JNK dependent AMPA-R regulation GM Thomas et al 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. removed from synapses by this putative Rap2-JNK pathway was GluR2L. We report here that JNK controls GluR2L re-insertion, but note several possible explanations for differences between our ﬁndings and those of Zhu et al. Firstly, the two studies used different experimental systems, in which JNK may play different roles. However, Zhu et al relied heavily on overexpression of constitutively active signaling proteins, AMPA-Rs or both, which may not reﬂect the endogenous situation. In particular, Zhu et al reported that NMDA treatment activates Rap2 and over- expression of constitutively active Rap2 increases phosphoJNK. However, they presented no data that NMDA treatment activates JNK (Zhu et al, 2005), and we observe no JNK activation following NMDA treatment. Indeed, JNK activation by NMDA in cortical neurons is induced only by pathologically high NMDA concentrations that induce excito- toxicity (e.g. Borsello et al, 2003). This suggests that the NMDA-Rap2-JNK link proposed (but not demonstrated) by Zhu et al is unlikely to function physiologically, and that the small changes in phospho-JNK following Rap2 overexpres- sion (Zhu et al, 2005) may be due to Rap2 effects on other pathways, which then indirectly affect JNK. Such indirect effects are well known when active small G proteins are overexpressed (e.g. McCarthy et al, 1995). Rap2-induced changes in AMPA-R phosphorylation (Zhu et al, 2005) are also likely to be indirect and not mediated by JNK. Although Rap2 overexpression affects ‘GluR2L-Ser842’ phosphorylation (GluR2L-Ser899 following standard notation; a site homologous to GluR4-Ser842) (Zhu et al, 2005), this site is not a likely direct JNK substrate. Firstly, GluR2L-Ser899 does not lie in a consensus sequence for JNK or for any JNK pathway kinase. Moreover, while the use of crude lysates by Zhu et al means that their Ser899 phos- phoantibody might also detect the homologous GluR4-Ser842 site, we ﬁnd that JNK inhibition affects neither GluR4-Ser842 nor GluR2L-Ser899 phosphorylation in neurons (data not shown). Rap2-induced GluR2L-Ser899 (and perhaps GluR4- Ser842) phosphorylation (Zhu et al, 2005) is thus best accounted for by a kinase(s) distinct from JNK. & 2008 European Molecular Biology Organization In contrast, our work mainly tracks endogenous AMPA-R phosphorylation in neurons and is supported by in vitro and heterologous cell studies. Both novel AMPA-R sites that we identify lie in excellent JNK consensus motifs and JNK phosphorylates both sites efﬁciently. Point mutation of each site eliminates both GluR2L and GluR4 phosphorylation by JNK. Phosphorylation of both GluR2L-Thr912 and GluR4- Thr855 is JNK-dependent in neurons and is most likely mediated by JNK1. This is consistent with other reports that basal neuronal JNK activity is high and is due mainly to JNK1 (Bjorkblom et al, 2005; Brecht et al, 2005). While a different JNK population (a different isoform or scaffold complex) may regulate the Rap2 pathway proposed (Zhu et al, 2005), some ﬁndings by Zhu et al may be due to indirect effects. The rapid and bi-directional modulation of GluR2L-Thr912 and GluR4-Thr855 phosphorylation following neuronal activ- ity changes is very interesting. Increases in neuronal activity promote dephosphorylation of both sites, while decreases in activity promote phosphorylation. This bi-directional modu- lation is similar to that recently reported for PSD-95 phos- phorylation at Ser-295, which was also ascribed to JNK1 (Kim et al, 2007) and thus supports our model of neuronal JNK substrate phosphorylation, rather than that described by Zhu et al. Importantly, bi-directional modulation of neuronal JNK substrates may reﬂect a common mechanism allowing rapid feedback control of phosphorylation and function of multiple proteins involved in neurotransmission. Feedback mechan- isms employed by neurons have drawn intense attention, most notably in the ﬁeld of homeostatic plasticity (O’Brien et al, 1998; Turrigiano et al, 1998; Thiagarajan et al, 2005; Shepherd et al, 2006). However, forms of homeostatic plas- ticity described thus far all occur over long time courses (hours to days). Our report of a rapid response system controlled by JNKs (and phosphatases that oppose them) reveals a new mechanism used by neurons to respond bi-directionally to activity changes on the timescale of only a few minutes. Regulatory feedback of this type has parallels to adaptation in sensory systems, where distinct mechanisms The EMBO Journal VOL 27 | NO 2 | 2008 369