Phorbol Ester Augments Bicuculline Induced GluA2 Ubiquitination

Phorbol ester augments Bicuculline induced GluA2 ubiquitination

ABSTRACT

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AMPA receptor (AMPARs) have recently been shown to undergo activity-dependent ubiquitination in mammalian neurons. GluA1 and GluA2 are the predominant AMPARs subunit that existed in hippocampal and cortical pyramidal neurons. It has been established that cross-talk of GluA2 ubiquitination and phosphorylation co-operatively act on its trafficking. However, such cross modulation and the underlying molecular mechanism remain poorly understood in GluA2 subunit. Here, we reveal that GluA2 is ubiquitinated at C-termini by bicuculline-induced ubiquitination, whereas the major sites of ubiquitination were mapped to K870 and K882 in GluA2. Interestingly, we found that K870 ubiquitination decrease Ser880 phosphorylation while K882 ubiquitination causes the increase of Ser880 phosphorylation. GRIP1 and PICK1 have been reported as GluA2 interacting protein that regulating the trafficking of GluA2. We report that the dissociation with GRIP1 and GluA2 interaction result in augment of ubiquitination. Remarkably, this enhancement on ubiquitination is disrupted by the dissociation of both GRIP1 and PICK1 interaction to GluA2. Lastly, we report that phorbol ester augments bicuculline-induced ubiquitination, in a way that is specific to GluA2 and independent from Ser880 phosphorylation. These data indicate that GluA2 ubiquitination is highly complex regulatory signal for controlling AMPAR trafficking and function, which may be essential for synaptic plasticity.

1. INTRODUCTION

Efficient neurotransmission is essential for information processing in the brain, which underpins all activities ranging from muscle movement and motor coordination to cognitive function such as learning and memory, perception and emotion. The strength and efficacy of neurotransmission can be dynamically modulated by neuronal activity at synapses, a process known as synaptic plasticity. One of the major factors that regulates synaptic plasticity is the dynamic modulation of the number of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) on neuronal plasma membrane. AMPARs are transmembrane ionotropic glutamate receptors that mediate the majority of fast excitatory neurotransmission in mammalian central nervous system. They are tetrameric assemblies of two dimers of various subunits. The predominant AMPAR subtype expressed in hippocampal and cortical pyramidal neurons is composed of two GluA1 and two GluA2 subunits. In general, the increase in the number of synaptic AMPARs result in long-term potentiation (LTP), whereas their removal leads to long-term depression (LTD) [1]. Alzheimer’s disease (AD) is characterized by the loss of synapses and the levels of surface AMPARs[2]. Despite this, the molecular mechanisms underlying the control of activity-dependent removal of AMPARs and their degradation remain poorly understood.

Surface expression of AMPARs is determined in most parts by their dynamic trafficking into and out of the plasma membrane and is tightly regulated by AMPAR interacting proteins and various post-translational modifications. The latter includes protein ubiquitination, phosphorylation, and palmitoylation on the C-terminal tails of AMPAR subunits. [3]. Ubiquitination is a reversible modification  that covalently attaches of one or more (mono- or poly-ubiquitination) highly conserved 76 amino acid ubiquitin moiety to target proteins [4]. Ubiquitination is known to regulate diverse physiological processes such as protein degradation, endocytosis and the intracellular sorting and trafficking of transmembrane protein [4]. All four subunits of AMPARs, GluA1-4, undergo activity-dependent ubiquitination which controls their trafficking towards late endosomes for degradation [5-9]. Several E3 ubiquitin ligases have also been demonstrated to mediate the ubiquitination of GluA1 and GluA2 subunits of AMPARs, including Nedd4-1 and RNF167 [6, 7, 10].

In cultured neurons, the ubiquitination of all four AMPA subunits is robustly induced by the application of the AMPAR agonist, AMPA, as well as bicuculline, a specific GABA_A receptor antagonist [5-9]. Although both AMPA- and bicuculline-induced ubiquitination of AMPARs are dependent on Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels (L-VGCCs), each of them engages slightly different signalling pathways. AMPA-induced ubiquitination acts by direct binding and activation of both synaptic and extrasynaptic AMPARs. In contrast, bicuculline-mediated ubiquitination relies on presynaptic release of glutamate and should therefore activates only synaptic AMPARs. In addition to AMPARs and L-VGCCs, bicuculline-induced ubiquitination of AMPARs also requires the NMDA and group I mGluR receptors-dependent signalling[9]. Although it is not clear, it is conceivably possible that AMPA- and bicuculline-induced ubiquitination of AMPARs differentially regulate the sorting of AMPARs in the intracellular endosomal compartments.

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The GluA1 subunit is phosphorylated at two major serine residues in the C-terminal tail, namely Ser-831 and Ser-845, by protein kinase C (PKC) or Ca²⁺/Calmodulin-dependent kinase II (CaMKII) and protein kinase A (PKA), respectively [11-15]. We have previously demonstrated the cross-talk between GluA1 ubiquitination and phosphorylation, in particular phosphorylation at Ser-845, which is crucial for the recycling of AMPARs[2]. In this study, we found that phosphorylation of Ser-845 reduces the binding of Nedd4-1 on GluA1. Consequently, ligand-induced ubiquitination of the GluA1 subunit is attenuated in the Ser-845 phospho-mimetic mutant, promoting the forward trafficking of AMPARs to the plasma membrane. Interestingly, the GluA1 ubiquitin-defective mutant also enhances forskolin-induced phosphorylation of Ser-845 in cultured neurons. Overall, these data provide new mechanistic insights into how cross-modulation of GluA1 phosphorylation and ubiquitination can fine-tune the dynamics of AMPAR intracellular trafficking and the sorting decision that ultimately determines the number of receptors on the cell surface.

The GluA2 is also phosphorylated in the C-terminal tail, notably on Tyr-876 and Ser-880 by the Src family tyrosine kinase and PKC, respectively, both of which act to promote GluA2 endocytosis [16, 17]. Mechanistically, phosphorylation of Ser-880 disrupts the interaction of GluA2 with glutamate receptor interacting protein 1/2 (GRIP1/2), which stabilises GluA2 containing receptors at synapses. The same phosphorylation also promotes GluA2 binding to protein interacting with C-kinase 1 (PICK1), which facilitates the endocytosis and intracellular retention of AMPARs. How the phosphorylation of GluA2 cross-modulate its ubiquitination status, and vice versa, remains an open question. Given the role of GluA2 phosphorylation in promoting GluA2 endocytosis, we hypothesise that the phosphorylation and ubiquitination GluA2 co-operatively act in the same pathway to facilitate AMPAR internalisation. In this study, we specifically focussed on the modulation of bicuculline-induced GluA2 ubiquitination by the PKC signalling pathway.

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RESULTS:

GluA2 are ubiquitinated on C-terminal lysine residues by bicuculline- To determine the specific bicuculline-induced ubiquitination of GluA2 site, we start with mapping the specific lysine that ubiquitin is attached on GluA2 C-terminal. All lysine on the C-terminal tails of GluA2 were mutated into arginine, either single mutation or in combination which were shown in Figure 1A.  High transfection efficient were ensured and achieved by electroporation of cortical neurons pH-GluA2(either WT or mutant) DIV0 (Figure 1A). At DIV13, neurons were treated with bicuculline, lysed (in denaturing condition to ensure complete dissociation of AMPAR subunit complex) and immunoprecipitated with anti-GFP antibodies. We found that the fully mutation of C-terminal lysine into arginine completely abolished bicuculline-induced ubiquitination of GluA2(GluA2-K838/844/847/850/870/882R, 10.8%±4.7% of WT, Figure 2B-2C). This suggest that the bicuculline-induced ubiquitination of GluA2 occurs in the C-terminal domain. The analysis of each individual lysine mutation of GluA2 reveal that K870(55.75%±10.14% of WT) and K882(49.50%±1.4% of WT) were the major ubiquitination sites (Figure 2B-2C). Furthermore, GluA2 K870/882R double mutant showed an additive effect in the reduction of bicuculline-induced ubiquitination (19.67%±5.0% of WT, Figure 2B-2C). There data provide us the fundamental molecular tool for examine the role of ubiquitination with GluA2 phosphorylation and regulation of GluA2 trafficking and function.

The cross-talk between GluA2 phosphorylation and ubiquitination- S880 phosphorylation of GluA2 is well characterised and known to be important for the trafficking of GluA2 and synaptic plasticity[17-20]. We previously established  that the GluA1 phosphorylation and ubiquitination are tightly cross modulated[2]. We proposed that there are cross-modulation of GluA2 phosphorylation and ubiquitination to fine-tune the trafficking and stability of GluA2. To address this hypothesise, we examined the effect of GluA2 ubiquitin-defective mutant, namely K870R, K882R and double mutants on S880 phosphorylation induced by PMA. We found that neurons overexpression of K882R and dKR abolished S880 phosphorylation (K882R, 34.96%±3.38% of WT-PMA; dKR, 44.36%±13.32% of WT-PMA). Interestingly, neurons that overexpressed pH-GluA2 K870R displayed significantly enhanced level of S880 phosphorylation (GluA2 K870R, ~150% of WT-PMA; Figure 2A-2B). Such cross-modulatory regulation between S880 phosphorylation with K870 and K882 ubiquitination illustrated the complexity and specificity regulation of GluA2 trafficking by post-translational modification. 

Disruption of GRIP1/GluA2 interaction enhance GluA2 ubiquitination- GRIP1 and PICK1 are PDZ-interacting protein that play role in the trafficking and stabilisation of GluA2 on plasma membrane. Phosphorylated S880 is known to dissociate GRIP1/GluA2 interaction and promote PICK1/GluA2 interaction which regulate the intracellular sorting of GluA2. We proposed that GRIP1 and PICK1 might be playing role in the bicuculline-induced ubiquitination. To test this possibility, we electroporated cortical neurons with pH-GluA2 (either WT or mutants) at DIV0. There are 4 construct that were used which are SVKI, the WT GluA2; AVKI, the S880 phospho-defective construct of GluA2 which remain interaction with GRIP1 and PICK1; EVKI, the S880 phospho-mimetic construct of GluA2 which lose interaction with GRIP1 while remain PICK1 interaction; and SVKE, PDZ dead GluA2 construct that lose both GRIP1 and PICK1 interaction. At DIV12, neurons were treated with bicuculline, lysed in denaturing condition and immunoprecipitated with anti-GFP antibodies. We found that there are significantly increase of ubiquitination in EVKI (195%±32.12% of WT, Figure 3A-3B). Interestingly, this increase is abolished with the eliminating of both GRIP1 and PICK1 interaction with GluA2 (95.71%±17.19% of WT, Figure 3A-3B). Collectively, those data show that ubiquitination of GluA2 is tightly regulated by GRIP1 and PICK1.

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Synergistic effect of phorbol ester and bicuculline on ubiquitination- Phorbol ester, the PKC activator has been showed to induce GluA2 internalization by S880 phosphorylation and decrease the surface expression of GluA2[17]. Our lab had previously demonstrated AMPAR are internalized without ubiquitinated but are subsequently ubiquitinated, either by E3-ligase that co-exist in endocytosis vesicle or at early endosome[9]. We hypothesised that PMA treatment might result in increase of GluA2 ubiquitination due to the increase of internalisation. To do this, we treat primary cortical neuron with either ACSF, bicuculline, PMA or PMA following bicuculline at DIV14, lysed and immunoprecipitated with anti-ubiquitin antibodies. In present study, we showed that activation of PKC-mediated phosphorylation by PMA alone does not affecting the ubiquitination (59.68%±9.21% of Bic-GluA2, Figure4A-4B). Interestingly, PMA and bicuculline act synergistically in the ubiquitnation of GluA2(210.60%±29.43% of Bic-GluA2, Figure 4A-4B). Remarkably, this phenomenon can only be observed in GluA2 subunit, whereas the pre-activation of PKC by PMA followed by bicuculline-induced ubiquitination level of GluA1(116.4%±23.79% of Bic-GluA1, Figure 4A-4C) remain unchanged. This indicate that PKC-mediated phosphorylation is involved in the signalling pathway of ubiquitination, in a way that specific and GluA2.

AVKI does not occlude synergistic effect of phorbol ester and bicuculline- To address the underlying mechanism for the synergistic effect of phorbol ester and bicuculline, we hypothesised that the well-studied PKC-mediated phosphorylation site of GluA2 S880 might play a key role. To test this possibility, we electroporated pH-GluA2 (either WT or AVKI mutant) on primary cortical neurons at DIV0. At DIV12, neurons were treated with either ACSF, bicuculline, PMA or PMA following bicuculline, lysed in denaturing condition and immunoprecipitated with anti-GFP antibodies. However, the robust increase of ubiquitination by synergistic effect of phorbol ester and bicuculline were observed in both WT (226.20%±31.66% of Bic-WT, Figure 5A-5B) and AVKI (226.40%±40.25% of Bic-AVKI, Figure 5A-5B). This suggest that the inhibition of PKC-mediated phosphorylation of S880 by AVKI does not block the synergistic effect of PMA and bicuculline on GluA2 ubiquitination that we revealed in figure 4A-4B.

DISCUSSION:

Post-translational modifications of AMPARs, such as phosphorylation and ubiquitination is a highly regulated process that control vital cellular processes in the brain, including synaptic plasticity, learning and memory. GluA2, the well-studied AMPA subunits has shown highly regulated by both phosphorylation and ubiquitination. AMPA, bicuculline, and PMA have been  widely used as an ubiquitination/PKC-mediated phosphorylation-inducer among those studies [9, 17, 21]. However, the differentiation of signalling pathways between AMPA and bicuculline and the cross-talk of GluA2 phosphorylation and ubiquitination remain controversial and need to examine systematically. Our lab suggested that GluA2 AMPA-induced ubiquitination happened on C-terminal while K870 and K882 are the major ubiquitination site under ligand-induced ubiquitination[9]. This finding is replicated in this study with bicuculline-induced ubiquitination and confirmed both AMPA- and bicuculline-induced ubiquitination promote GluA2 ubiquitination on K870 and K882, albeit their signalling for inducing ubiquitination is different. Furthermore, the bicuculline-induced ubiquitination was abolished on 6KR which further confirmed that GluA2 is ubiquitinated on C-terminal.

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Recent study has suggested that K882A ubiquitin mutant abolished the PKC-mediated phosphorylation of S880[21]. We reproduced the same finding by K882R ubiquitin-defective mutant and interestingly found that another ubiquitin-defective mutant, K870R enhances the phosphorylation of S880 while compare with the WT. This finding indicate that ubiquitination of GluA2 would not result in a absolute alteration of S880 phosphorylation. Depend on the site of ubiquitination, S880 phosphorylation either increase with the ubiquitination of K882 or decrease with the ubiquitination of K870.

It is well established that GluA2 phosphorylated at S880 by PKC disrupt its interaction with GRIP1 while the PICK1 interaction retained[17]. We illustrated the ubiquitination is increased with the dissociation of GRIP1 in EVKI. Given that GRIP1 function in GluA2 anchorage at plasma membrane and the dissociation of GRIP1 is required for GluA2 endocytosis[18, 22]. The increase of ubiquitination by EVKI is likely due to the enhance of GluA2 endocytosis and this further confirmed that ubiquitination of GluA2 happened after endocytosis. Notably, the increase of GluA2 ubiquitination by the dissociation with GRIP1 is eliminated in SVKE mutant. This might be an indication that the PICK1 is involved in GluA2 ubiquitination, perhaps by its interaction with GluA2 which result in enhancement of GluA2 endocytosis.

Our main result show that the synergistic effect of phorbol ester and bicuculline is specific for GluA2 ubiquitination. This is the first study to demonstrate that phorbol ester contribute to the ubiquitination of GluA2. To address the underlying mechanism of this finding, we first hypothesised that endocytosis of GluA2 by PKC-mediated S880 phosphorylation could play a role for this finding. However, our data on S880 phospho-defective mutant, AVKI suggest that this specific enhancement of GluA2 ubiquitination is independent from S880 phosphorylation. Next we question what would be the mechanism that contributes for the robust increase of GluA2 ubiquitination by PKC activation following bicuculline? We hypothesised that one of the key potential molecule responsible for  enhancement of ubiquitination is PKC-mediated phosphorylation of RNF167, an E3 ligase that known to regulate GluA2 ubiquitination[10].  The knockdown of RNF167 had shown reduction in GluA2 ubiquitination upon bicuculline treatment, however does not alter GluA1 ubiquitination levels[10]. Studies through kinase-specific phosphorylation prediction tool have shown that five serine sites within RNF167 PKC (S336, S340, S342, S344, and S345) could be phosphorylated [23]. We have previously demonstrated that mGluR1 is involved in bicuculline-induced ubiquitination [9]. It is understood that mGluR1 activation lead to diacylglycerol production which would result in the activation of PKC regulatory machinery in the presence of calcium. Together with those findings described above, we hypothesised a working model for the underlying mechanism of phorbol ester enhancement of GluA2 ubiquitination (Figure 6). First, introducing of phorbol ester greatly enhance activation of PKC which mimic the effect of mGluR1 activation by bicuculline. The raise of activated PKC subsequently activate RNF167 by RNF167phosphorylation that potentially lead to increase activity of ubiquitination. Next, the following stimulation of synaptic activity by bicuculline result in the increase of GluA2 internalisation. Surface GluA2 subunits are internalised without ubiquitination but subsequently ubiquitinated, either in GluA2 containing vesicle or early endosome by the vast activated RNF167. Ubiquitinated GluA2 then sorted into late endosome and degraded.  However, this hypothesis need to be addressed by further experiment.

EXERIMENTAL PROCEDURES

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DNA Constructs, antibodies and pharmacological agents:

DNA constructs encoding N-terminal pH-sensitive GFP (pHluorin)-tagged full-length GluA2, either wild-type, K870R, K882R and K870/882R double mutant (dKR), have been described previously [9]. pHluorin-tagged GluA2 phosphorylation mutants were generated by site-directed mutagenesis into alanine (S880A or AVKI) or glutamate (S880E or EVKI), which blocks or mimics GluA2 Ser-880 phosphorylation, respectively.

Specific antibodies against GluA2(JH1708), p-Ser880 GluA2, GluA1(4.9D) and GFP(RMO2) were generated in the R.L.H laboratory. The following antibodies were purchased from commercial source: anti-ubiquitin clone P4D1(Santa Cruz, Sc-8017) and p-Ser831 GluA1 (Millipore, MAB2263)

Bicuculline (abcam, product code: ab120109) were used to induce the ubiquitination of AMPARs. PMA (phorbol 12-myristate 13-acetate, Sigma), phorbol ester that were used to induce the PKC-mediated phosphorylation.

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Neuronal culture and electroporation

Primary cortical rat neurons culture were from E18 rat pups as described previously[9]. Briefly, they were seeded onto dishes coated with Poly-L-lysine in Neurobasal growth medium supplemented with 2% B27, 2 mM GlutaMAX, 50 units/ml penicillin, 50 μg/ml streptomycin, and 5% fetal bovine serum (FBS). After 24 hours post seeding, cortical neurons are switched to neurobasal medium with 1%FBS and fed twice a week. Ubiquitination assay on cortical neurons was performed on DIV14. At some experiments, neurons were electroporated by using Amaxa Nucleofector II system (Lonza) prior to plating at DIV0 and were used on DIV12-14.

Ubiquitination assay and immunoprecipitation

Ubiquitination of GluA1 and GluA2 were induced by incubating neurons in artificial cerebrospinal fluid(ACSF; 25mM HEPES, 120mM NaCl, 5mM KCl, 2mM

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CaCl2.2H2O, 2mM

MgCl2.6H2O, 30mM Glucose, pH to 7.4 and filtered before use) containing 40µM of Bicuculline  at 37°C for 10 minutes. In some experiments, neurons were either incubated with 1µM of PMA or incubated with 1µM PMA at 37°C for 10 minutes followed by 40µM of Bicuculline at 37°C for 10 minutes. Neurons were then lysed in warm 1%SDS (in PBS) and diluted in ten volumes of cold lysis buffer (1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM Na-pyrophosphate in PBS, pH to 7.4 and filtered before use) supplemented with 10mM of N-Ethylmaleimide (NEM, Sigma Lot No: 031M1734V) and Complete-EDTA free protease inhibitor cocktails (Roche). Lysates were sonicated for 10 second (amp-20%, zero pulse) and clear with protein A-(Sigma, product number: GE17-5280-01 Lot number: 10248703) or G-(Sigma, product number: GE17-0618-01 Lot number: 10246735) Sepharose beads. Part of precleared lysate were collected, denatured by 3x SDS and boiled prior to SDS-PAGE. The rest of precleared lysate were then incubated with either protein A- or G- Sepharose beads coupled with GFP or ubiquitin antibodies overnight at 4°C, washed for four times with RIPA buffer (50mM Tris, 100mM NaCl, 2mM EDTA, 2mM EGTA, 10mM Na-pyrophosphate, 50mM NaF, 1% TritonX-100, 0.5% Na-deoxycholate, 0.1% SDS, pH to 7.4 and filtered before used) and eluted in 2x SDS buffer. The immunoprecipitated proteins were resolved by SDS-PAGE and probed by western blot analysis with specific antibodies against ubiquitin, GluA1, p-S831, GluA2 and p-S880.

Phosphorylation assay

Phosphorylation of either pH-GluA2 or mutants were induced by incubating neurons in neurobasal media containing 10µM of PMA for 10 minutes at 37°C. Neurons were lysed immediately after the PMA treatment with 1x SDS and boiled for 10 minutes. Proteins were resolved by SDS-PAGE and probed by western blot analysis with specific antibodies against p-S880, GluA2 and Tubulin.

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Western Blot result analysis

The western blot band intensity was measure by Image Studio software. In the ubiquitination assay, the level of ubiquitinations are normalised with either endogenous or exogenous GluA2 signal. Quantification graph were then generated and analysed by GraphPad Prism 7.

References

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FIGURE LEGENDS

Figure 1. The GluA2 subunits of AMPARs are ubiquitinated on carboxyl tails lysine residues. (A)The alignment of GluA2 C-terminal amino acid sequence, with all lysine residues highlighted in red. (B) Cortical neurons were electroporated with either pH-GluA2 individual mutant constructs (K838, 844, 847, 850, 870, 882R), double mutants (K838, 870R; K838,882R; K870,882R), triple mutant(K838,870,882R) or all combination (K838, 844, 847, 850, 870, 882R), prior to plating. At DIV13, neurons were treated with bicuculline for 10 minutes at 37°C, lysed and immunoprecipitated with anti-GFP antibodies. Eluted proteins and total lysates were then proceeded to western blot and probed with anti-ubiquitin and anti-GluA2 antibodies. (C) The effects of lysine/arginine mutants on pH-GluA2(B) ubiquitination were quantified after normalizing against immunoprecipitated GluA2. Data represent mean± SEM of band intensities normalised to WT(control) neurons (Ordinary one-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n = 3 to 6 per group).

Figure 2.  Ubiquitination tightly regulate GluA2 phosphorylation at Ser-880. (A) Cortical neurons were electroporated with either pH-GluA2 WT, K870R, K882R or double mutant (K870,882R) prior to plating. At DIV14, neurons were treated with PMA for 10 minutes at 37°C and lysed immediately with 1xSDS. Lysates then subjected to western blot and probed with anti-pSer880 and GluA2 antibodies. (B) The effect of lysine/arginine mutants on S880 phosphorylation were quantified after normalising against total GluA2. Data represent mean± SEM of band intensities normalised to WT-PMA neurons (RM one-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n = 5 per group).

Figure 3.  EVKI increase GluA2 ubiquitination while SVKE block this augmentation.  (A) Cortical neurons were electroporated with either pH-GluA2 SVKI, AVKI, EVKI and SVKE prior to plating. At DIV12, neurons were treated with bicuculline for 10 minutes at 37°C, lysed with 1% SDS and immunoprecipitated with anti-GFP antibodies. Eluted proteins and total lysate were then subjected to western blot and probed with anti-ubiquitin and GluA2 antibodies. (B)The effect of pH-GluA2 mutant on GluA2 ubiquitination were quantified after normalising against immunoprecipitated GluA2. Data represent mean± SEM of band intensities normalised to WT-Bic neurons (Ordinary one-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n = 5 to 7 per group).

Figure 4. Robust increase of GluA2 ubiquitination by phorbol ester following bicuculline. (A) At DIV14, neurons were treated with either ACSF, bicuculline, PMA for 10 minutes at 37°C respectively or PMA for 10 minutes at 37°C following bicuculline for 10 minutes at 37°C. All neurons were lysed with 1% SDS and immunoprecipitated with anti-ubiquitin antibodies. Eluted proteins and total lysate were then subjected to western blot and probed with anti-GluA2, pSer880, GluA1 and pSer831 antibodies. (B-C) The effect of individual or double combination of drugs on GluA1 and GluA2 ubiquitination were quantified after normalising against total GluA2 or GluA2.  Data represent mean± SEM of band intensities normalised to WT-Bic neurons (Friedman test; ∗p < 0.05; ∗∗p < 0.01; n = 9 per GluA1 group; n=11 per GluA2 group).

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Figure 5. Synergistic effect of phorbol ester and bicuculline retain in AVKI. (A) Cortical neurons were electroporated with either pH-GluA2 SVKI or AVKI prior to plating. At DIV12, neurons were treated with either ACSF, bicuculline, PMA for 10 minutes at 37°C respectively or PMA for 10 minutes at 37°C following bicuculline for 10 minutes at 37°C. Neurons were lysed with 1% SDS and immunoprecipitated with anti-GFP antibodies. Eluted proteins and total lysates were then subjected to western blot and probed with anti-ubiquitin, pSer880 and GluA2 antibodies. (B)  The effect of either SVKI or AVKI on GluA2 ubiquitination were quantified after normalising against immunoprecipitated GluA2.  Data represent mean± SEM of band intensities normalised to WT-Bic neurons (RM one-way ANNOVA test; ∗p < 0.05;; n = 7 group).