Collapsin response-mediator protein 5 (CRMP5) phosphorylation at threonine 516 regulates neurite outgrowth inhibition
Collapsin response-mediator protein 5 (CRMP5) phosphorylation at threonine 516 regulates neurite outgrowth inhibition
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DOI：
10.1111/ejn.12674
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发表时间：
2014-10-01
影响因子：
3.4
通讯作者：
Honnorat, Jerome
中科院分区：
文献类型：
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作者：
Brot, Sebastien;Smaoune, Hinda;Honnorat, Jerome
塌陷反应介导蛋白 (CRMP) 是一种多功能蛋白，在大脑发育过程中高度表达，但在成人大脑中下调。它们参与轴突引导和神经突生长信号传导。其中，经过深入研究的CRMP2已被确定为轴突生长的重要参与者，这种活性通过CRMP2的磷酸化状态与细胞骨架蛋白的重组相关。另一个成员 CRMP5 通过抑制早期发育阶段的树突生长来限制 CRMP2 的生长促进作用。当 CRMP5 与微管蛋白和微管相关蛋白 MAP2 结合时，就会发生这种抑制作用，但 CRMP5 磷酸化的作用仍不清楚。在这里，我们通过突变分析研究了 CRMP5 磷酸化的作用。使用CRMP5的非磷酸化截短构建体，我们证明，在先前确定的四个CRMP5磷酸化位点（T509、T514、T516和S534）中，仅需要T516残基的磷酸化来抑制PC12细胞和培养的C57BL/6J小鼠海马神经元中的神经突生长。事实上，CRMP5非磷酸化形式的表达诱导了CRMP5的功能丧失，并且模仿磷酸化形式的突变体诱导了野生型CRMP5中所见的生长抑制功能。 T516 磷酸化是通过糖原合酶激酶 3 (GSK-3) 实现的，它可以磷酸化野生型蛋白，但不能磷酸化不可磷酸化的突变体。此外，我们还表明 T516 磷酸化对于 CRMP5 的微管蛋白结合特性至关重要。因此，CRMP5 诱导的生长抑制依赖于通过 GSK-3 途径的 T516 磷酸化。这些发现为神经突生长的机制提供了新的见解。
The collapsin response-mediator proteins (CRMPs) are multifunctional proteins highly expressed during brain development but down-regulated in the adult brain. They are involved in axon guidance and neurite outgrowth signalling. Among these, the intensively studied CRMP2 has been identified as an important actor in axon outgrowth, this activity being correlated with the reorganisation of cytoskeletal proteins via the phosphorylation state of CRMP2. Another member, CRMP5, restricts the growth-promotional effects of CRMP2 by inhibiting dendrite outgrowth at early developmental stages. This inhibition occurs when CRMP5 binds to tubulin and the microtubule-associated protein MAP2, but the role of CRMP5 phosphorylation is still unknown. Here, we have studied the role of CRMP5 phosphorylation by mutational analysis. Using non-phosphorylatable truncated constructs of CRMP5 we have demonstrated that, among the four previously identified CRMP5 phosphorylation sites (T509, T514, T516 and S534), only the phosphorylation at T516 residue was needed for neurite outgrowth inhibition in PC12 cells and in cultured C57BL/6J mouse hippocampal neurons. Indeed, the expression of the CRMP5 non-phosphorylated form induced a loss of function of CRMP5 and the mutant mimicking the phosphorylated form induced the growth inhibition function seen in wildtype CRMP5. The T516 phosphorylation was achieved by the glycogen synthase kinase-3 (GSK-3), which can phosphorylate the wildtype protein but not the non-phosphorylatable mutant. Furthermore, we have shown that T516 phosphorylation is essential for the tubulin-binding property of CRMP5. Therefore, CRMP5-induced growth inhibition is dependent on T516 phosphorylation through the GSK-3 pathway. The findings provide new insights into the mechanisms underlying neurite outgrowth.