Phosphorylation by extracellular signal-regulated kinase of a multidomain adaptor protein, vinexin, at synapses
Phosphorylation by extracellular signal-regulated kinase of a multidomain adaptor protein, vinexin, at synapses
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DOI：
10.1111/j.1471-4159.2006.04222.x
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发表时间：
2007-01-01
影响因子：
4.7
通讯作者：
Nagata, Koh-ichi
中科院分区：
文献类型：
--
作者：
Ito, Hidenori;Usuda, Nobuteru;Nagata, Koh-ichi
Vinexin是一种衔接蛋白，被认为在细胞粘附、细胞骨架组织和信号传导中起关键作用。迄今为止，已经报道了至少三种剪接变体，vinexin α、β和γ。尽管vinexin的可能的重要性，vinexin在神经元细胞中的性质和功能几乎是未知的。在这里，我们表明，vinexin亚型在大鼠大脑中表达的发育阶段依赖性的方式，和vinexin α是相对丰富的成年大鼠大脑的端脑区域。免疫组织化学研究显示，在大鼠大脑的神经元和神经胶质细胞定位vinexin α。在原代培养的大鼠海马神经元，vinexin被发现存在于突触和丝状伪足的生长锥的免疫荧光分析。生化分离显示了vinexin在突触体中的分布。电镜观察证实了vinexin的神经末梢定位。据报道，Vinexin β被细胞外信号调节激酶（ERK）在Ser189处磷酸化，其相当于vinexin α的Ser593。因此，我们构建了一个网站和磷酸化状态特异性抗体监测ERK介导的vinexin的磷酸化。在免疫荧光分析中，在培养的大鼠海马神经元之间形成的突触处观察到磷酸化，并且用PD 98059处理细胞可以减少磷酸化。免疫电镜观察发现，磷酸化信号主要位于大鼠海马神经元突触的突触后侧。由于活性ERK与vinexin在突触中共定位，因此ERK信号可能参与调节突触中vinexin依赖的细胞过程。另一方面，在培养3天的神经元中几乎没有检测到磷酸化，这表明在生长锥中存在尚未鉴定的vinexin调节机制。
Vinexin is an adaptor protein that is supposed to play pivotal roles in cell adhesion, cytoskeletal organization and signaling. At least three splice variants, vinexin alpha, beta and gamma, have so far been reported. In spite of the possible importance of vinexin, the properties and functions of vinexin in neuronal cells are almost unknown. Here we show that vinexin isoforms are expressed in rat brain in a developmental stage-dependent manner, and that vinexin alpha is relatively abundant in the telencephalon regions of the adult rat brain. An immunohistochemical study showed the localization of vinexin alpha in neurons and glia in the rat brain. In primary cultured rat hippocampal neurons, vinexin was found to be present at synapses and filopodia in growth cones by immunofluorescent analyses. Biochemical fractionation revealed the distribution of vinexin in synaptosomes. Nerve terminal localization of vinexin was confirmed by electron microscopy. Vinexin beta is reported to be phosphorylated by extracellular signal-regulated kinase (ERK) at Ser189, which is equivalent to Ser593 of vinexin alpha. We thus constructed a site- and phosphorylation state-specific antibody to monitor the ERK-mediated phosphorylation of vinexin. In immunofluorescent analyses, the phosphorylation was observed at synapses formed among cultured rat hippocampal neurons and it was reduced by treatment of the cells with PD98059. In an immunoelectron microscopic examination, the phosphorylation signal was mainly detected on the postsynaptic side of synapses in the rat hippocampal neurons. As active ERK was co-localized with vinexin in synapses, the ERK signal is likely to be involved in the regulation of vinexin-dependent cellular processes in synapses. On the other hand, the phosphorylation was hardly detected in neurons cultured for 3 days, suggesting the presence of a yet unidentified regulatory mechanism of vinexin at the growth cone.