Tyrosine phosphorylation regulates PI3K/Akt/Erk activation during synaptosomal oxidative injury

URANGA ROMINA; GIUSTO NORMA; SALVADOR

Mar del Plata

Congreso; . XLIII reunion anual de SAIB. Mar del Plata; 2007

Sociedad Argentina de Investigacion Bioquimica y Biologia Molecular

TYROSINE PHOSPHORYLATION REGULATES PI3K/Akt/Erk ACTIVATION DURING SYNAPTOSOMAL OXIDATIVE INJURY Uranga RM; Giusto NM; Salvador GA. Instituto de Investigaciones Bioquímicas de Bahía Blanca, B8000FWB, Bahía Blanca, ARGENTINA. E-mail: ruranga@criba.edu.ar. The aim of this work was to study the mechanisms involved in  phosphatidylinositol 3-kinase (PI3K)/Akt activation during oxidative injury in rat  cerebral cortex synaptosomes. We have previously demonstrated the activation of PI3K in synaptosomes exposed to FeSO4 (50 µM)  (Uranga et al., 2007). For evaluating downstream pathways activated by PI3K, Akt and Erk phosphorylation levels were determined by Western Blot. Akt phosphorylation in Ser473 and in Thr308 was increased upon a 5‑min exposure to Fe2+. However, pErk levels were increased after 30 min of oxidative insult exposure. Experiments with vanadate and herbimycin A let us determine that tyrosine phosphorylation is involved in the mechanism of PI3K activation by free iron.  PI3K activation was evident when we measured phosphatidylinositol bis-phosphate (PIP2) as product in whole synaptosomes. Additionally, immunoprecipitates (IPs) with anti-phosphotyrosine antibody showed an increase in PI kinase (namely PI-4 kinase) activity induced by iron.  Viability assays  demonstrated that  LY294002 ( a specific PI3K inhibitor)  was not able to prevent LDH  leakage and diminution of  MTT reduction  induced by Fe2+. Our results demonstrate that free iron provokes the activation of PI3K/Akt/Erk pathways in cerebral cortex synaptosomes. However, PI3K activation is not sufficient for protecting synaptic endings from oxidative damage.