Figure 4: CaMKv phosphorylation at Thr345 by Cdk5 and its role in spine morphogenesis.

(a) Schematic diagram showing the different phospho-serine and phospho-threonine residues of CaMKv. Thr345 is conserved in the human, mouse, and rat orthologues. (b) Phospho-specific antibody against CaMKv Thr345 (P-CaMKv) revealed the induction of Thr345 phosphorylation of FLAG-tagged CaMKv by recombinant Cdk5/p35. The signal was abolished in the T345A phospho-deficient CaMKv mutant. (c,d) Thr345 phosphorylation was significantly reduced in p35^−/− adult mouse brains. (p35^+/+: n=4 mice, p35^−/−: n=5 mice, *P<0.05, Student’s t-test). (e) CaMKv phosphorylation and expression levels in the mouse forebrain at different developmental stages. (f,g) CaMKv phosphorylation decreased significantly in the visual cortex after eye-opening (P13). CaMKv phosphorylation levels were normalized to that of total CaMKv (n=3 mice per condition, *P<0.05, Student’s t-test). (h,i) CaMKv phosphorylation significantly increased in the mouse visual cortex after DR (n=11 mice per condition, **P<0.01, Student’s t-test). (j,k) CaMKv phosphorylation increased on activity blockade. Cortical neurons (14–16 DIV) were treated with TTX (2 μM) for the indicated durations and quantified after normalization to total CaMKv (n=3 independent experiments). (l,m) The phospho-mimetic CaMKv-T345E mutant failed to rescue the spine defects on CaMKv knockdown by shRNA (scale bar, 10 μm; n=33 dendrites per condition, ***P<0.001, one-way analysis of variance, Tukey’s multiple comparison test).