JNK dependent AMPA-R regulation GM Thomas et al control rapid, short-term and long-term changes for multiple sensory modalities. Identiﬁcation of further physiological neuronal JNK substrates may provide key insights into mechanisms used by neurons to rapidly adjust to different levels of activity. Although GluR1-Ser845 dephosphorylation is implicated in GluR1 internalization (Ehlers, 2000; Lee et al, 2003), Thr912 dephosphorylation is unnecessary for GluR2L internalization. However, the status of GluR2L-Thr912 plays a key role in controlling GluR2L recycling to the plasma membrane following NMDA washout. Our ﬁnding that the non-phos- phorylatable GluR2L-T912A mutant and the putative phos- pho-mimetic GluR2L-T912D mutant both override the SP600125-dependent block of GluR2L recycling is initially surprising. A possible explanation is that NMDA treatment triggers retention of internalized, dephosphorylated GluR2L by a protein that speciﬁcally binds ‘Thr912-dephosphory- lated’ GluR2L. If this retention protein cannot bind the GluR2L-T912A and GluR2L-T912D mutants then these recep- tors would return to the cell surface. This explanation is not without precedent: for example, both S842A and S842D mutants of GluR4 trafﬁc to synapses in the presence of high magnesium concentrations that prevent synaptic delivery of wildtype GluR4 (Esteban et al, 2003). An interaction that retains ‘Ser842-dephosphorylated’ GluR4 was postulated to explain these ﬁndings. Indeed, proteins that bind only depho- sphorylated forms of other AMPA-Rs are already known; the multi-PDZ domain protein GRIP1 binds the GluR2 C-terminus only when GluR2 is dephosphorylated at Ser880 (‘dephosphoSVKI’; Matsuda et al, 1999; Chung et al, 2000). Ser880 phosphorylation or mutations to non-phosphorylata- ble (AVKI) or phospho-mimetic (EVKI) residues ablate GRIP binding (Dong et al, 1997; Chung et al, 2000). It is therefore very possible that a GluR2L-dephosphoThr912 interactor might not recognize GluR2L-T912A or GluR2L-T912D. The identiﬁcation of phospho-dependent AMPA-R interactors is an exciting area for future study, since such proteins likely play key roles in AMPA-R trafﬁcking. Our identiﬁcation of two novel AMPA-R phosphorylation sites and the pathways that regulate their phosphorylation and dephosphorylation, plus the characterization of a novel functional role for this phosphorylation cycle increases our knowledge of the speciﬁcity of AMPA-R regulation and re- veals new roles for neuronal JNK signaling. Since aberrant JNK signaling likely underlies several neuropathologies, the identiﬁcation of physiological neuronal JNK targets will greatly increase our understanding of cellular processes that may become disregulated during pathological JNK hyper- activation. With this knowledge the likelihood of improved treatments for JNK-dependent neuropathological conditions is greatly increased. Materials and methods Materials and Methods are described only brieﬂy here. Full details are available in Supplementary data. cDNA clones Full-length rat GluR4 and GluR2L, subcloned into the pRK5 mammalian expression vector, were used as PCR templates to generate GluR4 and GluR2L C-terminal tails, which were subcloned into the mammalian expression vector pCIS-GST. Point mutants of these constructs and full length GluR2L were generated by Quickchange (Stratagene, La Jolla, CA). The unique BspEI site 370 The EMBO Journal VOL 27 | NO 2 | 2008 in the rat GluR2 sequence was used to replace all except the C-terminus of GluR2L with a rat GluR2 ‘short’ construct N- terminally tagged with pH-sensitive GFP (pHluorin; Ashby et al, 2004). Mouse JNK1beta1 was subcloned into pRK5 in frame with an N-terminal myc epitope. The JIP1 JNK-binding domain (JBD: Dickens et al, 1997) was PCR-ampliﬁed from a rat EST (Genbank Accession CK475933) and subcloned into myc-pRK5. Puriﬁcation of GST-GluR tails GST-GluR tails were expressed in afﬁnity-puriﬁed. HEK 293T cells and In vitro JNK assay Kinase reactions contained 2 mM GST or GST-GluR tails plus 5 ng of His-JNK1 or blank buffer. Incorporation of 32P-radioactivity from [g]32P-ATP was detected by autoradiography. GluR phosphorylation in HEK293T cells HEK293T cells were transfected with full-length GluR2L or GluR4 cDNA. In some experiments, cells were co-transfected with GluR cDNA plus empty vector, myc-JBD or myc-JNK1 cDNAs. After 24 h recovery, cells were stimulated with 0.5 M Sorbitol for 30 min or left unstimulated. Kinase inhibitors were added to medium for 1 h prior to stimulation and remained during stimulation. Cells were lysed and GluR2L or GluR4 were immunoprecipitated. Immunoprecipi- tates were denatured in SDS sample buffer, subjected to SDS-PAGE, transferred to PVDF membrane and immunoblotted to examine AMPA-R phosphorylation. Bands were visualized by ECL. GluR phosphorylation in cortical neurons Cortical neurons were prepared from E18 rats. At 15–20 DIV, medium was replaced with artiﬁcial cerebrospinal ﬂuid (aCSF) containing, in mM: 10 HEPES pH 7.4, 150 NaCl, 3 KCl, 2 CaCl2, 1 Mg, 10 glucose and cells placed at 371C for 1 h. In some experiments kinase inhibitors were added to aCSF during this period. For NMDA stimulations, neurons were washed into aCSF plus 1 mM TTX for 1 h and then treated with NMDA for 10 min. For okadaic acid treatments, this compound or DMSO vehicle was added to aCSF 10 min prior to NMDA stimulation. For NMDA treatment and recovery experiments, cells were washed into aCSF for 1 h as above and then stimulated for 5 min with 20 mM NMDA. aCSF was removed, cells were washed twice with aCSF plus 1 mM TTX and then allowed to recover at 371C for 40 min. Following stimulations, cells were lysed and immunoprecipitates prepared as for HEK293T cells. For TTX and bicuculline treatments, cells remained in full medium and 1 mM TTX or 20 mM bicuculline (ﬁnal concentrations) were added for 20 min prior to cell lysis. Sucellular fractionation Adult rat PSD fractions were prepared as described (Cho et al, 1992) and normalized to equal protein concentrations using a BCA assay. Live-imaging of pH-GluR2L Rat hippocampal cultures (DIV 12) were transfected with pH- GluR2L or point mutants using lipofectamine (Lin and Huganir, 2007). Imaging was performed as described (Lin and Huganir, 2007). Images were collected at the rate of one image/min from a single optical section (o3 mm) near the surface of the neuron. A Zeiss MultiTime macro was used to correct for focus drift during imaging. Control experiments conﬁrming the speciﬁcity of the pHluorin ﬂuorescence signal are detailed in Supplementary data and presented in Supplementary Figure S12. Statistical analysis All biochemical experiments were performed at least three times. For neuronal experiments results were obtained from at least two individual platings of neurons. A ChemiDoc XRS system (Bio-Rad) was used for densitometric scanning of X-ray ﬁlms. Data are expressed as mean7s.e.m. from the indicated number of experi- ments. Statistical signiﬁcance was determined using an unpaired Student t-test and signiﬁcance level is noted in ﬁgure legends. & 2008 European Molecular Biology Organization