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. 2007 Dec 26;27(52):14349–14357. doi: 10.1523/JNEUROSCI.2969-07.2007

Figure 2.

Figure 2.

FMRP phosphorylation is dependent on group I mGluR and PP2A activity. A, FMRP phosphorylation is group I mGluR activity dependent. Primary hippocampal neurons were treated with 100 μm DHPG (group I mGluR agonist) or 10 μm MPEP (mGluR5 antagonist) for the time points indicated in the presence/absence of 0.5 nm OA (top and bottom panels, respectively) and analyzed by metabolic labeling; a DHPG-untreated (Unt.) sample was used as control. Western blotting for FMRP was used as a loading control. B, Time course study of FMRP phosphorylation in primary neurons reveals that FMRP is rapidly dephosphorylated after 1 min of (100 μm) DHPG-mediated group I mGluR stimulation in the presence of 0.5 nm OA. FMRP phosphorylation was monitored at 1, 2, 5, 10, and 30 min, and DHPG was washed out after 5 min for physiological relevance, as indicated by the gray arrowhead. Western blotting for FMRP was used as a loading control. In the bottom panel, P-FMRP signal was normalized to FMRP in the IPs, calculated as ± SEM, and represented as a histogram (n = 3; the asterisk denotes significance compared with untreated with a Student's t test, *p < 0.05). C, PP2A enzyme activity profiling after DHPG treatment. Primary hippocampal neurons were treated for 0.5, 1, 2, and 5 min with 100 μm DHPG; an untreated sample was used as a control. Neurons were harvested to examine enzyme activities of both PP1 and 2A after IPs using the serine-threonine phosphatase assay kit (Upstate Biotechnology). Error bars represent the SD between the three independent experiments, and asterisks indicate that the fold change as measured by two-tailed paired t tests at the 0.5 and 1 min time points (when compared with time point 0) was highly significant (<0.005). D, Protein phosphatase 2A association with FMRP is sensitive to group I mGluR activity changes. Western blot analyses of FMRP IPs from primary hippocampal neurons were conducted in the presence of DHPG (100 μm) applied for 1 or 10 min; a DHPG untreated sample was used as a control. As a control, the samples were probed for FMRP. In the right panel, PP2Ac immunoreactivity was normalized to FMRP in the IPs, calculated as ±SEM, and represented as a histogram (n = 4; the asterisk denotes significance compared with untreated with a Student's t test, *p < 0.05).