicate that Ang II can activate ERK1/2 both through nitration via reactive nitrogen speciessensitive
pathways and by phosphorylation through the Ras-Raf-MEK pathway.
Interestingly, ERK nitration appears to be a prerequisite for its activating phosphorylation by
MEK in response to Ang II in VSMC.
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