Phosphorylation of Homer3 by calcium/calmodulin-dependent kinase II regulates a coupling state of its target molecules in Purkinje cells
Phosphorylation of Homer3 by calcium/calmodulin-dependent kinase II regulates a coupling state of its target molecules in Purkinje cells
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DOI：
10.1523/jneurosci.4738-07.2008
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发表时间：
2008-05-14
影响因子：
5.3
通讯作者：
Mikoshiba, Katsuhiko
中科院分区：
文献类型：
--
作者：
Mizutani, Akihiro;Kuroda, Yukiko;Mikoshiba, Katsuhiko
荷马蛋白是突触后密度 (PSD) 的组成部分，在耦合不同靶分子中发挥着至关重要的作用。然而，Homer 介导的偶联的调控方面仅涉及 Homer1a 的显性负效应，这需要从头基因表达。在这里，我们提供的证据表明荷马介导的偶联是由其磷酸化状态调节的。我们发现，浦肯野细胞中的主要异构体 Homer3 在体外和体内均被钙/钙调蛋白依赖性蛋白激酶 II (CaMKII) 磷酸化。使用磷特异性抗体进行的生化分级显示，胞浆部分中存在磷酸化 Homer3，而 PSD 中非磷酸化 Homer3 水平较高。在 P/Q 型电压门控 Ca2+ 通道敲除小鼠中，CaMKII 激活减少，Homer3 磷酸化水平和 Homer 3 可溶形式显着降低。此外，Homer3 的强烈磷酸化及其与代谢型谷氨酸受体 1 α (mGluR1 α) 的解离都是由原代培养的浦肯野细胞中的去极化触发的，并且这些事件被 CaMKII 抑制剂抑制。体外结合动力学分析表明，这些磷酸化依赖性事件可归因于磷酸化 Homer3 与其配体的亲和力降低。在异源系统中，mGluR1 α 激活诱导的 Ca2+ 信号传导模式受到 Homer3 磷酸化状态的调节。总之，这些发现表明浦肯野细胞中的 Homer3 可能充当受 CaMKII 磷酸化调节的可逆偶联剂，并且磷酸化能够调节突触后分子结构以响应突触活动。
Homer proteins are components of postsynaptic density (PSD) and play a crucial role in coupling diverse target molecules. However, the regulatory aspect of Homer-mediated coupling has been addressed only about a dominant-negative effect of Homer1a, which requires de novo gene expression. Here, we present evidence that Homer-mediated coupling is regulated by its phosphorylation state. We found that Homer3, the predominant isoform in Purkinje cells, is phosphorylated by calcium/calmodulin-dependent protein kinase II( CaMKII) both in vitro and in vivo. Biochemical fractionation with phosphor-specific antibodies revealed the presence of phosphorylated Homer3 in the cytosolic fraction in contrast to high levels of nonphosphorylated Homer3 in PSD. In P/Q-type voltage-gated-Ca2+ channel knock-out mice, in which CaMKII activation was reduced, the levels of Homer3 phosphorylation and the soluble form of Homer 3 were markedly lower. Furthermore, both robust phosphorylation of Homer3 and its dissociation from metabotropic glutamate receptor 1 alpha (mGluR1 alpha) were triggered by depolarization in primary cultured Purkinje cells, and these events were inhibited by CaMKII inhibitor. An in vitro binding kinetic analysis revealed that these phosphorylation-dependent events were attributable to a decrease in the affinity of phosphorylated Homer3 for its ligand. In a heterologous system, the Ca2+ signaling pattern induced by mGluR1 alpha activation was modulated by the Homer3 phosphorylation state. Together, these findings suggested that Homer3 in Purkinje cells might function as a reversible coupler regulated by CaMKII phosphorylation and that the phosphorylation is capable of regulating the postsynaptic molecular architecture in response to synaptic activity.