Protein Kinase C-Dependent Phosphorylation of Synaptosome-Associated Protein of 25 kDa at Ser187 Potentiates Vesicle Recruitment
Protein Kinase C-Dependent Phosphorylation of Synaptosome-Associated Protein of 25 kDa at Ser187 Potentiates Vesicle Recruitment
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DOI：
10.1523/jneurosci.22-21-09278.2002
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发表时间：
2002-11
期刊：
影响因子：
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通讯作者：
G. Nagy;U. Matti;R. Nehring;T. Binz;J. Rettig;E. Neher;J. B. Sørensen
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文献类型：
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作者：
G. Nagy;U. Matti;R. Nehring;T. Binz;J. Rettig;E. Neher;J. B. Sørensen
蛋白激酶C(PKC)的激活是神经元和神经分泌细胞在广泛刺激下上调分泌强度的关键事件，可能是通过加快囊泡供应来实现的。然而，分子靶标及其作用方式仍然难以捉摸。我们研究了神经元可溶N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARE)复合体Ser187在25 kDa突触体相关蛋白(SNAP-25)中唯一依赖于PKC的磷酸化位点。该磷酸化位点位于SNAP-25的带负电荷的C末端，已被证明在钙触发的胞吐中起着至关重要的作用。我们将利用嗜铬细胞过度表达的突变研究与电容测量和笼养钙的闪光光解相结合，允许在刺激和测量胞吐过程中获得高时间分辨率。模拟磷酸化形式的Ser187突变体的过表达加速了可释放的囊泡池排空后的囊泡募集。过度表达模拟非磷酸化形式的突变体，或PKC的阻断，在类似程度上削弱了囊泡池的再充盈。生化研究证实，在细胞内钙浓度升高后，SNAP-25的一个亚群被磷酸化。其中一些突变导致快速胞吐猝发成分的适度减少，这似乎与SNAP-25的磷酸化状态无关。因此，SNAP-25的C末端对快速胞吐和囊泡募集都起作用，后一过程受PKC依赖的磷酸化调控。
Activation of protein kinase C (PKC) constitutes a key event in the upregulation of secretory strength in neurons and neurosecretory cells during extensive stimulation, presumably by speeding up vesicle supply. However, the molecular targets and their mode of action remain elusive. We studied the only PKC-dependent phosphorylation site in the neuronal solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Ser187, in synaptosome-associated protein of 25 kDa (SNAP-25). This phosphorylation site is located within the negatively charged C-terminal end of SNAP-25, which has been shown to be of critical importance in calcium-triggered exocytosis. We combined mutational studies that used overexpression in chromaffin cells with capacitance measurements and flash photolysis of caged calcium, allowing for high time resolution during both the stimulation and measurement of exocytosis. Overexpression of mutants simulating the phosphorylated form of Ser187 accelerated vesicle recruitment after the emptying of the releasable vesicle pools. Overexpression of mutants simulating the nonphosphorylated form, or block of PKC, impaired the refilling of the vesicle pools to similar extents. Biochemical studies verified the phosphorylation of a subpopulation of SNAP-25 after elevation of intracellular calcium concentrations. Some of the mutations led to a moderately decreased fast exocytotic burst component, which did not seem to be associated with the phosphorylation state of SNAP-25. Thus the C terminus of SNAP-25 plays a role for both fast exocytosis triggering and vesicle recruitment, and the latter process is regulated by PKC-dependent phosphorylation.