Rapid and bi-directional regulation of AMPA receptor phosphorylation and trafficking by JNK

Abstract

Jun N-terminal kinases (JNKs) are implicated in various neuropathological conditions. However, physiological roles for JNKs in neurons remain largely unknown, despite the high expression level of JNKs in brain. Here, using bioinformatic and biochemical approaches, we identify the AMPA receptor GluR2L and GluR4 subunits as novel physiological JNK substrates in vitro, in heterologous cells and in neurons. Consistent with this finding, GluR2L and GluR4 associate with specific JNK signaling components in the brain. Moreover, the modulation of the novel JNK sites in GluR2L and GluR4 is dynamic and bi-directional, such that phosphorylation and de-phosphorylation are triggered within minutes following decreases and increases in neuronal activity, respectively. Using live-imaging techniques to address the functional consequence of these activity-dependent changes we demonstrate that the novel JNK site in GluR2L controls reinsertion of internalized GluR2L back to the cell surface following NMDA treatment, without affecting basal GluR2L trafficking. Taken together, our results demonstrate that JNK directly regulates AMPA-R trafficking following changes in neuronal activity in a rapid and bi-directional manner.

Figure 1

JNK1 phosphorylates a novel site on AMPA-R C-terminal tails in vitro. (A) GluR2L and GluR4 sequences, from the GluR4 phosphorylation site Ser842 to the C-terminus, are aligned to indicate conserved potential MAPK phosphorylation sites (GluR2L-Thr912, GluR4-Thr855, shown in gray). These sequences show clear homology to sites on the JNK substrates cJun and MAP2 that are phosphorylated in vivo. A consensus (basic residue at N−3, aliphatic/polar/uncharged residues (Φ) at N−2 and N−1, proline at N+1, acidic residue at N+2, where N is the phosphorylated residue) is shown. A second potential MAPK site (Ser926) in GluR2L is underlined. (B) JNK1 phosphorylates GluR2L and GluR4 C-termini in vitro. Autoradiograph (top), immunoblots with GluR2LThr912(P) antibody (second panel) or GluR4Thr855(P) antibody (third panel) and Coomassie Blue staining (bottom) of purified GST, GST-GluR4 or GST-GluR2L used in kinase assays in the presence (+) or absence (−) of active JNK1. (C) JNK1 phosphorylates GST-GluR4 and GST-GluR2L at Thr-855 and Thr-912, respectively. GST, GST-GluR4, GST-GluR2L and point mutants (Thr-Ala) of these GluRs at the potential JNK phosphorylation site were incubated with active JNK1 and [γ]³²P-ATP. Autoradiograph (top), GluR2LThr912(P) immunoblot (second panel), GluR4Thr855(P) immunoblot (third panel) and Coomassie Blue staining (bottom) of duplicate determinations per condition.

Figure 2

Endogenous JNK phosphorylates GluR4-Thr855 and GluR2L-Thr912 in transfected cells. (A) HEK 293T cells transfected with vector (pRK5) or GluR2L cDNA were pre-incubated with DMSO vehicle (−) or the indicated inhibitors (SP10: 10 μM SP600125; SP20: 20 μM SP600125; SB: 10 μM SB 203580; U0: 10 μM U0126; Rosc: 10 μM Roscovitine) prior to stimulation with (+) or without (−) 0.5 M Sorbitol. Lysates were blotted for phosphoThr912 (top), total GluR2L (middle) and active, phosphorylated JNK (phosphoJNK, bottom). SP600125 blocks the kinase activity of JNK but not its phosphorylation by upstream kinases. Thus SP600125 minimally affects phosphoJNK signals but the catalytic activity of JNK itself is still inhibited. (B) As (A), except that cells were transfected with empty vector or GluR4 cDNA and GluR4 immunoprecipitates were blotted for phosphoThr855 (top) and total GluR4 (second panel). Lysates were blotted for phosphoJNK (bottom). (C) HEK293T cells were co-transfected with GluR2L cDNA plus either pRK5 vector, myc-tagged JNK-binding domain (myc-JBD) or myc-tagged JNK1 (myc-JNK1) prior to stimulation with (+) or without (−) 0.5 M Sorbitol for 30 min. GluR2L immunoprecipitates were blotted for phosphoThr912 (top), total GluR2L (middle) and lysates were blotted to detect myc-tagged proteins (bottom). (D) As (C), except that cells were transfected with empty vector or GluR4 cDNA plus pRK5 vector or myc-tagged JNK-binding domain (myc-JBD) or JNK1 (myc-JNK1) and GluR4 immunoprecipitates were blotted for phosphoThr855 (top), total GluR4 (middle) and lysates were blotted for myc-tagged proteins (bottom).

Figure 3

JNK-JIP1 complexes mediate high basal phosphorylation of GluR2L-Thr912 and GluR4-Thr855 in neurons. (A) Cortical neurons (DIV 15–20) were washed into aCSF containing DMSO vehicle control (‘Con') or the indicated inhibitors (SP10: 10 μM SP600125; SP20: 20 μM SP600125; SB: 10 μM SB 203580; U0: 10 μM U0126; Rosc: 10 μM Roscovitine) prior to lysis. GluR2L immunoprecipitates were blotted for phosphoThr912 (Top) and GluR2L (second panel). GluR2L-Thr912 phosphorylation relative to total GluR2L signal (normalized to control, 100%): SP10: 53.5±5.4%, N=6; SP20: 35.4±5.4%, N=6; SB: 95.7±18.2%, N=5; U0: 89.8±18.2%, N=6; Rosc: 86±24.5%, N=5). GluR4 immunoprecipitates were blotted for phosphoThr855 (third panel) or GluR4 (bottom). GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to control, 100%). SP10: 19.8±3.9%; SP20: 24.2±2.9%; SB: 93.7±9.1%; U0:86.9±4.8%; Rosc: 102.7±12.1% (N=4 for all treatments). (B) as A except that neurons were incubated for 1 h with the indicated concentrations of TAT-JBD peptide. (C) GluR2L immunoprecipitates (pre-incubated with or without antigenic peptide) were prepared from membrane fractions from juvenile (P17–20) rats and blotted for JNK pathway components. (D) As (C) except that GluR4 immunoprecipitates were prepared.

Figure 4

JNK dynamically regulates GluR2L-Thr912 and GluR4-Thr855 phosphorylation in neurons. (A) Cortical neurons were incubated in aCSF in the presence (+) or absence (−) of 10 μM SP600125 for 1 h and then stimulated for 10 min with 1 μM okadaic acid (+) or left unstimulated (−). GluR2L immunoprecipitates were blotted for phosphoThr912 (top), GluR2L (second panel), phosphoJNK (third panel) or panJNK (bottom). GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) SP: 53.5±5.4%, N=6; OA: 197.8±32.8%, N=4; OA+SP: 64.0±32.2%, N=4. (B) as A except that GluR4 immunoprecipitates were prepared and blotted for phosphoThr855 (top) or GluR4 antibody (second panel). GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to control, 100%) OA: 209.2±49.1%, N=6; OA+SP: 90.9±26.6%, N=5.

Figure 5

Neuronal activity changes rapidly and bi-directionally modulate GluR2L-Thr912 and GluR4-Thr855 phosphorylation in neurons. (A) Cortical neurons were incubated in aCSF for 1 h, treated for 10 min with 1 μM okadaic acid or DMSO vehicle, and then stimulated for 10 min with 30 μM NMDA, or were left unstimulated (Con). GluR2L immunoprecipitates were blotted for phosphoThr912 (top) or GluR2L (lower panel). GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) NMDA: 36.8±9.0%, N=6; NMDA+OA: 92.1±12%, N=6. GluR4 immunoprecipitates were blotted for phosphoThr855 (third panel) or GluR4 (bottom). GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to untreated control, 100%) NMDA: 28.5±6.3%, N=6; NMDA+OA: 89.3±17.4%, N=6. (B) Cortical neurons were stimulated with 20 μM Bicuculline (Bic) or 1 μM Tetrodotoxin (TTX) for 20 min. Immunoprecipitates were prepared and blotted as in (A). Lysates were blotted to detect phosphoJNK. GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) Bic: 61.9±12.8%, N=4; TTX: 142.2±16.9%, N=4. GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to control, 100%) Bic: 42.5±21.5%, N=4; TTX: 176.1±40.4%, N=4. (C) Cortical neurons were washed into aCSF in the presence (+) or absence (−) of 10 μM SP600125 for 1 h prior to stimulation for 5 min with 20 μM NMDA. Cells were lysed or washed into aCSF in the absence of NMDA for a further 40 min prior to lysis. Control cells were left unstimulated. GluR2L immunoprecipitates were blotted for phosphoThr912 (top), or GluR2L (second panel). GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) Con/SP: 75.2±3.3%, N=4; NMDA: 58.4±7.0%, N=4; NMDA/SP: 30.1±1.8%, N=4; Rec: 85.0±12.2%, N=4; Rec/SP: 27.5± 6.6%, N=4. GluR4 immunoprecipitates were blotted for phosphoThr855 (third panel), or GluR4 (bottom). GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to control, 100%) Con/SP: 65.6±22.0%, N=4; NMDA: 49.3±10.8%, N=4; NMDA/SP: 30.2±9.7%, N=4; Rec: 87.4±10.7%, N=4; Rec/SP: 38.3±9.9%, N=4. Lysates were blotted for phosphoJNK (bottom). Note that the effect of SP600125 on GluR2L-Thr912 and GluR4-Thr855 phosphorylation is more marked after NMDA washout than under basal conditions (compare lanes 1,2 with 3,4 and 9,10 with 11,12).

Figure 6

Live imaging of pHluorin-tagged GluR2L (pH-GluR2L) internalization and recycling. (A) Representative images of fluorescence change of a hippocampal neuron expressing pH-GluR2L, exposed at t=0 to 20 μM NMDA for 5 min and then allowed to recover following NMDA washout. Lower panels show enlargements of the dendritic region boxed in the upper panels. Scale bars=10 μm. As reported elsewhere (Ashby et al, 2004), NMDA perfusion predominantly internalizes diffuse, extra-synaptic AMPA-Rs. The signal:noise ratio of dendritic fluorescence change is thus less robust, due to the contribution of clustered/punctate pHGluR2L that does not internalize following NMDA treatment. (B) As A, but for a neuron incubated for 1 h in the presence of 10 μM SP600125 prior to NMDA treatment. SP600125 was present during NMDA treatment and recovery. (C) Fluorescence change (mean±s.e.m.) during initial incubation, perfusion with 20 μM NMDA for 5 min at t=0 min (blue bar) and washout, plotted for neurons in the presence of DMSO vehicle (red diamonds) or 10 μM SP600125 (black circles). SP600125 was present as indicated (black bar). (D) As C, but for neurons expressing pH-GluR2L-T912A. (E) As C but for neurons expressing pH-GluR2L-T912D. (F) As C, but for neurons expressing pH-GluR2(short). Data in A–E are plotted as mean±s.e.m. for the following number of individual cells, from at least two platings of neurons: GluR2L wt: N=8; GluR2L wt+SP: N=5; GluR2L T912A: N=3; GluR2L T912D: N=5; GluR2 (short): N=4 (G) Fluorescence recovery, quantitated 1 h after NMDA washout in B–E (^*P<0.01, Student t-test).

Figure 7

Phosphorylation of GluR2L by JNK controls activity-dependent reinsertion but is not required for basal recycling. NMDA-R activation triggers GluR2L internalization and Thr912 dephosphorylation. Following NMDA washout, high basal JNK activity catalyzes GluR2L-Thr912 rephosphorylation, allowing GluR2L re-insertion into the plasma membrane. JNK inhibition prevents rephosphorylation and the receptor cannot recycle. Basal (non-NMDA-dependent) recycling is unaffected by JNK inhibition.