Abstract: Both PKC δ and ShcA have been implicated in cell response to oxidative stress [Y. Hu, X. Wang, L. Zeng, D.Y. Cai, K. Sabapathy, S.P. Goff, E.J. Firpo, B. Li, Mol Biol Cell., 16 (2005) 3705–3718, B. Li, X. Wang, N. Rasheed, Y. Hu, S. Boast, T. Ishii, K. Nakayama, K.I. Nakayama, S.P., Goff, Genes Dev, 18 (2004) 1824–1837, E. Migliaccio, M. Giorgio, S. Mele, G. Pelicci, P. Reboldi, P.P. Pandolfi, L. Lanfrancone, P.G. Pelicci, Nature, 402 (1999) 309–313], yet their relationship in the response has not been studied. Here we report that PKC δ interacts with ShcA and this interaction is promoted by H2O2. PKC δ and ShcA are also colocalized in the cytoplasm and displayed co-translocation in response to H2O2. Activated PKC δ 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 PKC δ. This phosphorylation is critical in H2O2 induced ERK activation as reconstitution with ShcA Ser29A failed to rescue ERK activation of ShcA−/− MEFs, while ShcA could. In line with this conclusion, inhibition of PKC δ with inhibitors is able to diminish H2O2 induced ERK activation in MEFs. These results suggest that the interaction between PKC δ and ShcA and the phosphorylation of ShcA at Ser29 play important roles in ERK activation in cell response to H2O2. [Copyright &y& Elsevier]