Molecular Roles of Cdk5 in Neuronal Functions and Pain Signaling

炎症过程中CDK5活性的调节： 鉴于我们早期关于CDK5在炎症诱导的疼痛中的重要作用的发现，我们继续我们的研究，以进一步确定在炎症过程中释放的哪些分子可以诱导CDK5的活性。使用我们实验室开发的基于PC12细胞的分析方法，我们首先筛选了已知的促炎趋化因子、白三烯和细胞因子对CDK5激酶活性的影响。通过这些研究，我们发现肿瘤坏死因子-α诱导CDK5的辅活化子p35持续而强劲的表达，从而增加CDK5的活性。肿瘤坏死因子-α激活ERK1/2导致Egr-1表达增加，随后p35表达上调。此外，我们还证明了小鼠足爪注射角叉菜胶引起的实验性炎症增加了肿瘤坏死因子-α的表达，进而诱导了更多的CDK5活性，导致疼痛信号的增强。 CDK5靶标的磷酸蛋白质组学分析： 人类基因组编码500多种不同的蛋白激酶，这些蛋白激酶是关键的调节酶，催化大约100,000个不同位点的蛋白质磷酸化，以可逆地控制其功能活动。特定蛋白激酶的缺陷与400多种疾病有关，目前约25%的制药工业研究和开发集中于发现和评估用于治疗应用的蛋白激酶抑制剂。CDK5因其在神经元动态平衡中的关键作用而成为制药业关注的靶点。到目前为止，已经鉴定了40多种不同的CDK5底物，并且异常的CDK5活性与一些疾病过程有关，包括神经退行性疾病、癌症和糖尿病。然而，CDK5介导的蛋白质磷酸化的全球图谱仍然不可用。我们目前对这类图谱的了解来自于在不同实验室进行的实验，主要是基于二维凝胶电泳法或酵母双杂交筛选。由于这些技术在验证目标蛋白方面的局限性，我们采取了不同的方法来解决这个问题。我们通过对CDK5-/-和WT野生型脑进行简单的蛋白质印迹分析，比较了258种不同蛋白质的磷酸化状态和总蛋白水平。这项分析中使用的抗体已经被证明在不同的生化途径中对其靶点具有高度的特异性。我们根据CDK5的一些已知和预测的功能来选择这些蛋白质，并进一步将它们分为以下几类：细胞凋亡、激酶、细胞周期和神经退行性变。我们现在已经通过进一步的测试验证了其中一些蛋白质，我们将继续研究它们在细胞中的作用。 现在很明显，CDK5在许多神经元功能中发挥着重要作用，特别是在疼痛信号转导中。我们未来工作的总体战略是描述CDK5在疼痛信号中的分子作用，重点是面部和口腔区域。

Regulation of Cdk5 activity during inflammation: In light of our earlier findings on the important role of Cdk5 in inflammation-induced pain, we continued our studies further to identify which molecules released during inflammation induce Cdk5 activity. Using a PC12 cell-based assay developed in our laboratory, we first screened known pro-inflammatory chemokines, leukotrienes and cytokines for their effects on Cdk5 kinase activity. Through these studies, we identified that tumor necrosis factor-alpha (TNF-alpha) induces a sustained and robust expression of p35, a coactivator of Cdk5, thereby increasing Cdk5 kinase activity. The activation of ERK1/2 by TNF-alpha leads to an increase in Egr-1 expression and the subsequent elevation of p35 expression. Furthermore, we demonstrated that the experimental inflammation induced by injecting carrageenan in mouse paw increases expression of TNF-alpha which in turn induces more Cdk5 activity, resulting in elevated pain signaling. Phosphoproteomic analysis of Cdk5 targets: The human genome encodes over 500 different protein kinases, which are the key regulatory enzymes that catalyze the phosphorylation of proteins at about 100,000 different sites to reversibly control their functional activities. Defects in specific protein kinases have been linked to over 400 diseases, and about 25% of all pharmaceutical industry research and development is now focused on the discovery and evaluation of protein kinase inhibitors for therapeutic applications. Cdk5 has become a target of high interest to the drug industry because of its key role in neuronal homeostasis. So far more than 40 different Cdk5 substrates have been identified, and abnormal Cdk5 activity has been implicated in several disease processes, including neurodegenerative disorders, cancer, and diabetes. However, a global profiling of protein phosphorylation mediated by Cdk5 is still not available. Our current knowledge about such profiling comes from experiments that are performed in different laboratories and are mainly based on 2-dimensional gel electrophoresis or yeast 2-hybrid screening. Because of the limitations of these techniques at the point of validation of targeted proteins, we took a different approach to resolve this issue. We compared the phosphorylation status and the total protein levels of 258 different proteins through simple Western blotting analysis of Cdk5-/- and WT wild-type brains. The antibodies used in this analysis had already proven to be highly specific for their targeted sites in different biochemical pathways. We based our selection of these proteins on some known and predicted functions of Cdk5, and further categorized them into the following groups: apoptosis, kinases, cell cycle, and neurodegeneration. We have now validated some of these proteins by further testing, and we will continue to study them for their cellular roles. It is clear now that Cdk5 plays an important role in many neuronal functions and especially in pain signaling. The overall strategy for our future work is focused on delineating the molecular roles of Cdk5 in pain signaling, with an emphasis on the facial and oral region.