Proteomics of phospho-proteins during brain development using mutant mice

目的与方法：在本提案中，我试图通过分析在大脑发育过程中重要的蛋白质磷酸化来了解大脑发育的机制。脑发育缺陷小鼠与正常小鼠的磷酸化蛋白比较，将为脑发育的分子机制提供新的发现。为此，我们采用2D-DIGE法和MS分析法对蛋白进行鉴定。结果：2006年，我们分析了Cdk5 KO小鼠和它们的同窝对照在e18.5时的大脑皮层样本。Cdk5是一种神经元特异性丝氨酸/苏氨酸蛋白激酶，Cdk5 KO小鼠表现出大脑发育缺陷，特别是在皮层结构的层形成中。采用2D-DIGE方法对磷酸化蛋白进行蛋白质组学分析，在23个差异点中鉴定出CRMP1、2和4。我们之前报道过Cdk5磷酸化CRMP2的Ser522位点。我们使用pThr509 CRMP1的磷酸化特异性抗体，证实了Cdk5在体内磷酸化CRMP1的Thr509位点。然而，我们没有检测到CRMP4 Ser522磷酸化的减少，表明有其他激酶的冗余磷酸化。我们还发现镇静蛋白Ser38的磷酸化降低（Hayashi等人，2006）。2007年，我们分析了Dab 1突变体yotari和小脑特异性Cdk5 KO小鼠的小脑以及它们的同窝对照。我们发现了几个不同的斑点，现在我们的实验室正在对这些斑点进行进一步的分析。我们还报道了Cdk5通过Dabl的磷酸化抑制Reelin信号传导（Ohshima等，2007）。

Purpose and Method : In this proposal, I attempt to understand the mechanism of brain development by analyzing protein phosphorylations which are important for such processes. Comparison of phospho-proteins between controls and mutant mice which have defective brain development will provide us new findings about molecular mechanism of brain development. For this purpose, we conducted 2D-DIGE method followed by MS analysis to identify the proteins.Results: In 2006, we analyzed the cerebral cortex samples from Cdk5 KO mice and their littermate controls at E 18.5. Cdk5 is a neuron-specific Ser/Thr protein kinase and Cdk5 KO mice exhibit defective brain development particularly in layer formation of cortical structures. We conducted protetomics of phospho-proteins by 2D-DIGE method, and identified CRMP1, 2 and 4 among 23 differential spots. We previously reported that Cdk5 phosphorylates CRMP2 at Ser522. We confirmed that Cdk5 phosphorylates CRMP1 at Thr 509 in vivo using phosphor-specific antibody for pThr509 CRMP1. However, we couldn't detect the reduction of phosphorylation of CRMP4 Ser522, indicating the redundant phosphorylation by other kinase (s). We also found the reduced phosphorylation of stathmin Ser38 (Hayashi, et. al., 2006).In 2007, we analyzed cerebella from Dab 1 mutant yotari and cerebellum-specific Cdk5 KO mice along with their littermate controls. We identified several differential spots and further analysis of these spots is now on going in our laboratory. We also reported that Cdk5 inhibits Reelin signaling through the phosphorylation of Dabl (Ohshima, et. al., 2007).