JNK dependent AMPA-R regulation GM Thomas et al 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 mM SP600125; SP20: 20 mM SP600125; SB: 10 mM SB 203580; U0: 10 mM U0126; Rosc: 10 mM Roscovitine) prior to stimulation with ( þ ) 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 ( þ ) 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). JNK activator MAP kinase kinase-7 (MKK7) in GluR2L and GluR4 immunoprecipitates from rat brain membranes (Figure 3C and D). Other JNK isoforms (JNK2 and JNK3) were not detected (data not shown). This suggests that JNK pathway kinases, scaffolded by JIP1, associate with AMPA-Rs in neu- rons and thus provides a molecular explanation for how JNK might phosphorylate GluR2L/GluR4. The speciﬁc presence of JNK1 in such complexes is consistent with reports that JNK1 accounts for much of the basal JNK activity in brain (Coffey et al, 2002; Bjorkblom et al, 2005; Brecht et al, 2005). This isoform-speciﬁc enrichment of JNK1 in AMPA-R complexes led us to examine the subcellular distribution of JNK pathway components and AMPA-Rs (Supplementary Figure S5). Synaptosomal and Post-synaptic density (PSD) fractions showed clear presence of JNK1, JNK3, MKK7 and JIP1. In contrast, JNK2 was largely excluded from PSD 364 The EMBO Journal VOL 27 | NO 2 | 2008 & 2008 European Molecular Biology Organization