Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Dec 26;27(52):14349-57.
doi: 10.1523/JNEUROSCI.2969-07.2007.

FMRP phosphorylation reveals an immediate-early signaling pathway triggered by group I mGluR and mediated by PP2A

Usha Narayanan  1 , Vijayalaxmi Nalavadi, Mika Nakamoto, David C Pallas, Stephanie Ceman, Gary J Bassell, Stephen T Warren
Affiliations

FMRP phosphorylation reveals an immediate-early signaling pathway triggered by group I mGluR and mediated by PP2A

Usha Narayanan et al. J Neurosci. .

Abstract

Fragile X syndrome is a common form of inherited mental retardation and is caused by loss of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that influences the translation of target messages. Here, we identify protein phosphatase 2A (PP2A) as an FMRP phosphatase and report rapid FMRP dephosphorylation after immediate group I metabotropic glutamate receptor (mGluR) stimulation (<1 min) in neurons caused by enhanced PP2A enzymatic activity. In contrast, extended mGluR activation (1-5 min) resulted in mammalian target of rapamycin (mTOR)-mediated PP2A suppression and FMRP rephosphorylation. These activity-dependent changes in FMRP phosphorylation were also observed in dendrites and showed a temporal correlation with the translational profile of select FMRP target transcripts. Collectively, these data reveal an immediate-early signaling pathway linking group I mGluR activity to rapid FMRP phosphorylation dynamics mediated by mTOR and PP2A.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
PP2A is a major FMRP phosphatase in non-neuronal cells. A, Pharmacological inhibitors of serine-threonine phosphatase reveal PP2A as a candidate FMRP phosphatase. L cells stably expressing the Flag-tag (VC) or Flag-FMRP (WT) were used in metabolic labeling assays with 32P orthophosphate in the presence of the phosphatase inhibitors deltamethrin, OA, and microcystin, either applied individually (# 2–4, respectively) or as a mixture (# 1). P-FMRP was identified by autoradiography, and Western blotting for FMRP was used as a loading control. B, Only wild-type FMRP (WT) and not nonphosphorylated FMRP (S499A) was found to coimmunoprecipitate with the catalytic subunit of PP2A, PP2Ac. Western blotting for FMRP was used as a loading control. C, FMRP becomes hyperphosphorylated after treatment with 100 nm OA compared with 0.5 nm OA (left panel) in a manner similar to well-known PP2A substrate, Raf1 (right panel). Western blotting for FMRP and Raf1 were used as loading controls. D, WT or dominant-negative mutant (L199P) of PP2Ac, the catalytic subunit of PP2A, was transfected into L cells; untransfected L cells were used as a control (mock). Metabolic labeling analyses were conducted in the presence of 0.5 nm OA, and Western blotting for PP2Ac was used as a transfection control. E, PP2A is a major FMRP phosphatase in primary hippocampal neurons. Metabolic labeling assays were conducted in primary rat hippocampal neurons in the presence/absence of 0.5 nm OA, as indicated, and IPs were conducted using FMRP antibodies. Western blotting for FMRP was used as a loading control. All the biochemical assays were repeated three times, unless otherwise stated.
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).
Figure 3.
Figure 3.
Extended (1–5 min) group I mGluR stimulation results in FMRP phosphorylation though mTOR-dependent PP2A suppression. A, B, Time course study of FMRP phosphorylation in the presence of 100 μm DHPG alone or after 20 μm rapamycin treatment (B). Metabolic labeling assays conducted in primary neurons reveal a delay in FMRP phosphorylation. 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. The DHPG-untreated (Unt.) samples contained 0.5 nm OA so that basal P-FMRP could be detected; Western blotting for FMRP was used as an IP control. In the bottom panels, P-FMRP signal was normalized to FMRP in the IPs, and P-mTOR was normalized to total mTOR, 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 in the presence/absence of mTOR activation. Primary hippocampal neurons were treated with 20 μm rapamycin or an inactive chemical analog, ascomycin, and followed by treatment for 0.5, 1, 2, 5, and 10 min with 100 μm DHPG/DHPG stimulation; a DHPG-untreated sample was used as a control. Neurons were harvested to examine enzyme activities of PP2A after IPs using the serine-threonine phosphatase assay kit. Error bars represent the SD between three independent experiments, and asterisks indicate that the fold change as measured by two-tailed paired t tests at the 2 and 5 min time points between DHPG plus rapamycin (red) and DHPG alone (blue) was highly significant (<0.005).
Figure 4.
Figure 4.
Rapid FMRP dephosphorylation was also detected in dendrites after DHPG-mediated group I mGluR activation. A, Phosphorylated FMRP is distributed throughout neuronal dendrites and spines. Wild-type rat hippocampal neurons fixed, labeled with a rabbit antibody to the phosphopeptide NSEAS*NAS*ETES*DHRDE (S* denotes phosphorylated serine) revealing abundant P-FMRP (red) in dendrites and spines (arrows) in the form of granules; actin (blue) was used as a cytoskeletal marker. B, 3-D reconstructed isosurfaces of double labeling assays from 14 DIV, wild-type rat hippocampal neurons showing the total FMRP (green), P-FMRP (red), and colocalized (yellow) granules in a distal dendrite stained with phalloidin (gray). C, Representative images of distal dendrites from hippocampal neurons at steady state, treated with 50 μm DHPG for 30 s in the presence/absence of 10 nm OA before fixation and immunofluorescence. D, Average percentage of immunofluorescence intensity of P-FMRP present in the distal dendrites was quantified using IP laboratory software. The table shows that percentage of basal and DHPG-treated (±OA) P-FMRP intensities in distal dendrites, and the SD was calculated using 10 neurons each from three different experiments, and the p value was found to be significant (p < 0.005) as measured by ANOVA.
Figure 5.
Figure 5.
Group I mGluR activity-dependent FMRP phosphorylation correlates with translation of FMRP ligand SAPAP3. A, Neurons were treated with 100 μm DHPG for 0 [untreated (Unt.)], 2, or 10 min, and protein expression levels of the FMRP ligands were examined by Western blotting cytoplasmic cell lysates. A synapse associated protein, SAPAP3, shows changes in protein expression that correlate with the time course of FMRP phosphorylation. B, Densitometry analyses of the changes in FMRP ligand expression in the presence of 100 μm DHPG in primary hippocampal neurons. Error bars represent the SD between sample triplicates in the assay. C, Neurons were treated with 100 μm DHPG for 0 (untreated), 2, or 10 min in the presence of DHPG alone/DHPG stimulation in the presence of 10 nm OA or 20 μm rapamycin. SAPAP3 protein expression levels as measured by Western blotting of cytoplasmic cell lysates show PP2A activity-sensitive changes in protein expression. In the right panel, SAPAP3 immunoreactivity was normalized to β-III tubulin under the conditions studied, 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).
Figure 6.
Figure 6.
Model depicting a novel, early signaling cascade linking group I mGluR stimulation to FMRP phosphorylation through mTOR and PP2A. Immediate (<1 min) DHPG-induced group I mGluR activation results in rapid FMRP dephosphorylation mediated by increased PP2A activity correlating with a burst of translation (FMRP ligands). Extended group I mGluR activation (1–5 min) reveals that FMRP is rephosphorylated by mTOR-dependent PP2A suppression.
See this image and copyright information in PMC

References

    1. Adams DG, Coffee RL, Jr, Zhang H, Pelech S, Strack S, Wadzinski BE. Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes. J Biol Chem. 2005;280:42644–42654. - PubMed
    1. Antar LN, Afroz R, Dictenberg JB, Carroll RC, Bassell GJ. Metabotropic glutamate receptor activation regulates fragile x mental retardation protein and FMR1 mRNA localization differentially in dendrites and at synapses. J Neurosci. 2004;24:2648–2655. - PMC - PubMed
    1. Bear MF, Huber KM, Warren ST. The mGluR theory of fragile X mental retardation. Trends Neurosci. 2004;27:370–377. - PubMed
    1. Bottorff D, Stang S, Agellon S, Stone JC. RAS signalling is abnormal in a c-raf1 MEK1 double mutant. Mol Cell Biol. 1995;15:5113–5122. - PMC - PubMed
    1. Brown V, Small K, Lakkis L, Feng Y, Gunter C, Wilkinson KD, Warren ST. Purified recombinant Fmrp exhibits selective RNA binding as an intrinsic property of the fragile X mental retardation protein. J Biol Chem. 1998;273:15521–15527. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources