Protein phosphatase 2A dephosphorylates SNAP-25 through two distinct mechanisms in mouse brain synaptosomes
Protein phosphatase 2A dephosphorylates SNAP-25 through two distinct mechanisms in mouse brain synaptosomes
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DOI：
10.1016/j.neures.2013.01.002
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发表时间：
2013-03-01
影响因子：
2.9
通讯作者：
Takahashi, Masami
中科院分区：
文献类型：
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作者：
Iida, Yuuki;Yamamori, Saori;Takahashi, Masami
突触体相关蛋白25 （SNAP-25）作为t-SNARE蛋白在胞外神经递质释放中起重要作用。SNAP-25以蛋白激酶C （PKC）依赖的方式在丝氨酸（187）位点磷酸化，但其去磷酸化的机制尚不清楚。我们通过将SNAP-25与生长相关蛋白43（另一种pkc依赖性突触前磷酸化蛋白GAP-43）进行比较，研究了SNAP-25在小鼠脑突触体粗制剂中的去磷酸化作用。在PKC激活剂phorbol 12,13 -dibutyrate （PDB）处理后，SNAP-25和GAP-43的磷酸化水平显著增加，而离子霉素处理以时间依赖性的方式诱导了显著的降低。这种去磷酸化仅发生在细胞外Ca2+存在的情况下，表明参与了Ca2+依赖性磷酸酶。钙调磷酸酶/PP2B抑制剂（如FK506和环孢素A）不抑制Ca2+依赖性去磷酸化，然而，SNAP-25去磷酸化被calyculin A （PP1和PP2A的非选择性抑制剂）和冈田酸（PP2A选择性抑制剂）抑制，但不被PP1选择性的他霉素（tautomycin）抑制。相反，这些抑制剂都没有抑制GAP-43的去磷酸化。冈田酸以浓度依赖性的方式增强了pdb诱导的SNAP-25磷酸化。这些结果表明PP2A通过Ca2+依赖性和Ca2+非依赖性机制参与SNAP-25的去磷酸化，但不参与GAP-43的去磷酸化。(C) 2013爱思唯尔爱尔兰有限公司和日本神经科学学会。版权所有。
Synaptosomal-associated protein 25 (SNAP-25) plays an essential role in exocytotic neurotransmitter release as a t-SNARE protein. SNAP-25 is phosphorylated at Ser(187) in a protein kinase C (PKC)-dependent manner, but the mechanism for dephosphorylation has yet to be clarified. We investigated SNAP-25 dephosphorylation by comparing it to growth associated protein 43 (GAP-43), another PKC-dependent presynaptic phosphoprotein, in crude mouse brain synaptosome preparations. Phosphorylation levels for both SNAP-25 and GAP-43 increased significantly after treatment with PKC activator phorbol 12, 13-dibutyrate (PDB), and ionomycin treatment induced a striking reduction in a time-dependent manner. This dephosphorylation occurred only in the presence of extracellular Ca2+, indicating involvement of a Ca2+-dependent phosphatase. Ca2+-dependent dephosphorylation was not suppressed by calcineurin/PP2B inhibitors such as FK506 and cyclosporine A. SNAP-25 dephosphorylation, however, was suppressed by calyculin A, a non-selective inhibitor of PP1 and PP2A, and okadaic acid selective for PP2A, but not by tautomycin selective for PP1. In contrast, none of these inhibitors suppressed GAP-43 dephosphorylation. PDB-induced SNAP-25 phosphorylation was enhanced by okadaic acid in a concentration-dependent manner. These results suggest that PP2A participates in SNAP-25 dephosphorylation through Ca2+-dependent and Ca2+-independent mechanisms but is not involved in GAP-43 dephosphorylation. (C) 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.