Fig. 3.
Phosphorylations of MeCP2 at S80 and S421 are dynamically regulated in response to neuronal activity and do not cross-talk with each other. (A) Brain extracts of normal mice or mice that have undergone seizures induced by kainic acid were blotted first with phospho-S80 antibody and then MeCP2 full-length antibody. The asterisk marks the upper shifted band. (B and C) E15 + 3DIV cultured mouse cortical neurons were treated either with 0.5 μM tetrodotoxin (TTX) or 50 mM KCl for 1 h before harvested for protein assays. Western blot showed that upon depolarization, S80 phosphorylation decreased (B) while S421 phosphorylation increased (C) with phospho-serine 133-Creb as a positive control for depolarization. (D) E14 cultured Mecp2 −/y mouse cortical neurons were infected with lentiviruses expressing wild-type, serine 421 to alanine mutant (S421A) or serine 421 to aspartate mutant (S421D) MeCP2. Western blot was done with the antibody recognizing full-length MeCP2. (E) Flag tagged wild-type MeCP2, serine 80 to alanine mutant (S80A) and serines 80 to aspartate mutant (S80D) were expressed in E15 mouse cortical neurons by lentiviral infection. Protein lysates were blotted with flag antibody. Densitometry analysis of the upper band (phospho-S421 MeCP2) intensity versus both bands (total MeCP2) showed little difference (n = 3, 1-way ANOVA analysis, P = 0.839). (F) Similarly, neurons overexpressing flag-tagged MeCP2 were treated with TTX for 1 h before harvested for the protein lysates. Exogenous MeCP2 were immunoprecipitated with flag antibody and subjected to Western blot with phospho-S80 antibody and flag antibody respectively. Densitometry analysis of phospho-S80 intensity versus flag intensity showed that S421D did not change phospho-S80 intensity compared with wild-type MeCP2. S421A showed a slight but not significant increase of S80 phosphorylation (n = 3, 1-way ANOVA analysis, P = 0.205). (G) Nimodipine (5 μM) was applied in E15 mouse cortical neuronal culture for 1 h before depolarizing the neurons with 50 mM KCl. (H) E15 mouse cortical neurons were treated with KN62 (5 μM) for 1 h before depolarization with 50 mM KCl for 1 h and subsequently underwent Western blot. (I) SMARTPOOL CaMK IV siRNA and control siRNA (Dharmacon) were transfected into E15 mouse cortical neurons with nucleofector (Amaxa). Protein lysates were harvested 72 h after transfection and undergone Western blot. (J) Densitometry quantification of Western blots showed CaMK IV knockdown decreased S421 phosphorylation by 50% (P < 0.05, n = 3, paired t test).