Neuroscience Letters 444 (2008) 245–249 Contents lists available at ScienceDirect Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet Amphiphysin I phosphorylation on residue threonine 260 in a pentylenetetrazole-induced seizure model Mowdood Choudhury, Oliver Kleiner, Raymond Chung, Laura Barden, Louise Morgan, Toshal R. Patel, James M. Staddon ∗ Eisai London Research Laboratories Limited, Bernard Katz Building, University College London, Gower Street, London WC1E 6BT, United Kingdom article info Article history: Received 13 June 2008 Received in revised form 17 July 2008 Accepted 11 August 2008 Keywords: Amphiphysin Threonine 260 Pentylenetetrazole Phosphorylation Epilepsy abstract A method to evaluate kinase inhibitor action was reported [L. Morgan, S.J. Neame, H. Child, R. Chung, B. Shah, L. Barden, J.M. Staddon, T.R. Patel, Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system, Neurosci. Lett. 395 (2006) 143–148]. In this, acute administration of the GABA antagonist pentylenetetrazole triggers seizures through glutamate- dependent pathways. Under such conditions, activation of the c-Jun N-terminal kinase (JNK) pathway was detected in hippocampal extracts. Phosphorylation of the upstream JNK kinase MKK4 was also revealed through use of a phospho-MKK4-specific antibody. Here, this antibody is shown to also react with a protein of ∼125kDa which underwent increased phosphorylation in response to pentylenetetrazole treatment. The present study aimed to identify the ∼125kDa protein as it may provide novel insight into signalling, neuronal activity and seizures. Using chromatographic methods and mass spectrometry, the protein was identified as amphiphysin I. This was confirmed by 2D gel analysis and immunoblot with amphiphysin I- specific antibodies. Although the phospho-MKK4 antibody was raised against an MKK4-specific peptide, partial sequence homology between this sequence and a region of amphiphysin was discerned. New antibodies raised against the phospho-threonine 260-amphiphysin-specific sequence detected increased phosphorylation in response to pentylenetetrazole treatment. This particular phosphorylation site does not seem to have been described before, possibly reflecting a novel regulatory aspect of amphiphysin biology. As amphiphysin is involved in the regulation of endocytosis, phosphorylation at this site may play a role in the regulated re-uptake of synaptic vesicles after neurotransmitter release. © 2008 Elsevier Ireland Ltd. All rights reserved. A pentylenetetrazole (PTZ)-induced seizure model to evaluate effects of inhibitors of c-Jun N-terminal kinases (JNKs) on c- Jun phosphorylation in the central nervous system was reported [10]. PTZ, a non-competitive GABA antagonist, leads to overac- tive glutamate signalling, resulting in seizures. Excessive glutamate signalling triggers activation of stress pathways in neurons, and phosphorylation of c-Jun (reviewed by [2,7]). JNKs are activated through phosphorylation by upstream kinases, MKK4 and MKK7 which are also activated by phosphorylation [19]. In the earlier PTZ study [10], robust phosphorylation of MKK4 was detected through use of a specific anti-phospho-MKK4 antibody. This par- ticular antibody also cross-reacted with a 125kDa protein, and immunoreactivity, presumably reflecting increased phosphoryla- tion, was also increased following PTZ treatment. As signalling pathways are not completely defined in PTZ-induced seizures, it Abbreviation: PTZ, pentylenetetrazole. ∗ Corresponding author. Tel.: +44 20 7388 4746; fax: +44 20 7413 1121. E-mail address: james staddon@eisai.net (J.M. Staddon). was decided to attempt to identify this protein. This is reported here and implications discussed. Animal procedures were carried out following approval and guidance of the U.K. Home Office (UK Animals (Scientific Pro- cedures) Act 1986). PTZ treatment (1h) was carried out exactly as described [10]. Hippocampi were dissected rapidly and homogenised immediately in 0.5 ml of ice-cold HPI buffer (see [10]). Samples were snap-frozen and stored at -80 ◦ C. For blotting, samples were mixed with an equal volume of 2× Laemmli sample buffer [8] and adjusted to 2 mg protein/ml. Pro- tein (60 g) was resolved by SDS-PAGE (8% polyacrylamide, large format), transferred to nitrocellulose (Hybond ECL, GE) and stained with Ponceau S. Membranes were blocked for 2 h in PBST (PBS containing 0.05% Tween 20) containing 1% BSA, probed with pri- mary antibody in block overnight at 4 ◦ C, rinsed in PBST and then incubated with HRP-conjugated secondary antibody in 1% pow- dered milk in PBST for 1 h at room temperature. After rinsing the membrane using PBST, HRP was detected by luminography (ECL, GE). Band intensities were quantified by scanning densitometry (Phoretix 1D software, Version 2003.01, Nonlinear Dynamics Ltd.). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.055