The EMBO Journal (2008) 27, 361–372 | & 2008 European Molecular Biology Organization | Some Rights Reserved 0261-4189/08 www.embojournal.org Rapid and bi-directional regulation of AMPA receptor phosphorylation and trafﬁcking by JNK THE EMBO JOURNAL EMBO open This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without speciﬁc permission. Gareth M Thomas, Da-Ting Lin, Mutsuo Nuriya1 and Richard L Huganir* Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA Jun N-terminal kinases (JNKs) are implicated in various neuropathological conditions. However, physiological roles for JNKs in neurons remain largely unknown, despite the high expression level of JNKs in brain. Here, using bioinformatic and biochemical approaches, we iden- tify the AMPA receptor GluR2L and GluR4 subunits as novel physiological JNK substrates in vitro, in hetero- logous cells and in neurons. Consistent with this ﬁnding, GluR2L and GluR4 associate with speciﬁc JNK signaling components in the brain. Moreover, the modulation of the novel JNK sites in GluR2L and GluR4 is dynamic and bi-directional, such that phosphorylation and de-phos- phorylation are triggered within minutes following decreases and increases in neuronal activity, respectively. Using live-imaging techniques to address the functional consequence of these activity-dependent changes we demonstrate that the novel JNK site in GluR2L controls reinsertion of internalized GluR2L back to the cell surface following NMDA treatment, without affecting basal GluR2L trafﬁcking. Taken together, our results demon- strate that JNK directly regulates AMPA-R trafﬁcking following changes in neuronal activity in a rapid and bi-directional manner. The EMBO Journal (2008) 27, 361–372. doi:10.1038/ sj.emboj.7601969; Published online 10 January 2008 Subject Categories: neuroscience; signal transduction Keywords: LTD; LTP; MAPK; synaptic transmission Introduction Mitogen-activated protein kinases (MAPKs) mediate cellular responses to diverse signals, including growth factors, pro- inﬂammatory cytokines and high osmolarity (Pearson et al, 2001). However, MAPKs are also highly expressed in brain, where they regulate various forms of synaptic plasticity. For example, the extracellular-signal regulated kinases (ERKs) *Corresponding author. Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, 725 N Wolfe Street, Hunterian 1001, Baltimore, MD 21205, USA. Tel.: þ 1 410 955 4050; Fax: þ 1 410 955 0877; E-mail: rhuganir@jhmi.edu 1Present address: Department of Pharmacology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan Received: 5 November 2007; accepted: 3 December 2007; published online: 10 January 2008 & 2008 European Molecular Biology Organization play key roles in several forms of Long-term potentiation (LTP), while p38 MAPK may mediate certain forms of Long-term depression (LTD) (Zhu et al, 2002; Thomas and Huganir, 2004). Another major MAPK subgroup is the cJun N-terminal kinases (JNKs), named for their ability to phosphorylate the transcription factor cJun. The JNK family comprises three related gene products: JNK1 and JNK2 are widely expressed, while JNK3 is restricted to brain, plus a limited set of non- neuronal tissues (Barr and Bogoyevitch, 2001; Pearson et al, 2001). Much work on JNKs in neurons has focused on their roles in neuropathology. In particular, mice lacking JNK3 show diminished excitotoxic cell death (Yang et al, 1997) and reduced effects of cerebral ischemia-hypoxia (Kuan et al, 2003), while JNK inhibitors show therapeutic promise by reducing cell death in many neuropathological models (Xia et al, 2001; Waetzig and Herdegen, 2005). Despite this focus on their neuropathological roles, the high neuronal expres- sion of JNKs suggests that these kinases play important physiological roles. This hypothesis is strengthened by reports that JNKs, particularly the JNK1 isoform, exhibit high constitutive activity in brain (Lee et al, 1999; Coffey et al, 2002; Kuan et al, 2003; Bjorkblom et al, 2005; Brecht et al, 2005). Furthermore, neuronal JNK-dependent effects in both physiological and pathological conditions correlate poorly with cJun phosphorylation, suggesting key roles for additional JNK substrates (Bjorkblom et al, 2005; Besirli et al, 2005). Recently, JNKs have been reported to regulate synaptic plasticity (Chen et al, 2005; Zhu et al, 2005). However, direct JNK substrates that might control synaptic transmission remain to be identiﬁed. The lack of deﬁnitive physiological neuronal JNK substrates has also hindered investigation into how JNKs, and the phosphatases that oppose them, regulate downstream targets in response to changes in neuronal activity. AMPA-type glutamate receptors (AMPA-Rs) mediate the majority of fast excitatory neurotransmission in the brain. Regulation of AMPA-Rs plays critical roles in forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) (Malinow and Malenka, 2002; Song and Huganir, 2002). Native AMPA-Rs are multimers, formed from combinations of four different subunits, named Glutamate Receptors (GluRs) 1–4. GluRs are grouped, accord- ing to length and homology of their intracellular C-terminal tails, into ‘long’ and ‘short’ classes. Alternative splicing of the GluR2 and GluR4 mRNAs generates both long and short- tailed versions of these AMPA-Rs while GluR1 and GluR3 have only a long-tailed and a short-tailed version, respec- tively. Phosphorylation sites and interaction motifs present only in speciﬁc AMPA-R subunits allow regulation of speciﬁc receptor subpopulations, which then play key roles in speciﬁc forms of plasticity. For example, AMPA-Rs containing the GluR1 and GluR2 (short form) subunits are reported to play The EMBO Journal VOL 27 | NO 2 | 2008 361