S6K1 Phosphorylates and Regulates Fragile X Mental Retardation Protein (FMRP) with the Neuronal Protein Synthesis-dependent Mammalian Target of Rapamycin (mTOR) Signaling Cascade

Abstract

Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a group I metabotropic glutamate receptor (mGluR) activity-dependent manner following brief protein phosphatase 2A (PP2A)-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthesis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus, finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mammalian target of rapamycin (mTOR), ERK1/2, and PP2A. Further, the loss of hippocampal S6K1 and the subsequent absence of phospho-FMRP mimic FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for proteinsynthesis-dependent synaptic plasticity.Fragile X syndrome is the most common form of inherited mental retardation and is caused by a functional absence of the RNA-binding protein, fragile X mental retardation protein, FMRP.3 The protein, FMRP, is known to associate with ϳ3% of the mammalian brain mRNAs, repressing their translation; many of these target mRNAs indeed appear overtranslated in the absence of FMRP (1). Electrophysiology of the fragile X mouse model has revealed a synaptic deficit in the hippocampus with elevated group I metabotropic glutamate receptor (mGluR)-mediated long term depression. Accordingly, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization is enhanced in the absence of FMRP, all thought due to constitutive overtranslation of an long-term depression (LTD)-required FMRP target mRNA(s) (2-4). Group I mGluR activity is also known to influence FMRP transport and synthesis (5, 6); however, little is known about regulation of FMRP itself.Post-translational modifications are known regulators of activity-dependent protein synthesis (7), and FMRP is known to be phosphorylated at a highly conserved serine at position 499. The effects of FMRP phosphorylation on translation by ribosomal run-off assays suggested that non-phosphorylated FMRP associates with actively translating ribosomes, whereas phosphorylated FMRP is found in the context of potentially stalled ribosomes (8). We recently determined that FMRP is dephosphorylated by protein phosphatase 2A (PP2A) and, immediately following mGluR stimulation, PP2A dephosphorylates FMRP, corresponding with the translation of SAPAP3, an FMRP target mRNA (9). SAPAP3 is a post-synaptic scaffolding protein associated with PSD95, whose message was previously identified as an FMRP target mRNA in the mouse brain following FMRP immunoprecipitation and microchip analyses (10,35,36). Less than 2 min following mGluR activation, FMRP is rephosphorylated in a PP2A-and mamm...

Fragile X Mental Retardation Protein Regulates the Levels of Scaffold Proteins and Glutamate Receptors in Postsynaptic Densities

“…and synaptic levels of the corresponding transcripts, SAPAP1 and SAPAP3 are more abundant at neocortical and hippocampal synapses of adult Fmr1 Ϫ/Ϫ mice, respectively. Consistently, an elevated SAPAP3 concentration was observed in hippocampal lysates of FMRP-deficient mice (68). Here we also show that mRNAs encoding SAPAP1-3 are in vivo brain targets of FMRP.…”

Genotype and cognitive phenotype of patients with tuberous sclerosis complex

“…[7][8][9][10][11][12][13] A bimodal intellectual quotient (IQ) distribution in the total TSC population has been suggested 7 and was recently refined as being observed only in the TSC2 population. 10 TSC patients with germline TSC1 and TSC2 mutations have only one fully functional TSC2 allele in all their cells, and this condition could lead to neurocognitive dysfunction through the mechanism of haplo-insufficiency, [14][15][16] similar to Fragile-X syndrome and Neurofibromatosis type 1. [14][15][16] However, in TSC there are additional factors which may contribute to cognitive impairment, including loss of heterozygosity, which may contribute to tuber development, 17,18 and effects of early onset and refractory epilepsy.…”

“…10 TSC patients with germline TSC1 and TSC2 mutations have only one fully functional TSC2 allele in all their cells, and this condition could lead to neurocognitive dysfunction through the mechanism of haplo-insufficiency, [14][15][16] similar to Fragile-X syndrome and Neurofibromatosis type 1. [14][15][16] However, in TSC there are additional factors which may contribute to cognitive impairment, including loss of heterozygosity, which may contribute to tuber development, 17,18 and effects of early onset and refractory epilepsy. Thus far, no associations have been found between specific TSC mutation types and cognitive outcomes, 10,19 although there are reports on associations with epilepsy and psychiatric features.…”

Dendritic mRNA Targeting of Jacob and N-Methyl-d-aspartate-induced Nuclear Translocation after Calpain-mediated Proteolysis

“…RNA extracted from immunoprecipitates was probed for Jacob, SAPAP3, and BC1 RNAs by RT-PCR and real time RT-PCR. SAPAP3-and BC1-specific amplifications served as positive and negative controls for FMRP-associated RNAs, respectively (19,24,25). RT-PCR data showed that Jacob and SAPAP3 mRNAs were present in both the WT-F-IP and WT-P-IP but were basically absent or only weakly detected in the KO-F-IP and WT-IgG-IP (Fig.…”

“…5B). Similar to SAPAP3 mRNAs, known FMRP targets (24,25), Jacob transcripts were found to specifically associate with FMRP (Jacob mRNA enrichment: WT-F-IP versus KO-F-IP ϭ 6.3 Ϯ 1.8 and WT-F-IP versus WT-IgG-IP 11.1 Ϯ 0.3; SAPAP3 mRNA enrichment: WT-F-IP versus KO-F-IP ϭ 9.6 Ϯ 3.6 and WT-F-IP versus WT-IgG-IP ϭ 10.8 Ϯ 0.9; n ϭ 4); and PABP (Jacob and SAPAP3 mRNA enrichment: WT-P-IP versus WT-IgG-IP ϭ 32.4 Ϯ 3.3 and 28.5 Ϯ 2.1, respectively; n ϭ 4). To analyze whether FMRP plays a role in dendritic targeting of Jacob mRNAs, we performed fluorescent in situ hybridization on cultured cortical mouse neurons.…”