Oxidative stress induces activation of the transcription factor CREB in neuronal cells

PREGI N.; BYK L.; MARAZITA M.; CÁNEPA E.T.

Carlos Paz, Córdoba, Argentina

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigación en; 2008

CB-P12. OXIDATIVE STRESS INDUCES ACTIVATION OF THE TRANSCRIPTION FACTOR CREB IN NEURONAL CELLS Pregi N, Byk L, Marazita MC, Cánepa ET. Laboratorio de Biología Molecular - Departamento de Química Biológica – FCEN – UBA – Buenos Aires. E-mail: npregi@qb.fcen.uba.ar Cyclic AMP response element binding (CREB) protein, a transcription factor, mediates responses to a number of physiological and pathological signals such as neurotransmitters, mitogens, and stress factors. Phosphorylation of CREB at Ser133, a well-characterized modification site, seems to be necessary but not sufficient to trigger its activation, suggesting a requirement for additional independent mechanisms including other phosphorylation sites and pathways. The aim of this work is to study the role of CREB in the DNA damage response in neuronal cells. In particular, the modification of CREB at phosphorylation sites in addition to Ser133 will be explored. Metabolic labeling of cells with 32P-ATP followed by treatment with H2O2 showed that CREB becomes phosphorylated starting at 30 minutes post-treatment. However, unlike treatment with cAMP, addition of H2O2 did not induce phosphorylation of CREB at Ser133, as detected by Western blot. H2O2 also activated CREB transcriptional activity 3.5-fold in reporter assays using p4xCRE-CAT. This effect was partially blocked by inhibitors of Ras (PD152440) and MEK (PD98059) indicating that the Ras/ERK pathway is involved in the activation of CREB by H2O2. Taken together, these results suggest that H2O2 activates CREB by phosphorylation at sites other than the canonical Ser133 through a pathway that requires Ras and MEK activity. P-ATP followed by treatment with H2O2 showed that CREB becomes phosphorylated starting at 30 minutes post-treatment. However, unlike treatment with cAMP, addition of H2O2 did not induce phosphorylation of CREB at Ser133, as detected by Western blot. H2O2 also activated CREB transcriptional activity 3.5-fold in reporter assays using p4xCRE-CAT. This effect was partially blocked by inhibitors of Ras (PD152440) and MEK (PD98059) indicating that the Ras/ERK pathway is involved in the activation of CREB by H2O2. Taken together, these results suggest that H2O2 activates CREB by phosphorylation at sites other than the canonical Ser133 through a pathway that requires Ras and MEK activity. well-characterized modification site, seems to be necessary but not sufficient to trigger its activation, suggesting a requirement for additional independent mechanisms including other phosphorylation sites and pathways. The aim of this work is to study the role of CREB in the DNA damage response in neuronal cells. In particular, the modification of CREB at phosphorylation sites in addition to Ser133 will be explored. Metabolic labeling of cells with 32P-ATP followed by treatment with H2O2 showed that CREB becomes phosphorylated starting at 30 minutes post-treatment. However, unlike treatment with cAMP, addition of H2O2 did not induce phosphorylation of CREB at Ser133, as detected by Western blot. H2O2 also activated CREB transcriptional activity 3.5-fold in reporter assays using p4xCRE-CAT. This effect was partially blocked by inhibitors of Ras (PD152440) and MEK (PD98059) indicating that the Ras/ERK pathway is involved in the activation of CREB by H2O2. Taken together, these results suggest that H2O2 activates CREB by phosphorylation at sites other than the canonical Ser133 through a pathway that requires Ras and MEK activity. P-ATP followed by treatment with H2O2 showed that CREB becomes phosphorylated starting at 30 minutes post-treatment. However, unlike treatment with cAMP, addition of H2O2 did not induce phosphorylation of CREB at Ser133, as detected by Western blot. H2O2 also activated CREB transcriptional activity 3.5-fold in reporter assays using p4xCRE-CAT. This effect was partially blocked by inhibitors of Ras (PD152440) and MEK (PD98059) indicating that the Ras/ERK pathway is involved in the activation of CREB by H2O2. Taken together, these results suggest that H2O2 activates CREB by phosphorylation at sites other than the canonical Ser133 through a pathway that requires Ras and MEK activity.