Nerve growth factor-induced phosphorylation of SNAP-25 in PC12 cells: A possible involvement in the regulation of SNAP-25 localization
Nerve growth factor-induced phosphorylation of SNAP-25 in PC12 cells: A possible involvement in the regulation of SNAP-25 localization
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DOI：
10.1046/j.1471-4159.2000.0742058.x
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发表时间：
2000-05-01
影响因子：
4.7
通讯作者：
Takahashi, M
中科院分区：
文献类型：
--
作者：
Kataoka, M;Kuwahara, R;Takahashi, M
25 kDa 突触体相关蛋白 (SNAP-25) 是一种神经递质释放所必需的 t-SNARE 蛋白，在用佛波醇 12-肉豆蔻酸酯 13-乙酸酯处理激活细胞蛋白激酶 C 后，在 Ser(187) 处磷酸化。然而，尚不清楚神经元活动或内源配体是否诱导 SNAP-25 磷酸化。在这里，我们使用针对 Ser(187) 磷酸化 SNAP-25 的特异性抗体研究了 PC12 细胞中 SNAP-25 的磷酸化。当细胞在缺乏神经生长因子 (NGF) 的情况下生长时，一小部分 SNAP-25 会被磷酸化。足以激活丝裂原激活蛋白激酶信号转导途径的 NGF 短暂处理不会增加 SNAP-25 的磷酸化；然而，与 NGF 长时间孵育后，磷酸化作用上调。上调是暂时的，并且在添加 NGF 后 36 至 48 小时内达到最大磷酸化（比基础磷酸化增加四倍）。免疫荧光显微镜显示 SNAP-25 主要位于质膜中，尽管细胞质中也存在大量的 SNAP-25。定量显微荧光测定法显示，长时间使用 NGF 处理会导致 SNAP-25 在质膜中优先定位。使用带有绿色荧光蛋白的融合蛋白作为标签的突变研究表明，Ser(187) 点突变为 Ala 消除了 NGF 依赖性重定位。 Ser(187) 点突变为 Glu 后，质膜中 SNAP-25 的数量并未增加；然而，长期使用 NGF 处理会增加这种磷酸化，这表明 SNAP-25 磷酸化对于 NGF 诱导的向质膜的重新定位是必要的，但还不够。我们的结果表明，SNAP-25 磷酸化的上调及其重新定位与 NGF 诱导的 PC12 细胞分化之间存在密切的时间关系。
Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein essential for neurotransmitter release, is phosphorylated at Ser(187) following activation of cellular protein kinase C by treatment with phorbol 12-myristate 13-acetate. However, it remains unclear whether neuronal activity or an endogenous ligand induces the phosphorylation of SNAP-25. Here we studied the phosphorylation of SNAP-25 in PC12 cells using a specific antibody for SNAP-25 phosphorylated at Ser(187). A small fraction of SNAP-25 was phosphorylated when cells were grown in the absence of nerve growth factor (NGF). A brief treatment with NGF that was enough to activate the mitogen-activated protein kinase signal transduction pathway did not increase the phosphorylation of SNAP-25; however, phosphorylation was up-regulated after a prolonged incubation with NGF. Up-regulation was transitory, and maximum phosphorylation (a fourfold increase over basal phosphorylation) was achieved between 36 and 48 h after the addition of NGF. Immunofluorescent microscopy showed that SNAP-25 was localized primarily in the plasma membrane, although a significant population was also present in the cytoplasm. Quantitative microfluorometry revealed that prolonged treatment with NGF resulted in a preferential localization of SNAP-25 in the plasma membrane. A mutational study using a fusion protein with green fluorescent protein as a tag indicated that the point mutation of Ser(187) to Ala abolished the NGF-dependent relocalization. A population of SNAP-25 in the plasma membrane was not increased by a point mutation at Ser(187) to Glu; however, it was increased by prolonged treatment with NGF, indicating that the SNAP-25 phosphorylation is essential, but not sufficient, for the NGF-induced relocation to the plasma membrane. Our results suggest a close temporal relationship between the up-regulation of SNAP-25 phosphorylation and its relocation, and NGF-induced differentiation of PC12 cells.