1. CK2 phosphorylates mammalian FMRP S499.
CK2 phosphorylates mammalian FMRP S499. A, Immunoblots for 3-h treatment of N2a cells with vehicle (DMSO), D4476 (25 µm), IC261 (20 µm), PHA (5 µm), DRB (50 µm), TBB (25 µm), or βARK (200 µm) followed by Western blot for protein indicated on the left. B, Quantification of pFMRP/tFMRP signal in A. ns, not significant (Kruskal–Wallis one-way ANOVA analysis [H(8) = 4.56, p = ], n = 4). C, Immunoblot for 24-h treatment of N2a cells with the same agents listed in A. D, Quantification of pFMRP/tFMRP signal in C (Kruskal–Wallis one-way ANOVA [H(8) = 7.239, p = ], n = 4, error bars = SEM). E, Baseline, untreated N2a cells were collected at time 0, and the remainder of the cells were treated with either DMSO or CX-4945 (5 or 1 µm) for 24 h. tFMRP and pFMRP S499 signals increased in DMSO-treated samples; however, only tFMRP increased in CX-treated samples, thereby causing a significant reduction in relative FMRP S499 phosphorylation. All immunoblot signals are from the same membrane; however, intervening lanes have been removed for clarity. F, Quantification of pFMRP S499 to tFMRP ratio from D; one-way ANOVA, n = 4, error bars = SEM, *p < G, HEK293 cells were collected at baseline (time 0) or treated for 24 h with DMSO or CX H, CX-4945 significantly reduced FMRP S499 phosphorylation compared with DMSO (one-tailed Mann–Whitney test, p = ). I, J, Mouse cortical neurons at 7 d in vitro treated with 1 µm CX-4945 for 24 h exhibited a significant reduction in FMRP S499 phosphorylation compared with DMSO-treated neurons (one-tailed Mann–Whitney test, p = 0.05).
Christopher M. Bartley et al. eNeuro 2016;3:ENEURO
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