in synaptic regulation (see Discussion). In particular, the cJun/MAP2 motif perfectly matched regions within the C-terminal tails of two AMPA-Rs, GluR4 and the long splice form of GluR2 (GluR2L). The GluR4 (Thr855) and GluR2L (Thr912) JNK consensus motifs are conserved across species (data not shown). We therefore examined whether GST-fusions of GluR4 and GluR2L tails could serve as JNK substrates in in vitro kinase assays. Indeed, JNK1 phosphorylated both GST-GluR2L and GST-GluR4 extremely effectively in vitro (Figure 1B), with Km values approaching those of the known JNK substrate ATF2 (data not shown). Mutation of GluR2L-Thr912 or GluR4- Thr855 to alanine abolished JNK1 phosphorylation of these tails (Figure 1C), indicating that Thr855 and Thr912 were the sites phosphorylated by JNK1. In contrast, other MAPK family enzymes with known roles in synaptic regulation (ERK2, p38alpha and the MAPK-related cyclin family kinase CDK5) barely phosphorylated these GluR tails in vitro, though each kinase efﬁciently phosphorylated its known substrates (data not shown). Two phospho-antibodies were developed to monitor GluR2L-Thr912 and GluR4-Thr855 phosphorylation. Both antibodies were highly phospho-speciﬁc, recognizing their GluR antigens only after phosphorylation with JNK1 in vitro (Figure 1B). Neither antibody recognized the GluR2L or GluR4 alanine mutants (Figure 1C). The GluR4-Thr855(P) antibody also recognized GluR2L-Thr912(P) and the GluR2L- Thr912(P) antibody weakly recognized GluR4-Thr855(P) (Figure 1C). This was not unexpected since these sites are highly similar (Figure 1A). To examine whether JNKs could regulate GluR2L phos- phorylation in mammalian cells we transfected HEK293T cells with GluR2L cDNA and added 0.5 M sorbitol, an osmotic shock that activates JNK (Bagowski et al, 2003). Blotting of lysates with GluR2L-Thr912(P) antibodies revealed immuno- reactivity only in GluR2L-transfected cells (Figure 2A). Immunoreactivity was weak in unstimulated cells but was increased dramatically by sorbitol treatment (Figure 2A). A JNK inhibitor, SP600125 (Bennett et al, 2001), prevented the sorbitol-induced increase in phosphoThr912 signal. Inhibitors of other MAPK pathways (SB203580, which inhi- bits p38/SAPK2, and U0126, which prevents ERK activation) or Roscovitine, a cyclin-dependent kinase (cdk) inhibitor did not affect sorbitol-induced GluR2L-Thr912 phosphorylation (Figure 2A). This suggests that endogenous HEK293T cell JNKs phosphorylate GluR2L-Thr912. Using similar methods we examined GluR4 phosphory- lation in transfected HEK293T cells. Due to cellular bands recognized by the GluR4-Thr855(P) antibody we immuno- precipitated GluR4 (Supplementary Figures S1, S2) to examine its phosphorylation in isolation. PhosphoThr855 immunoreactivity (Figure 2B) was only detected in immuno- precipitates from GluR4-transfected cells. PhosphoThr855 immunoreactivity was weak in unstimulated cells, was increased dramatically by sorbitol treatment, and was greatly reduced by SP600125 but not by ERK pathway, p38 or cdk inhibitors. These data suggest that endogenous HEK293T cell JNKs phosphorylate GluR4 at Thr855. As a complementary method to modulate JNK signaling we used the JNK-binding domain (JBD) of the scaffold protein JIP1 (JNK-interacting protein-1, also known as Islet-brain-1; Dickens et al, 1997; Whitmarsh et al, 1998; & 2008 European Molecular Biology Organization JNK dependent AMPA-R regulation GM Thomas et al Xia et al, 2001). JIP proteins scaffold multiple JNK pathway kinases and increase efﬁcacy of signaling via this pathway. JBD over-expression draws JNK away from its upstream activators and prevents phosphorylation of downstream JNK substrates (Dickens et al, 1997; Xia et al, 2001). Consistent with this model, myc-JBD co-transfection greatly reduced sorbitol-induced GluR2L-Thr912 phosphorylation. In contrast, sorbitol-induced GluR2L-Thr912 phosphorylation was dramatically higher in cells co-transfected with myc-JNK1 (Figure 2C). Sorbitol-induced GluR4-Thr855 phosphorylation was also reduced by myc-JBD, and augmen- ted by myc-JNK1 (Figure 2D), supporting the hypothesis that JNKs phosphorylate GluR2L and GluR4 in heterologous cells. To examine endogenous GluR2L and GluR4 phosphory- lation we immunoprecipitated these AMPA-Rs from cultured cortical neurons and from rat brain membrane fractions using speciﬁc antibodies (Supplementary Figures S1, S2). GluR2L and GluR4 immunoprecipitates showed strong immuno- reactivity with GluR2L-Thr912(P) and GluR4-Thr855(P) antibodies, respectively, and lambda phosphatase treat- ment conﬁrmed that both signals were phospho-speciﬁc (Supplementary Figure S2). GluR2L is not detected in GluR4 co-immunoprecipitates (or vice versa) from either cultured neurons or rat brain membrane fractions (Supplementary Figure S1). Thus, GluR2L and GluR4 are phosphorylated, at Thr912 and Thr855 respectively, in cultured neurons and in rat brain. JNK activity, particularly attributed to JNK1, is constitu- tively high in brain (Lee et al, 1999; Coffey et al, 2002). Consistent with this, basal phosphorylation of GluR2L- Thr912 and GluR4-Thr855 was high in unstimulated neurons. However, basal GluR2L-Thr912 and GluR4-Thr855 phosphor- ylation were both lowered substantially by SP600125, but not by other MAPK pathway inhibitors, or by a cdk5 inhibitor (Figure 3A). A structural analog of SP600125 that poorly inhibits JNK affected neither GluR2L-Thr912 nor GluR4- Thr855 phosphorylation (Supplementary Figure S3). These results suggest that high basal JNK activity (Lee et al, 1999; Coffey et al, 2002; Kuan et al, 2003; Bjorkblom et al, 2005; Brecht et al, 2005) mediates the majority of basal GluR2L- Thr912 and GluR4-Thr855 phosphorylation in neurons. Interestingly, membrane and synaptosomal fractions from juvenile rat brain showed markedly higher phosphoJNK signals, though JNK levels were similar across fractions (Supplementary Figure S4). This suggests that active JNKs are enriched in membrane fractions, where they are ideally situated to maintain high basal phosphorylation of AMPA-Rs and other synaptic substrates. As in heterologous cells, we used a JBD-based approach to modulate JNK signaling and examined GluR2L/GluR4 phosphorylation. A cell-permeant peptide consisting of the HIV-1 TAT sequence fused to a JBD fragment (TAT-JBD) also dramatically reduced both GluR2L-Thr912 and GluR4-Thr855 phosphorylation (Figure 3B). In contrast, a TAT-JBD peptide with mutations in two residues critical for JNK binding did not affect phosphorylation of either GluR2L-Thr912 or GluR4- Thr855 (Supplementary Figure S3). The reduction of GluR2L/GluR4 phosphorylation by JIP1-based intervention provided further evidence that JNK phosphorylates these AMPA-Rs in neurons and implicated JIP-dependent JNK pathway scaffolds in regulation of these AMPA-Rs. Indeed, we detected JNK1, JIP1 and the upstream The EMBO Journal VOL 27 | NO 2 | 2008 363