JNK dependent AMPA-R regulation GM Thomas et al speciﬁc roles in hippocampal LTP and LTD, respectively (Malinow and Malenka, 2002; Song and Huganir, 2002). Using bioinformatics techniques we identiﬁed consensus sites for JNK family MAPKs on the intracellular regions of two long-tailed AMPA-R subunits; GluR4 and the long-tailed splice form of GluR2, GluR2L. Speciﬁc mechanisms that regulate GluR2L and GluR4 are largely unknown, despite the fact that these receptors contribute signiﬁcantly to AMPA-R transmission during development (Zhu et al, 2000; Esteban et al, 2003; Kolleker et al, 2003). We therefore investigated the possibility that GluR2L and GluR4 might be novel neuronal JNK substrates, and that JNK-dependent phosphorylation might speciﬁcally regulate these AMPA-Rs. Here we show that GluR2L and GluR4 are indeed JNK substrates in vitro, in heterologous cells and in neurons. We demonstrate that in neurons, basal JNK activity maintains high constitutive phosphorylation of both GluR2L and GluR4, but several stimuli that increase glutamatergic signaling trigger rapid dephosphorylation of these receptors at JNK sites. Conversely, blocking neuronal activity with TTX rapidly increases JNK-mediated phosphorylation of both GluR2L and GluR4. This suggests that JNKs, and the phosphatases that oppose them, form a bi-directional system to rapidly respond to neuronal activity changes. Using live-imaging methods to investigate the functional consequence of GluR2L phosphorylation by JNKs we report that the novel JNK phosphorylation site on GluR2L plays a key role in control- ling activity-dependent GluR2L trafﬁcking. These ﬁndings identify novel physiological JNK substrates in mature neurons, revealing a previously unappreciated role for these kinases in regulation of speciﬁc AMPA-Rs. Results One of the few known neuronal JNK substrates, the micro- tubule-associated protein MAP2, is phosphorylated by JNK1 at unspeciﬁed sites (Chang et al, 2003; Bjorkblom et al, 2005). A large-scale proteomic study revealed vast numbers of in vivo phosphorylated sites in synaptic prepara- tions (Trinidad et al, 2006) and we inspected this report for MAP2 in vivo phosphorylation sites. As expected, we found phosphorylated MAP2 sites homologous to phos- phorylated sites on the well-characterized JNK substrate, cJun. This suggested that further JNK substrates might contain sequences homologous to these cJun/MAP2 sites (Figure 1A). We therefore probed the Scansite database (http://scansite.mit.edu) with a consensus motif derived from the conserved MAP2/cJun phospho-motif (Figure 1A). Strikingly, the ‘hits’ included several proteins with key roles 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 (F) at N 2 and N 1, proline at N þ 1, acidic residue at N þ 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 ( þ ) 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 [g]32P-ATP. Autoradiograph (top), GluR2LThr912(P) immunoblot (second panel), GluR4Thr855(P) immunoblot (third panel) and Coomassie Blue staining (bottom) of duplicate determinations per condition. 362 The EMBO Journal VOL 27 | NO 2 | 2008 & 2008 European Molecular Biology Organization