Neuroscience Letters 407 (2006) 258–262 Development- and activity-dependent regulation of SNAP-25 phosphorylation in rat brain Masakazu Kataoka a , Reiko Kuwahara b , Ryota Matsuo c , Mariko Sekiguchi b , Kaoru Inokuchi b , Masami Takahashi d,∗ a Department of Environmental Science and Technology, Faculty of Engineering, Shinshu University, Wakasato 4-17-1, Nagano-shi, Nagano 380-8553, Japan b Mitsubishi Kagaku Institute of Life Sciences, Minamiooya 11, Machida, Tokyo 194-8511, Japan c Laboratory of Functional Biology, Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Shido, Sanuki-City, Kagawa 769-2193, Japan d Department of Biochemistry, Kitasato University School of Medicine, Kitasato 1-15-1, Sagamihara-shi, Kanagawa 228-8555, Japan Received 17 May 2006; received in revised form 21 August 2006; accepted 22 August 2006 Abstract Synaptosomal-associated protein of 25 kDa (SNAP-25), a member of the SNARE proteins essential for neurotransmitter release, is phosphorylated at Ser 187 in PC12 cells and in the rat brain in a protein kinase C-dependent manner. It remains unclear how the phosphorylation of SNAP-25 is regulated during development and by neuronal activity. We studied the mode of SNAP-25 phosphorylation at Ser 187 in the rat brain using an anti-phosphorylated SNAP-25 antibody. Both the expression and phosphorylation of SNAP-25 increased remarkably during the early postnatal period, but their onsets were quite different. SNAP-25 expression was detected as early as embryonic Day 18, whereas the phosphorylation of SNAP-25 could not be detected until postnatal Day 4. A delay in the onset of phosphorylation was also observed in cultured rat hippocampal neurons. The phosphorylation of SNAP-25 was regulated in a neuronal activity-dependent manner and, in the rat hippocampus, decreased by introducing seizures with kainic acid. These results clearly indicated that the phosphorylation of SNAP-25 at Ser 187 is regulated in development- and neuronal activity-dependent manners, and is likely to play important roles in higher brain functions. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: SNAP-25; Phosphorylation; Development; Activity-dependent Neurotransmitter release is regulated by several different mech- anisms, and these regulations are some of the important mecha- nisms of synaptic plasticity that underlie learning and memory. In the short-term, many protein kinases regulate neurotransmit- ter release both positively and negatively, for which various protein substrates have been identified [2,11,24,32,40]. How- ever, little is known about the precise mechanisms and functional relevance to brain functions. Synaptosomal-associated protein of 25 kDa (SNAP-25) is a membrane protein expressed in neurons and endocrine cells [29]. In neurons, SNAP-25 is localized primarily in axons Abbreviations: BAP, bacterial alkaline phosphatase; HFS, high frequency stimulation; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMA, phorbol-12-myristate-13-acetate; SDS, sodium dodecyl sulfate; SNAP-25, synaptosomal-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive fusion protein receptor ∗ Corresponding author. Tel.: +81 42 778 8626; fax: +81 42 778 9548. E-mail address: masami@med.kitasato-u.ac.jp (M. Takahashi). and nerve terminals, with a large part associated with the plasma membrane through palmitoylated cysteine residues. A significant population of SNAP-25 is also localized in cytoplasmic vesicular membranes [43]. SNAP-25 is one of the so-called soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE) proteins and plays an essential role in neu- rotransmitter release through the formation of a core complex with other SNARE proteins, VAMP-2/synaptobrevin 2 and syntaxin 1 [4,5,17,33,37–39]. In addition to its essential role in neurotransmitter release, the possible involvement of SNAP-25 in neurite extension and sprouting [3,28], regulation of ion channel functions [13,14,20,44,46], and neurotransmitter- receptor incorporation into the plasma membrane [18,19] has been suggested. Thus, SNAP-25 is likely to play multiple roles in synapses, and the elucidation of its regulatory mechanisms will contribute to the understanding of synaptic plasticity. Previously, we showed that the treatment of clonal rat pheochromocytoma PC12 cells with phorbol-12-myristate- 13-acetate (PMA), a potent protein kinase C (PKC) activator, 0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.08.055