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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]

In the context of fibroblast growth factor (FGF) signaling, Sprouty2 (Spry2) is the most profound inhibitor of the Ras/ERK pathway as compared with other Spry isoforms. An exclusive, necessary, but cryptic PXXPXR motif in the C terminus of Spry2 is revealed upon stimulation. The activation of Spry2 appears to be linked to sequences in the N-terminal half of the protein and correlated with a bandshifting seen on SDS-PAGE. The band-shifting is likely caused by changes in the phosphorylation status of key Ser and Thr residues following receptor stimulation. Dephosphorylation of at least two conserved Ser residues (Ser-112 and Ser-115) within a conserved Ser/Thr sequence is accomplished upon stimulation by a phosphatase that binds to Spry2 around residues 50–60. We show that human Spry2 coimmunoprecipitates with both the catalytic and the regulatory subunits of protein phosphatase 2A (PP2A-C and PP2A-A, respectively) in cells upon FGF receptor (FGFR) activation. PP2A-A binds directly to Spry2, but not to Spry2Δ50-60 (Δ50-60), and the activity of PP2A increases with both FGF treatment and FGFR1 overexpression, c-Cbl and PP2A-A compete for binding centered around Tyr-55 on Spry2. We show that there are at least two distinct pools of Spry2, one that binds PP2A and another that binds c-Cbl, c-Cbl binding likely targets Spry2 for ubiquitin-linked destruction, whereas the phosphatase binding and activity are necessary to dephosphorylate specific Ser/Thr residues. The resulting change in tertiary structure enables the Pro-rich motif to be revealed with subsequent binding of Grb2, a necessary step for Spry2 to act as a Ras/ERK pathway inhibitor in FGF signaling. [ABSTRACT FROM AUTHOR]