Amphiphysin I phosphorylation on residue threonine 260 in a pentylenetetrazole-induced seizure model

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• 作者：Mowdood Choudhury ; Oliver Kleiner ; Raymond Chung ; Laura Barden ; Louise Morgan ; Toshal R. Patel ; James M. Staddon
• 关键词：Amphiphysin ; Threonine 260 ; Pentylenetetrazole ; Phosphorylation ; Epilepsy
• 刊名：Neuroscience Letters
• 出版年：2008
• 出版时间：31 October 2008
• 年：2008
• 卷：444
• 期：3
• 页码：245-249
• 全文大小：537 K

文摘

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 125 kDa which underwent increased phosphorylation in response to pentylenetetrazole treatment. The present study aimed to identify the 125 kDa 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.