JNK dependent AMPA-R regulation GM Thomas et al Figure 3 JNK-JIP1 complexes mediate high basal phosphorylation of GluR2L-Thr912 and GluR4-Thr855 in neurons. (A) Cortical neurons (DIV 15–20) were washed into aCSF containing DMSO vehicle control (‘Con’) 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 lysis. GluR2L immunoprecipitates were blotted for phosphoThr912 (Top) and GluR2L (second panel). GluR2L-Thr912 phosphorylation relative to total GluR2L signal (normalized to control, 100%): SP10: 53.575.4%, N ¼ 6; SP20: 35.475.4%, N ¼ 6; SB: 95.7718.2%, N ¼ 5; U0: 89.8718.2%, N ¼ 6; Rosc: 86724.5%, N ¼ 5). GluR4 immunoprecipitates were blotted for phosphoThr855 (third panel) or GluR4 (bottom). GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to control, 100%). SP10: 19.873.9%; SP20: 24.272.9%; SB: 93.779.1%; U0:86.974.8%; Rosc: 102.7712.1% (N ¼ 4 for all treatments). (B) as A except that neurons were incubated for 1 h with the indicated concentrations of TAT-JBD peptide. (C) GluR2L immunoprecipitates (pre-incubated with or without antigenic peptide) were prepared from membrane fractions from juvenile (P17–20) rats and blotted for JNK pathway components. (D) As (C) except that GluR4 immunoprecipitates were prepared. Figure 4 JNK dynamically regulates GluR2L-Thr912 and GluR4-Thr855 phosphorylation in neurons. (A) Cortical neurons were incubated in aCSF in the presence ( þ ) or absence ( ) of 10 mM SP600125 for 1 h and then stimulated for 10 min with 1 mM okadaic acid ( þ ) or left unstimulated ( ). GluR2L immunoprecipitates were blotted for phosphoThr912 (top), GluR2L (second panel), phosphoJNK (third panel) or panJNK (bottom). GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) SP: 53.575.4%, N ¼ 6; OA: 197.8732.8%, N ¼ 4; OA þ SP: 64.0732.2%, N ¼ 4. (B) as A except that GluR4 immunoprecipitates were prepared and blotted for phosphoThr855 (top) or GluR4 antibody (second panel). GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to control, 100%) OA: 209.2749.1%, N ¼ 6; OA þ SP: 90.9726.6%, N ¼ 5. fractions, consistent with data suggesting that JNK2 functions pre-synaptically (Chen et al, 2005). Phosphorylation is a rapid and reversible process and so we investigated whether GluR2L-Thr912 or GluR4-Thr855 phosphorylation are dynamically regulated in neurons. Treatment of neurons with the phosphatase inhibitor okadaic acid increased levels of phosphoJNK, GluR2L-Thr912(P) (Figure 4A) and GluR4-Thr855(P) (Figure 4B). Okadaic & 2008 European Molecular Biology Organization acid-induced increases in both GluR2L-Thr912(P) and GluR4-Thr855(P) were JNK inhibitor-sensitive. Pathological stimuli alter neuronal JNK substrate phos- phorylation (Yang et al, 1997; Fogarty et al, 2003), but it is less clear how more subtle changes, particularly in glutama- tergic signaling, might regulate neuronal JNK substrates. We therefore examined whether glutamatergic signaling regulates GluR2L/GluR4 phosphorylation. Bath application The EMBO Journal VOL 27 | NO 2 | 2008 365