(Lee et al, 1998; Ehlers, 2000). Subsequent washout of NMDA allows GluR1-Ser845 re-phosphorylation and GluR1 returns to the plasma membrane (Ehlers, 2000). We therefore examined whether NMDA treatment and washout might induce a similar dephosphorylation/re-phosphorylation cycle for GluR2L-Thr-912 and/or GluR4-Thr855. As previously, NMDA treatment led to dephosphorylation of both GluR2L- Thr-912 and GluR4-Thr855. Both sites were rephosphory- lated, essentially to basal levels, following NMDA washout (Figure 5C). Importantly, rephosphorylation of GluR2L- Thr912 and GluR4-Thr855 was highly sensitive to JNK in- hibition (Figure 5C). This suggests that JNKs are critical for rapid rephosphorylation of GluR2L-Thr912 and GluR4- Thr855 following NMDA washout. Changes in GluR1 phosphorylation following NMDA treat- ment and washout accompany alterations in GluR1 trafﬁck- ing (Ehlers, 2000). The strikingly similar dephosphorylation/ re-phosphorylation of GluR2L-Thr912 (and GluR4-Thr855) (Figure 5C) led us to examine the role of Thr912 phosphor- ylation in GluR2L trafﬁcking. We therefore modiﬁed GluR2L with a pH-sensitive GFP (super-ecliptic pHluorin) at its N- terminus. The pHluorin tag ﬂuoresces at neutral pH (e.g. when present on the cell surface and exposed to extracellular medium), but at acidic pH (as encountered when the receptor internalizes to acidic endosomes) ﬂuorescence is quenched (Ashby et al, 2004; Lin and Huganir, 2007). pH-GluR2L expressed efﬁciently in neurons and was efﬁciently targeted to the cell surface (Supplementary Figure S7). Expression levels of pH-GluR2Lwt, pH-GluR2L-T912A (a non-phos- phorylatable mutant) and pH-GluR2L-T912D (a putative phospho-mimetic mutant) were similar in neurons (data not shown). In addition, pH-GluR2Lwt, pH-GluR2L-T912A and pH-GluR2L-T912D surface expression levels were similar, as judged by immunostaining (Supplementary Figures S7, S8) or by biotinylation methods to isolate cell surface fractions (data not shown). Furthermore, pH-GluR2Lwt, pH-GluR2L- T912A and pH-GluR2L-T912D were similarly targeted to excitatory synapses (Supplementary Figure S7). These results suggested that the status of GluR2L-Thr912 affects neither expression of this AMPA-R, nor its targeting to the cell surface or to synapses. Moreover, SP600125 addition during live- imaging experiments caused no run-up or rundown of ﬂuorescence for either pH-GluR2Lwt, pH-GluR2L-T912A or pH-GluR2L-T912D, suggesting that although JNK inhibi- tion triggers GluR2L-Thr912 dephosphorylation (Figure 3A), modulation of Thr912 phosphorylation does not affect basal GluR2L trafﬁcking. This hypothesis was supported by Fluore- scence Recovery After Photobleach (FRAP) experiments (Supplementary Figure S9). Following brief NMDA treatment the robust basal ﬂuore- scence of pH-GluR2L decreased dramatically in both the cell soma and in dendrites (Figure 6A, Supplementary Figure S10), consistent with receptor internalization from the cell surface to acidic endosomes as reported for pH-GluR2 (Ashby et al, 2004; Lin and Huganir, 2007). NMDA-induced decreases in dendritic pHGluR2L ﬂuorescence occurred with similar kinetics to those in the cell soma (Supplementary Figure S10). As reported elsewhere (Ashby et al, 2004; Lin and Huganir, 2007), NMDA perfusion predominantly internalizes diffuse, extra-synaptic AMPA-Rs (Supplementary Figure S10). Following NMDA washout, pH-GluR2L ﬂuorescence recov- ered, reaching levels observed prior to NMDA perfusion & 2008 European Molecular Biology Organization JNK dependent AMPA-R regulation GM Thomas et al (Figure 6A and C). Fluorescence recovery was largely due to recycling of endocytosed pH-GluR2L back to the plasma membrane as recovery was largely (475%) prevented by photobleach of surface pH-GluR2L prior to NMDA treatment (data not shown). Interestingly, pH-GluR2Lwt, pH-GluR2L-T912A and pH- GluR2L-T912D internalized at similar rates and to similar degrees following NMDA perfusion, suggesting that the status of GluR2L-Thr912 did not inﬂuence receptor endocytosis (Supplementary Figure S11). Moreover, NMDA-induced pH- GluR2Lwt internalization was unaltered by SP600125 (Figure 6C). However, SP600125 dramatically attenuated re-insertion of pH-GluR2Lwt to the plasma membrane, with o40% of initial ﬂuorescence recovered, even 60 min after NMDA washout in both the cell soma and in dendrites (Figure 6B, C and G, Supplementary Figure S10). This suggested that JNK signaling is critical for pH-GluR2L recycling to the plasma membrane. This might be due to direct modulation of pH-GluR2L-Thr912 by JNK, or because SP600125 prevents JNK from phosphorylating a trafﬁcking protein that controls pH-GluR2L recycling. Strikingly, how- ever, SP600125 did not prevent either pH-GluR2L-T912D (Figure 6D) or pH-GluR2L-T912A (Figure 6E) recycling, sug- gesting that the status of Thr912 was indeed critical for recycling. The speciﬁcity of SP600125 was further conﬁrmed by examining recycling of pH-GluR2 (short form), which differs from pH-GluR2L only in its intracellular C-terminus. pH-GluR2 ﬂuorescence reduced dramatically in response to NMDA (consistent with internalization) and recovered, essentially to initial levels, following NMDA washout in the presence of SP600125 (Figure 6F). Thus SP600125 prevents recycling of pH-GluR2Lwt, but not recycling of pH-GluR2L- T912A, pH-GluR2L-T912D or pH-GluR2 (Figure 6G). Discussion Despite the high neuronal expression of JNKs, physiological JNK targets in brain and their modes of regulation by neuronal activity have remained obscure. Here, using a bioinformatics-based approach, we identiﬁed two AMPA-R subunits as novel JNK substrates in developed neurons. We further showed that both AMPA-R subunits are regulated in a novel JNK-dependent manner in response to neuronal acti- vity changes, and revealed a previously unappreciated role for JNK signaling in the control of AMPA-R trafﬁcking. A schematic summarizing these results is shown in Figure 7. Our initial interest in the novel GluR2L/GluR4 sites was aroused by their homology to phosphorylated sites in the JNK substrates cJun and MAP2 (Figure 1A, consensus sequence K/R-F-F-S/T-P-D/E, where F is an aliphatic/non-polar resi- due). Our ﬁndings that GluR2L and GluR4 are also phos- phorylated by JNK at such sites suggest that K/R-F-F-S/T-P- D/E is a novel JNK consensus motif. We note that other synaptic proteins with key roles in AMPA-R regulation (e.g. SAP97, 4.1N, liprin-alpha3; Malinow and Malenka, 2002; Song and Huganir, 2002; Wyszinski et al, 2002) also contain K/R-F- F-S/T-P-D/E motifs. The possibility that such proteins may also be in vivo JNK substrates is intriguing. To this point, roles for JNK in synaptic transmission have gone largely unreported. However, the GTPase Rap2 report- edly signals via JNK to regulate synaptic depotentiation (Zhu et al, 2005). Zhu et al reported that one AMPA-R subtype The EMBO Journal VOL 27 | NO 2 | 2008 367