Identification and functional analysis of Cdk5-mediated phosphorylation of the proteins that are related to brain formation and development

目的和方法：CDK5是一种神经元特异性Ser/Thr蛋白激酶，CDK5 KO小鼠表现出脑发育缺陷，尤其是皮质结构的层形成。CDK5KO小鼠的大脑发育异常是由大脑发育相关蛋白的磷酸化缺陷引起的。利用CDK5KO小鼠的大脑，我们进行了一项筛选，以确定CDK5底物(S)。进一步对新鉴定的底物S进行了功能分析。我们还研究了CDK5介导的Reelin信号转导因子Dab1的磷酸化的功能意义。结果：通过筛选，我们确定CRMP2是CDK5的底物。在横滨市立大学医学院与Goshima博士的研究小组的合作下，我们发现CDK5在Ser522处使CRMP2磷酸化。这种磷酸化是丝氨酸509被GSK3β磷酸化所必需的。CDK5和GSK3β的这种顺序磷酸化降低了CRMP2和微管蛋白之间的亲和力。我们还证明了CRMP2的这种顺序磷酸化与3F4反应的产生有关，特别是在阿尔茨海默病患者的大脑中。在另一项研究中，我们发现CDK5在其C末端的多个位置磷酸化Dab1。CDK5介导的Dab1丝氨酸/苏氨酸磷酸化抑制Reelin诱导的Dab1酪氨酸磷酸化。这一结果表明CDK5对Reelin信号的负调制。

Purpose and Method :Cdk5 is a neuron-specific Ser/Thr protein kinase and Cdk5 KO mice exhibit defective brain development particularly in layer formation of cortical structures. Abnormalities of brain development in Cdk5 KO mice are caused by defects in the phosphorylations of brain development related-proteins. Using brains from Cdk5 KO mice, we conducted a screening to identify Cdk5 substrate(s). Functional analysis of newly identified substrate(s) is further performed. We also studied the functional significance of Cdk5-mediated phosphorylation of Dab1 which is a intracellular mediator of Reelin signaling.Results :Through the screening, we identified CRMP2 as a substrate of Cdk5. Under the collaboration with Dr.Goshima's group in Yokohama City University School of Medicine, we found Cdk5 phosphorylates CRMP2 at Ser522. This phosphorylation is required for Ser509 phosphorylation by GSK3β. This sequential phosphorylation by Cdk5 and GSK3β reduces the affinity between CRMP2 and tubulins. We also demonstrate that this sequential phosphorylation of CRMP2 is related to the production of 3F4 reactivity specifically in the brains from Alzheimer's patients. In another study, we showed that Cdk5 phosphorylates Dab1 at multiple sites in its C-terminal. Cdk5-mediated Ser/Thr phosphorylations of Dab1 inhibit tyrosine phosphorylation of Dab1 induced by Reelin. This result indicates a negative modulation of Reelin signaling by Cdk5.