The roles of ShcA proteins in response to oxidative stress

Abstract

ShcA proteins have important roles in oxidative stress response.Mice deficient for p66ShcA represent an animal model that link oxidative stress with aging. p66ShcA is implicated in oxidative stress response and mitogenic signaling. Phosphorylation of p66ShcA on Ser36 is critical for its function in oxidative stress response. I identified ERK as the protein kinase phosphorylating p66ShcA at Serine36 in response to H2O2 treatment. Activation of ERKs was necessary and sufficient for Ser36 phosphorylation in oxidative stress. p66ShcA interacted with ERK and was demonstrated to be a substrate for ERK, with Ser36 being the major phosphorylation site. Furthermore, in response to H2O2, chemical inhibition of ERK activativity repressed p66ShcA-dependent phosphorylation of FOXO3a and cause the transcriptional down regulation of its target gene p27kip1. As a feedback regulation, Ser36 phosphorylated p66shcA attenuated H2O2-induced ERK activation, whereas p52/46ShcA facilitated ERK activation, which required tyrosine phosphorylation of CH1 domain. p66ShcA formed a complex with p52/46ShcA, which may provide a platform for efficient signal amplify. My study suggests that there exists interplay between ERK and ShcA proteins, which modulates the expression of p27 and cell response to oxidative stress. PKCI' , which is implicated in cell response to oxidative stress , was found to interact with ShcA and this interaction was promoted upon H2O2 treatment. PKCI' and ShcA are also colocalized in the cytoplasm and displayed co-translocation to plasma membrane in response to H2O2. Activated PKCI' was able to phosphorylate ShcA at Ser29, as determined by mass spectrometry. These results suggest that ShcA, p66 and p52, are substrates that interact with PKCI'. This speciality is critical in H2O2 induced ERK activation as reconstitution with ShcA is able to restore ERK activation in ShcA-/- MEFs, while ShcA Ser29A failed to do so. In line with this conclusion, inhibition of PKCI' activity with chemical inhibitors is able to diminish H2O2-induced ERK activation in MEFs. These results suggest that the interaction between PKCI' and ShcA and phosphorylation of ShcA at Ser29 play important roles in ERK activation in cellular response to H2O2.