Sub-cellular trafficking of ERK2 is driven by redox-regulated phosphorylation in mitochondria

Y. ALIPPE1, M.E. ELGUERO, H. PÉREZ, M.C. CARRERAS, J.J. PODEROSO

San Carlos de Bariloche

Congreso; Second South American Spring Symposium in Signal Transductionnand Molecular Medicine; 2012

Previously, we showed that ERK2 translocation to nucleus was regulated by the previous passage to mitochondria in response to proliferative or anti-proliferative H2O2 conditions; also, ERK2 oxidation increased the binding to MEK1/2 in the organelles. Our aim is to demonstrate that mitochondrial trafficking of ERK2 depends on its phosphorylation status and on mitochondrial redox conditions, and that kinase oxidation by H2O2 modulates the kinetics of ERK2 phosphorylation. By WB analysis, we followed the distribution of ERK2 phospho-forms in tumor LP07 cells. Under proliferative conditions (1 µM H2O2), we observed that pTyrERK peaked early in cytosol (1 fold at 5-15 min), before the increase in mitochondrial pTyr/pThrERK (3 fd at 15 min); mitochondrial levels of pTyrERK remained constant, and pTyr/pThrERK dropped at control level (60 min). Under anti-proliferative conditions (50 µM H2O2) pTyrERK also peaked early in citosol (2 fold at 5 min) and increased in mitochondria (2 fold at 30 min), but pTyr/pThrERK slowly accumulated in the organelle. Distribution of non-phosphorylatable variants of ERK2 was analyzed by WB and confocal microscopy. Y185A mutant accumulated in cytosol at the expense of its entry into mitochondria (+100% vs -25%), while T183A increased in mitochondria as it decreased in cytosol (+50% vs -20%). Finally, in vitro recombinant ERK2 phosphorylation analysis showed that pre-treatment of the kinase with 0.1-1 µM H2O2 decrease the lag between Tyr and Thr phosphorylations. All these findings support a tri-compartment model for ERK2 activation, where mitochondrial entrance is driven by cytosolic phosphorylation on Tyr185, and redox state regulates the rate of Thr183 phosphorylation inside the organelle and the subsequent nuclear translocation.