Figure 7. CaMKII Mediates the Phosphorylation of MeCP2 at S421.
(A and B) Western blot analysis of extracts from E18 + 10 DIV hippocampal neurons that were pretreated for 60 min with the CaMKII inhibitor KN93, the inactive analog KN92, or vehicle control (DMSO), followed by stimulation with 55 mM KCl (A) or 20 μM NMDA (B) for 30 min.
(C) E18 + 5 DIV cortical neurons were cotransfected with FLAG-tagged MeCP2 and vector control, ca-CaMKK, dn-CaMKK, ca-CaMKII, or CaMKII-N. Extracts were prepared from these neurons left unstimulated (−) or membrane depolarized for 1 hr (+) 2 days after transfection and probed with antibodies specific to the FLAG epitope. ca, constitutive-active; dn, dominant-negative.
(D) Western blot analysis using anti-MeCP2 pS421 or anti-FLAG antibodies of extracts from HEK293T cells cotransfected with FLAG-MeCP2 and vector control, ca-CaMKI, ca-CaMKII, ca-CaMKIV, or ca-Akt.
(E) CaMKII phosphorylates MeCP2 in vitro. FLAG-tagged wild-type, S421A mutant MeCP2, or C5A mutant MeCP2 (S341A, S350A, S360A, S385A, S399A) were purified from HEK293T cells, coincubated with recombinant constitutively active CaMKII (left panel) and 32P-ATP, and separated on an SDS-PAGE gel. The right panel shows the results of a control reaction in the absence of any added kinase.
(F) BDNF-dependent induction of MeCP2 S421 phosphorylation is likely mediated by CaMKII. Western blot analysis of whole-cell extracts prepared from E18 + 12 DIV hippocampal neurons that were treated with indicated agents for 60 min followed by BDNF treatment for 60 min. K252a is an inhibitor of the BDNF receptor TrkB.