Project PXD001074

PRIDE Assigned Tags:
Biological Dataset Biomedical Dataset

Summary

Title

BDNF stimulation of protein synthesis in cortical neurons requires the eIF4E kinase Mnk1

Description

Although the Mnks have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here we explore how BDNF stimulates protein synthesis in cortical neurons. Using a combination of pharmacological and genetic approaches, we show that this effect depends on MEK/ERK signaling and on the downstream kinase, Mnk1, which phosphorylates eIF4E. Translation initiation is mediated by the interaction of eIF4E with the m7GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins such as CYFIP1, which together with its partner, FMRP acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on Mnk1. Finally, using a novel combination of BONCAT and SILAC, we identify the a subset of proteins whose synthesis is upregulated by BDNF signalling via Mnk1 in neurons. The This subset of Mnk1-sensitive proteins are enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of Mnk1 Mnk1-sensitive proteins and proteins encoded by known FMRP-binding mRNAs. Taken together, our data implicate Mnk1 as a key component of BDNF-mediated translational regulation in neurons.

Sample Processing Protocol

Primary cortical neurons were isolated and cultured with Neurobasal SILAC medium containing either 89 mg/L heavy-arginine (13C6 + 15N4) and 154 mg/L heavy-lysine (13C6 + 15N2) or 87 mg/L medium-arginine (13C6) and 150 mg/L medium-lysine (2H4). At 10-12 DIV cells were starved of methionine for 30 min, then stimulated with 25 ng/mL of BDNF in the presence of 2 mM azidohomoalanine (AHA) for 2 hr. Cells were washed twice in cold PBS and lysed in urea lysis buffer (300 mM Tris pH 8, 6.2% CHAPS, 1.5 m NaCI, 0.8 g/mL) containing 2X concentration of protease inhibitors (Roche). Lysates were then sonicated on ice and protein concentrations were determined using a 660 assay (Pierce). Equal amounts of protein from medium and heavy labelled neurons were then mixed prior to performing click-chemistry to an alkyne agarose resin overnight with agitation at room temperature following the manufacturer’s instructions (Invitrogen; C-10416). After incubation, the resin was washed once in H2O and re-suspended in 1 mL of SDS wash buffer (100 mM Tris pH 8, 1% SDS, 250 mM NaCI, 5 mM EDTA) with 10 mM DTT and incubated at 70 C for 15 min. The resin was then centrifuged at 1,000 x g for 5 min, supernatant discarded, and resuspended in SDS wash buffer with 40 mM iodoacetamide for 30 min in the dark at room temperature. The resin was then transferred to a spin column (Thermo, 89868) and washed eight times each with SDS wash buffer, urea wash buffer (8M urea/100 mM Tris (pH 8)) and 20 % acetonitrile followed by three washes with 50 mM ammonium bicarbonate. After the last wash, the resin was re-suspended in 500 µL ammonium bicarbonate, transferred to a fresh tube, and urea was added (~1.5 M final concentration). The resin was centrifuged, supernatant removed and 0.1 µg Lys-C (Roche) protease was added and the resin incubated at 37 C for 4 h with agitation. Then calcium chloride (final concentration 0.1 mM) and 1 µg sequencing grade trypsin (Promega) were added and the resin incubated at 37 C overnight with agitation. The resin digest was then added to a spin column, centrifuged at 1,000 xg for 2 min, and the flow through was kept. Formic acid was added (~1 % final concentration). Peptide mixtures were cleaned with 100 µL C18 tips (Thermo Scientific) with three iterations, eluting into 2-98 % HPLC grade CH3CN (Fisher Scientific), 0.1 % analytical grade formic acid (Fisher Scientific). Eluates were lyophilised in a vacuum concentrator for 4 h at room temperature (Eppendorf). Lyophilised peptides were reconstituted in 30 µl of loading solution (2 % acetonitrile, 0.1 % formic acid) and 7 µl were loaded by a Dionex Ultimate 3000 (Thermo Scientific) at 20 µL/min for 6 min onto a C18 PepMap100 trapping cartridge (100 μm × 300 µm internal diameter, 5 μm particle) (Thermo Scientific). Peptides were eluted at 300 nl/min over a gradient of 2-18 % (355 min), 18-35 % (45 min) and 35-85 % (25 min) organic phase (95% acetonitrile, 5% DMSO (Sigma), 0.1% formic acid) in aqueous phase (2% acetonitrile, 5% DMSO, 0.1% FA) and resolved on an Acclaim PepMap 100 column (C18 75 μm × 50 cm, 2 μm particle) retrofitted to a PicoTip nESI emitter (New Objective). Electrospray ionisation was conducted at 2.4 kV and ions were characterised with an Orbitrap Elite (Thermo Scientific) at 240,000 mass resolution. The top 12 +2 and +3 precursor ions MS scan (minimum intensity 1000) were characterised by HCD (30,000 mass resolution, 1.2 Da isolation window, 40 keV collision energy) and CID (ion trap MS, 2 Da isolation window 35 keV) with a dynamic exclusion (±5 ppm) of 200 seconds.

Data Processing Protocol

Peptide spectrum matching and quantifications were performed with Proteome Discoverer 1.4 (Thermo Scientific) with SequestHT against the UniprotKB SwissProt mouse proteome (downloaded 03/2014). For matching and quantitation, precursor tolerance was set at 10 and 3 ppm, respectively. Fragment matching was set at 0.02 and 0.5 Da for high-energy collisional dissociation (HCD) and collision induced dissociation (CID), respectively. Target-decoy searching allowed for 1 missed cleavage, a minimum length of 6 amino acids and a maximum of 3 variable (1 equal) modifications of; Met->Aha (M), deamidation (Asn, Gln) or phosphorylation (Ser,Thr or Tyr). Carbamidomethyl (Cys), were set as fixed modifications with Lys (+4 Da), Arg (+6 Da) and Lys (+8 Da), Arg (+10 Da) searched to determine medium and heavy labelled peptides, respectively. False discovery rate (FDR) was estimated with the percolator and results were filtered <5 % FDR. All three replicates were analysed separately and opened in unison to enable paralleled protein grouping and quantitation.

Contact

Harvey Johnston, University of Southampton
Spiro D. Garbis, Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK ( lab head )

Submission Date

23/06/2014

Publication Date

06/11/2014

Publication

    Genheden M, Kenney JW, Johnston HE, Manousopoulou A, Garbis SD, Proud CG. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1. J Neurosci. 2015 Jan 21;35(3):972-84 PubMed: 25609615