Summary of the proposed mechanistic design for double ERK phosphorylation in cytosol and mitochondria under different redox conditions.

<p>After EGF or 1 μM H₂O₂ stimulation, which exert a well described and robust proliferative action, ERK2 is rapidly phosphorylated by cytosolic MEK in Tyr185 (pYERK2), allowing a further association with mitochondria and phosphorylation of Thr183, by a mitochondrial MEK pool. This second and compartmentalized phosphorylation drives fully active-ERK2 (2pERK2) translocation to the nucleus to promote gene transcription and cellular division (A). Panel B shows mitochondrial pThr ERK2 kinetics in response to 1 and 50 μM H₂O₂. At 1 μM H₂O₂ Thr183 is rapidly phosphorylated in mitochondria with a transient localization and fast translocation to the nucleus (white circles); on the other hand, in the presence of 50 μM H₂O₂ arresting cell cycle, Thr183 phosphorylated signal is retained in the mitochondrial milieu and ERK translocation to the nucleus is impeded.</p

Double phosphorylation of ERK by MEK relies almost completely on mitochondrial phospho-Thr183.

<p>LP07 cells were transiently transfected with wild type ERK2-V5 (WT), Y183A or T183A ERK mutants, using Lipofectamine 2000 reagent. Forty-eight hours post-transfection LP07 were stimulated with 1 μM H₂O₂ during 0–30 min and then incubated in the presence or absence of 1 μM H₂O₂ for the indicated times. Then, the cytosolic, mitochondrial and nuclear fractions were obtained and the detection of ERK2 was analyzed by western blot using anti-V5 antibody. Representative images of cytosolic and mitochondrial fractions ERK2-WT (Panel A) and nuclear fraction ERK2-WT (panel D) distribution and representative images of Y185A and T185A distribution in the cytosolic (Panel B), mitochondrial (Panel C) and nuclear (Panel D) fractions, from three independent experiments. Relative levels of ERK mutants in cytosol and mitochondria relative to basal conditions, arbitrarily defined as 1 (Panels B and C). Data are expressed as the mean ± SD of three independent experiments * p<0.05, **p<0.01 vs. 0 min with H₂O₂.</p

Mitochondrial ERK localization depends on differential phosphorylation.

<p>Representative confocal images of LP07 tumor lung transfected cells with wild type ERK/V5 WT (left panel), Y183A/V5 (middle panel) and T183A/V5 (right panel). Forty-eight hours post transfection, cells were incubated with 1 μM H₂O₂ during 0–30 min. From top to bottom of the panels images show increasing H₂O₂ incubating times. Mitochondria were visualized in red by staining with Mitotracker Deep Red. Cells were fixed and incubated with anti-V5 antibody and a secondary antibody conjugated with Cy2, and analyzed in an Olympus FV1000 confocal microscope. Co-localization was highlighted in green on the grayscale images (last column of each panel). Images directly exported from Olympus Fluoview acquisition program. Fold changes relative to control images of combined MitoTracker and Cy2 intensity for ERK2 WT (Panel A), Y185A (Panel B) and T183A (Panel C). Basal conditions without H₂O₂ arbitrarily defined as 1. Bar = 10 μm.</p

Redox status modulates ERK cellular traffic through differential phosphorylation.

<p>LP07 cells were incubated in the presence or absence of 1 μM or 50 μM H₂O₂ for the indicated times. Then, mitochondrial, nucleus and cytosolic fractions were obtained and the detection of pTyr (pYERK) and Thr/Tyr phosphorylated ERK1/2 (2pERK) was analyzed by Western blot. Representative images for the cytosolic (Panel A), mitochondrial (Panel B) and nuclear fraction (Panel C) of three independent experiments. Actin, complex III (CIII) and TFIID protein antibodies were used as loading control markers to obtain relative phosphorylated ERK levels. Levels of pYERK and 2pERK relative to actin (Panel A), CIII (Panel B) and TFDII (Panel C) expressed in arbitrary units (A.U.). Panel D shows representative immunoblots with specific antibodies for each subcellular fraction. Data are expressed as the mean ± SD of three independent experiments * p<0.05, **p<0.01 vs. 0 min with H₂O₂.</p

ERK differential phosphorylation depends on H₂O₂ in normal murine lung tissue.

<p>Normal male FVB mice (<i>n</i> = 3) were treated with saline solution or 6 mM H₂O₂, both administered intravenously. The detection of pTyr (pYERK) and Thr/Tyr phosphorylated ERK1/2 (2pERK) was analyzed by Western blot. Representative images of the mitochondrial (Panel A) and cytosolic fractions (Panel B) of three independent experiments. Actin and complex III (CIII) antibodies were used as loading control markers to obtain relative phosphorylated ERK levels. Levels of pYERK and 2pERK relative to actin or to CIII expressed in arbitrary units (A.U.). Saline solution-treated mice results were defined as 1 and were used to normalize H₂O₂ samples (inset below images).</p

ERK2 is compartmentalized upon EGF stimuli in LP07 tumor lung cells.

<p>LP07 cells were transiently transfected with wild type ERK2-V5 (wt), using Lipofectamine 2000 reagent. Twenty-four hours before the experiment, serum was removed from the culture media to avoid any impact on ERK phosphorylation and localization due to serum effect. Forty-eight hours post-transfection, LP07 were stimulated with 10ng/ml EGF peptide for the indicated times. Temporal activation and distribution of ERK2-V5 in the subcellular fractions were analyzed by western blot using antibodies against V5 tag. Representative images of three independent experiments (Panel A). Protein loading was determined with antibodies anti-TOM40 for mitochondria, actin for cytosol, and polymerase II (POLII) for nuclei. In Panel B, the graph indicates amounts of ERK2-V5 respective to control cells (100%). Panel C shows a representative immunoblot with specific antibodies for each subcellular fraction (M = mitochondria; C = cytosol; N = nucleus).</p