Skip to main content
. Author manuscript; available in PMC: 2018 Jan 27.
Published in final edited form as: Mol Psychiatry. 2016 Oct 18;23(2):271–281. doi: 10.1038/mp.2016.163

Figure 5. NRG1 signaling leads to ubiquitination and degradation of STEP61 in rat cortical neurons and hiPSC neurons.

Figure 5

(a-c) Ubiquitination of STEP is decreased in SZ hiPSC neurons. Ubiquitinated protein were pulled down from SZ1-FB hiPSC neurons (a), SZ2-FB hiPSC neurons (b) or SZ2-GLU hiPSC neurons (c) using tandem ubiquitin binding entities (TUBE) agarose beads as described in the SI Methods, followed by detecting using anti-STEP antibody. (d) Ubiquitination of STEP is decreased in Nrg1+/− mice. Male WT and Nrg1+/− mice (9 months old) were used to isolate ubiquitinated STEP using TUBE. (e) NRG1β treatment results in decreased STEP61 level and increased Tyr phosphorylation of GluN2B and ERK1/2 in cultures. Rat cortical neurons (DIV 14) were pretreated with vehicle (0.1% DMSO) or pan-ErbB receptor antagonist (PD158780, 10 μM) for 30 min, followed by NRG1β (10 nM) for 30 min. Neuronal lysates were analyzed for STEP61, pSTEP61 and tyrosine phosphorylation of STEP substrates. (f) NRG1β treatment leads to increased surface NMDARs in cultures. Cortical neurons were treated with NRG1β (10 nM) for 30 min. Cells were incubated with 1.5 mg/ml Sulfo-NHS-SS-Biotin for 20 min at 4°C, lysed in 1xRIPA buffer. Equal amount of lysates (300 μg) was incubated with NeutrAvidin agarose to isolate biotinylated proteins including surface receptors. Total lysates were used to measure total protein levels. (g) NRG1β administration normalizes STEP61 level and restored Tyr phosphorylation of GluN2B and ERK1/2 in Nrg1+/− mice. Male WT and Nrg1+/− littermates (6-9 months old) were administered vehicle (saline) or NRG1β ECD (50 ng/kg, i.p.) daily for 5 days. Frontal cortices were collected 3 h after the last injections and P2 fractions were used for biochemical analysis. (h) NRG1β treatment results in decreased STEP61 level and increased Tyr phosphorylation of GluN2B and ERK1/2 in human neurons. SZ1-FB and control neurons were treated with NRG1β (10 nM) for 6 h. STEP61 and phosphorylation of STEP substrates were assessed on WB. (i) NRG1 signaling leads to increased ubiquitination of STEP61 in rat cortical neurons. Cultures were pretreated with MG-132 (10 μM) for 1 h, followed by NRG1β treatment (10 nM, 30 min). All STEP species were immunoprecipitated using anti-STEP antibody and probed with anti-ubiquitin antibody. (j) NRG1β treatment leads to increased ubiquitination of STEP61 in human neurons. SZ1-FB and control neurons were pretreated with MG-132 (10 μM, 1 h), followed by NRG1β treatment (10 nM, 6 h). Ubiquitinated STEP species were pulled down using TUBE and detected by anti-STEP antibody. All data were expressed as mean ± SEM and statistical significance was determined using one-way ANOVA with Bonferroni test (*P < 0.05, **P < 0.01, n = 6 each group).