Dephosphorylation-induced ubiquitination and degradation of FMRP in dendrites: a role in immediate early mGluR-stimulated translation

Abstract

Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP), which represses and reversibly regulates the translation of a subset of mRNAs in dendrites. Protein synthesis can be rapidly stimulated by mGluR-induced and protein phosphatase 2a (PP2A)-mediated dephosphorylation of FMRP, which is coupled to the dissociation of FMRP and target mRNAs from miRNA-induced silencing complexes. Here, we report the rapid ubiquitination and ubiquitin proteasome system (UPS)-mediated degradation of FMRP in dendrites upon DHPG (3,5-dihydroxyphenylglycine) stimulation in cultured rat neurons. Using inhibitors to PP2A and FMRP phosphomutants, degradation of FMRP was observed to depend on its prior dephosphorylation. Translational induction of an FMRP target, postsynaptic density-95 mRNA, required both PP2A and UPS. Thus, control of FMRP levels at the synapse by dephosphorylation-induced and UPS-mediated degradation provides a mode to regulate protein synthesis.

Figure 1.

Rapid degradation of FMRP in dendrites and synapses by UPS in response to mGluR activation requires PP2A activity. A, Representative images of FMRP detected by immunofluorescence (red) in hippocampal neurons with and without DHPG treatment. Boxes indicate regions of interest for quantitation of dendritic FMRP fluorescence. Phalloidin staining (green) for F-actin labels neuronal morphology. B, C, E, Quantitation of endogenous FMRP intensity from neurons treated with DHPG for 30 s with or without pretreatment with MG132 (B), okadaic acid (OA) (C), or bortezomib (E). Histograms represent normalized mean intensities for dendritic FMRP of multiple neurons per experiment. (n = 3, p < 0.0001, one-way ANOVA with Bonferroni's test, mean ± SEM). D, Mean ± SEM of relative intensity of phospho-FMRP signal in distal dendrites after DHPG treatment with or without MG132 (n = 3, p = 0.01, One-way ANOVA). F, Cortical SNSs were stimulated with DHPG for given time intervals in the presence of the protein synthesis inhibitor anisomycin, with or without MG132, and FMRP was quantified by Western blotting with an anti-FMRP antibody. G, Mean ± SEM of normalized FMRP band intensities in anisomycin-treated SNSs as a function of time without (n = 4, p < 0.001, one-way ANOVA with Bonferroni's test) or with (n = 4, p = 0.89) pretreatment of the SNSs with MG132. H, Synaptoneurosomes were pretreated with anisomycin only or anisomycin and MPEP before stimulation with DHPG for indicated times, and FMRP was quantified by western blot. I, Mean ± SEM of FMRP levels in synaptoneurosomes treated with DHPG, and with or without MPEP (n = 3, p < 0.05). J–M, mGluR stimulation of hippocampal neurons with DHPG induced a rapid UPS- and PP2A-dependent loss of locally photoactivated dendritic PAGFP–FMRP signal. J, Neurons were cotransfected with PAGFP-FMRP and mCherry (to reveal morphology), and were locally photoactivated to reveal the GFP fluorescence only in the distal dendrites before activation with DHPG and imaged every 10 s for 8 min. K, Sequential time-lapse images of the activated region indicated in J) at indicated times of DHPG activation. The FMRP granules were tracked automatically as spots with variable areas (ImarisTrack). Bottom, Sequential images of a heat map of the spot intensities of selected granules. L, Sample traces of the relative intensities of selected PAGFP particles in K as a function of time after DHPG treatment. M, Temporal variation of cumulative relative fluorescence intensities of multiple PAGFP-FMRP granules (n = 60–80) under basal condition, DHPG treatment, or a pretreatment with okadaic acid or MG132 for 30 min were fitted to exponential decay as a function of time after DHPG, and the rate constants were extracted and are depicted in Table 1.

Figure 2.

Rapid mGluR-induced polyubiquitination of FMRP requires dephosphorylation by PP2A at serine 499. A, Western blots performed with antibodies to different epitopes of FMRP in FMRP immunoprecipitates from cortical neurons pretreated with MG132 followed by DHPG for 1 min. *High-molecular-weight FMRP species; arrow, the unubiquitinated protein. B, Western blots of FMRP immunoprecipitates from WT or Fmr1 KO neurons pretreated with MG132 and with or without pretreatment with okadaic acid. *High-molecular-weight FMRP species; arrow, the unubiquitnated band. C, Western blots of lysates from Neuro2a cells expressing FLAGmCherry-FMRP or FLAGmCherry in the presence of MG132 with or without treatment with DHPG. *High-molecular-weight FMRP species; arrow, the unubiquitinated protein. D, Histogram of mean ± SEM of ubiquitinated FMRP band intensities from C normalized to nonubiquitinated FMRP (n = 4, p = 0.01, t test). E, F, Mutation constructs of the primary phosphorylation site S499 (S499D, phosphomimic; S499A, dephosphomimic) and HA ubiquitin were cotransfected in Neuro2a cells. E, Immunoprecipitated FMRP extracts screened by western blot with anti-HA antibody. F, Histogram of mean ± SEM of HA–ubiquitin smear intensities in the IP normalized first to FMRP in IP and then to HA ubiquitin in the lysate (n = 4, p = 0.0332, one-way ANOVA with Bonferroni's test).

Figure 3.

DHPG induction of PSD-95 expression is rapid and requires UPS and PP2A activity. A, Western blot of endogenous PSD-95 (top lane) in synaptoneurosomes treated with 50 μm DHPG for indicated times. Tubulin is detected as loading control for normalization (bottom lanes). B, Mean ± SEM of normalized PSD-95 levels in SNSs as seen in A) at indicated times after DHPG stimulation (n = 6, p = 0.0029, one-way ANOVA with Bonferroni's test). C, Relative luciferase activity from cortical neurons transfected with f-luciferase–PSD-95 3′ UTR construct. Neurons were stimulated for indicated times with 50 μm DHPG with or without pretreatment with okadaic acid and MG132 for 30 min and relative luciferase activity was measured. The graph represents mean ± SEM as a function of time after DHPG stimulation (n = 5, p = 0.0044, one-way ANOVA with Bonferroni's test).