Fig 2: Subcellular distribution of KAT proteins and acetylation of CaM by SRC3 in HEK293 cells.A, the family and representative proteins of KAT. B, expression of Histone H3 (H3), α-tubulin, and synapsin 1 (SYN1) in the nuclear (N), cytoplasmic (S2), and P2 fractions. The bottom image is the Ponceau staining. C, distribution of different KAT proteins in the nuclear (N), cytoplasmic (S2) and P2 fraction of mouse forebrain. Histone H3 (H3), α-tubulin, and Synapsin1 (SYN1) are markers for the N, S2, and P2 fractions. D, heat map of KAT protein levels in the nuclear (N), cytoplasmic (S2), and P2 fractions of mouse forebrain. Original western blots were shown in panel C. E, overexpression of SRC3 induces acetylation of CaM in HEK293 cells. The total lysates from HEK293 cells treated with 1 μM TSA or transfected with different KAT plasmids for 36 h were probed with the indicated Abs. F, expression of Flag-tagged KAT constructs in HEK 293 cells. The total lysates of HEK293 cells transfected with different Flag-tagged KAT constructs were probed with anti-Flag antibodies. The arrows indicate the main bands for different KAT proteins. G, SRC3 has a higher capacity to acetylate CaM than other KATs. Shown were acetylated CaM levels normalized by the intensity of Flag signal in panel F. Data were represented as mean ± SD. NS, not significant, ∗∗p = 0.0024, ∗∗∗p < 0.0001, compared with control, one-way ANOVA, n = 6, data were normalized by control.

Fig 3: CaM-SRC3 interaction in a Ca2+and NMDAR-dependent manner.A, increased CaM-SRC3 interaction by Ca2+. His-SRC3-KAT and GST-CaM were purified from bacteria and their interaction was investigated by GST pull-down during which acetylation could not occur. B, quantification of CaM-SRC3 interaction as His/GST in panel A. Data were represented as mean ± SD. ∗∗∗p < 0.0001, one-way ANOVA, n = 6, data were normalized to 0.01 mM Ca2+. C, the binding curve of GST-CaM and His-SRC3-KAT under different Ca2+ concentrations. The Kd of Ca2+ for the interaction between GST-CaM and His-SRC3-KAT is 2.734 μM, n = 3. Data were represented as mean ± SD. D, CaM-SRC3 interaction in HEK293 cells depends on Ca2+. The immunoprecipitation (IP) of Flag-SRC3 was probed with anti-CaM antibodies. The CaM-SRC3 interaction was abolished when the IP buffer contained 1 mM EGTA. E, increased CaM-SRC3 interaction by cLTP, which is dependent on NMDAR. The CaM-SRC3 interaction was assessed by coimmunoprecipitation (co-IP) from total lysate of hippocampal slices with different treatment. The co-IP was performed in RIPA buffer without EGTA or detergent. F, quantification of CaM-SRC3 interaction as CaM/SRC3 in panel E. Data were represented as mean ± SD. ∗∗∗p < 0.0001, one-way ANOVA, n = 6, data were normalized to control.

Fig 4: No effects of SRC3 inhibition or downregulation on locomotion.A, equal distance traveled during the first 30 min in open field for mice treated with vehicle or SI-2. Data were represented as mean ± SD. Genotype F (1,22) = 0.3879, p = 0.5398, two-way ANOVA, n = 12 for each group. B, similar time staying in the center and margin of the open field during the first 5 min for mice treated with vehicle or SI-2. Data were represented as mean ± SD. NS, not significant, p = 0.5613, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 12 for each group. C, equal distance traveled during the first 30 min in open field for Rosa26-Cas9 mice receiving hippocampal injection of the indicated AAV. Data were represented as mean ± SD. NS, not significant, Genotype F (1,22) = 0.9887, p = 0.3309, two-way ANOVA, n = 12 for each group. D, similar time staying in the center and margin of the open field during the first 5 min for Rosa26-Cas9 mice receiving hippocampal injection of the indicated AAV. Data were represented as mean ± SD. NS, not significant, p = 0.9811, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 12 for each group.

Fig 5: A critical role of SRC3 in novel object recognition (NOR). A Distance travelled during the first 30 min in the open field by WT and Src3+/− mice (n = 16/group; mean ± SEM; Genotype F(1, 30) = 0.4119, P = 0.5259). B Time spent in the center and margin of the open field during the first 5 min by WT and Src3+/− mice (n = 16/group; mean ± SEM; NS, not significant, two-sided t-test. C Occupancy plot of the heads of WT and Src3+/− mice around a novel (N) and a familiar (F) object. D Total exploration time with N and F in WT and Src3+/− mice (n = 15/group; mean ± SEM; NS, not significant, two-sided t-test. E Discrimination indexes showing impaired NOR in Src3+/− mice (n = 15/group; mean ± SEM; *P = 0.0218, two-sided t-test. F Occupancy plot of the heads of Src3+/− mice receiving different AAV treatment around a novel (N) and a familiar (F) object. G Total exploration time with N and F in Src3+/− mice receiving different AAV treatments (n = 15/group; mean ± SEM; NS, not significant, one-way ANOVA. H Discrimination index showing the KAT domain but not the RID of SRC3 rescues the NOR deficit of Src3+/− mice (n = 15/group; mean ± SEM; NS, not significant, **P = 0.0017, one-way ANOVA vs GFP group).