Proteomics of phospho-proteins during brain development using mutant mice

目的和方法：在这个提议中，我试图通过分析蛋白质磷酸化来了解大脑发育的机制，这对这些过程很重要。比较正常小鼠和脑发育缺陷突变小鼠的磷酸化蛋白质，将为脑发育的分子机制提供新的发现。为了这个目的，我们进行了二维DIGE方法，然后通过MS分析，以确定proteins.Results：在2006年，我们分析了大脑皮层样品Cdk 5基因敲除小鼠及其同窝对照在E 18.5。Cdk 5是神经元特异性的Ser/Thr蛋白激酶，并且Cdk 5 KO小鼠表现出缺陷的脑发育，特别是在皮质结构的层形成中。利用2D-DIGE技术对磷酸化蛋白质组学进行了研究，在23个差异点中鉴定出CRMP 1、2和4。我们先前报道了Cdk 5在Ser 522磷酸化CRMP 2。我们使用pThr 509 CRMP 1的磷特异性抗体证实Cdk 5在体内磷酸化CRMP 1的Thr 509。然而，我们不能检测到CRMP 4 Ser 522磷酸化的减少，表明其他激酶的冗余磷酸化。我们还发现肌球蛋白Ser 38的磷酸化减少（Hayashi，et.例如，2007年，我们分析了来自Dab 1突变型yotari和小脑特异性Cdk 5 KO小鼠的小脑沿着它们的同窝对照。我们确定了几个差异点，我们的实验室正在进一步分析这些点。我们还报道了Cdk 5通过Dabl的磷酸化抑制Reelin信号传导（Ohshima，et.例如，2007年）。

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).