Fig. 6

CaMKIV activation mediates IGF1-induced histone acetylation. (a, b) IGF1R interacts with CaMKIV, and GEE enhances the association of IGF1R with CaMKIV in E19 embryo extracts, as measured by co-immunoprecipitation and western blotting. (c, d) Inhibition of IGF1R reverses GEE-increased CaMKIV phosphorylation at tyrosine residues (n = 3, one–way ANOVA, ***P < 0.001 vs. Ctrl; ^###P < 0.001 vs. GEE). (e) Mutation of CaMKIV at Y136 and Y172 dose-dependently decreases the IGF1-induced CaMKIV phosphorylation at tyrosine residues. HEK293 cells transfected with EGFP-CaMKIV-WT, EGFP-CaMKIV-Y136F, EGFP-CaMKIV-Y172F or EGFP-CaMKIV-Y136F/Y172F for 24 h were subsequently treated with IGF1 (100 ng/μl) for 2 h, and the cell extracts were immunoprecipitated using anti-GFP and blotted with anti-p-Tyr and CaMKIV antibodies. (f, g) Mutation of CaMKIV at Y136 and Y172 abolishes IGF1-induced histone acetylation and CaMKIV phosphorylation. HEK293 cells transfected with EGFP-CaMKIV-WT, EGFP-CaMKIV-Y136F, EGFP-CaMKIV-Y172F or EGFP-CaMKIV-Y136F/Y172F for 24 h were subsequently treated with IGF1 (100 ng/μl) for 2 h, and levels of histone acetylation and CaMKIV phosphorylation in cell extracts were measured by western blotting (n = 4, one–way ANOVA, *P < 0.05, **P < 0.01,***P < 0.001 vs. WT; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. IGF1 + WT; & P < 0.05, &&P < 0.01, &&&P < 0.001 vs. IGF1 + Y136F; $$ P < 0.01 vs. IGF1 + Y172F). (h) IGF1 induces nuclear translocation of CaMKIV, while mutation of CaMKIV at Tyr172 abolishes the effect of IGF1. Data are presented as the mean ± s.e.m.