Fig. 3
De novo point mutation of CAMK2G impairs CaM trapping and shuttling by γCaMKII. a Schematic of γCaMKII protein, highlighting the putative CaM-trapping region (aa ~286–315 within the regulatory region). Amino acids shaded in gray directly bind Ca2+/CaM41,58,63. Arg292 to Pro292 mutation (R292P, red) identified as a candidate basis for intellectual disability via family-based exome-sequencing28. b L-LTP induction in hippocampal slices from γCaMKII KO mice (black) or exc-KO mice injected with lentivirus expressing γCaMKII (blue) or γCaMKII R292P (red). Note overlap between KO and exc-KO + γCaMKII R292P traces. Potentiation at 180 min significantly greater for exc-KO + γCaMKII (198.9 ± 26.8%), than exc-KO + CaMKII R292P (107.3 ± 5.7%) (n = 4 mice for each group). Superimposed representative EPSPs show basal EPSP (bold color) and EPSP at 165 min (muted color) after L-LTP induction. Calibration bars, 1 mV and 10 ms. c CaM overlay analysis (Experimental Procedures). Purified HA-tagged γCaMKII or γCaMKII R292P was run on SDS-PAGE gel and membrane exposed to 0.5 μg/ml CaM for 1 h in the presence of 1 mM CaCl2. d Western blot analysis of phospho-Thr287 using antibodies against pCaMKII and HA tag, following incubation of purified HA-tagged γCaMKII or γCaMKII R292P with 25 μM ATP for 1 min in presence of 1 mM CaCl2 and 1 μM CaM. e In vitro phosphorylation of syntide-2 by purified recombinant HA-tagged γCaMKII or γCaMKII R292P was measured using the CycLex CaM kinase II activity ELISA kit. f Dissociation kinetics of phosphorylated γCaMKII and γCaMKII R292P. Traces show that dissociation of CaM(C75)IAE from complexes with phospho-γCaMKII (blue) is 1500-fold faster (p = 0.002) than from complexes with phospho-γCaMKII R292P (red). Curves generated by fitting data to two phase decay (Y = A1*exp(−t/τ1) + A2*exp(−t/τ2) + Y0)41. Data normalized so intensity difference between kinase-bound and free Ca2+/ CaM(C75)IAE would be 1. g–j Western blot probing for nuclear γCaMKII, CaM, and c-Fos in isolated nuclei of cultured hippocampal and cortical neurons from WT or γCaMKII KO mice stimulated with 40 mM KCl for 1 h. Both γCaMKII (WT) and γCaMKII R292P translocated to the nucleus (g, h); however, only re-introduction of transgenic γCaMKII (WT), not γCaMKII R292P, into γCaMKII KO neurons rescued CaM translocation (g, i) and c-Fos expression (g, j). k c-Fos response in cultured hippocampal and cortical neurons from γCaMKII KO mice stimulated with 40 mM KCl for 1 h. γCaMKII (WT) but not γCaMKII R292P (no HA tag) could restore c-Fos response. Co-expressing γCaMKII R292P with NLS-Nrgn but not NLS-Nrgn S36D was able to restore the c-Fos response, consistent respectively with an ability or inability to harbor CaM for release upon nuclear Ca2+ elevation19. Scale bar, 10 μm. Statistical analysis performed with one-way ANOVA followed by Holm–Sidak post hoc test unless otherwise noted. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Error bars represent SEM
