Molecular Roles of Cdk5 in Neuronal Functions and Pain Signaling

CDK5调节疼痛信号：我们目前的研究主要集中在描绘CDK5在中枢和外周神经系统中的作用，特别强调伤害性和疼痛。我们早些时候在伤害性神经元中发现了CDK5和p35的表达，并发现这种表达在外周炎症过程中受到调节。我们还发现，诱导的炎症会导致感觉神经元中钙蛋白酶活性的增加，从而激活p35到p25的裂解，从而增加CDK5的活性。与野生型对照相比，p35-/-小鼠表现出显著的CDK5活性降低，对疼痛的热刺激的反应延迟。相比之下，过度表达p35且CDK5活性水平升高的小鼠比对照组对疼痛的热刺激更敏感。我们分析了CDK5对TRPV1的潜在磷酸化作用，发现CDK5可以在苏氨酸407位直接磷酸化TRPV1，进而调节激动剂诱导的钙内流。我们还发现，抑制CDK5活性可导致辣椒素引起的培养背根神经节神经元钙内流的减弱，且这种减弱是可逆的。这些观察结果表明，CDK5介导的TRPV1的磷酸化对于辣椒素通过该受体介导的钙内流是重要的。由于CDK5-/-小鼠在围产期死亡，我们建立了初级伤害性受体特异性的CDK5条件性基因敲除(CoKO)小鼠，以确定CDK5在初级传入疼痛信号中的确切作用。在基础状态下，条件性基因敲除小鼠有显著的痛觉减退，证实了正常的CDK5活性在初级传入中的直接作用。总之，我们的发现显示了一种新的CDK5调节TRPV1功能的分子机制，并提示CDK5/p35可能是开发止痛药的一个靶点。 我们目前在08财年进行的研究的目的是确定调节CDK5/p35活性的促炎分子对炎症的反应。我们构建了一个含有小鼠p35启动子的载体，该启动子驱动荧光素酶的表达。我们将该载体瞬时导入PC12细胞，以检测几种细胞因子对p35转录活性和CDK5活性的影响。我们的结果表明，肿瘤坏死因子-α以剂量和时间依赖的方式激活p35启动子的活性，并伴随着上调CDK5的活性。由于已知肿瘤坏死因子-α可激活ERK1/2、p38MAPK、JNK和NF-kB信号通路，因此我们研究了它们在激活p35启动子活性中的作用。抑制ERK激活的MEK抑制剂降低了p35启动子的活性，而p38MAPK、JNK和NF-kB的抑制剂则增加了p35启动子的活性，表明这些途径对p35表达的调控是不同的。早期基因反应1(Egr-1)是一种转录因子，经肿瘤坏死因子-α处理后，早期基因反应1(Egr-1)的mRNA和蛋白水平均增加，且这种增加依赖于ERK信号转导。在一种炎症性疼痛的小鼠模型中，角叉菜胶注射到后爪引起对热刺激的超敏反应，注射部位的肿瘤坏死因子-αmRNA增加。这些发现表明，肿瘤坏死因子-α介导的CDK5活性调节在炎症诱导的疼痛信号中起着重要作用。 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，这些关键调控酶催化大约100,000个不同位点的蛋白质磷酸化，从而可逆地控制它们的功能活动。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。我们目前对这种图谱的了解来自于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野生型大脑的简单蛋白质印迹分析，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6个不同的组。第一组由25个不同的蛋白组成，第二组由77个不同的其他蛋白组成，第三组由39个不同的底物组成，第四组由43个参与细胞周期的不同蛋白组成，第五组由37个不同的蛋白组成，这些不同的蛋白参与多种神经生物学功能，第六组由37个不同的蛋白控制不同的激酶通路。这种磷酸蛋白质组的筛选使我们对参与这些生化途径和细胞过程的CDK5靶标有了一个广泛的概述。 我们将制定策略，帮助我们确定CDK5在牙齿疼痛中的作用，并开发潜在的止痛药。与CDK5相关的其他潜在重要问题的工作已经转移到与一些神经生物学领域的领先实验室正在进行的重大合作上。这一转变将使我们在CDK5和疼痛信号方面日益占据主导地位的计划受益。

Cdk5 regulates pain signaling: Our present studies are primarily focused on delineating the roles of Cdk5 in the central and peripheral nervous system with a special emphasis on nociception and pain. We have earlier identified expression of Cdk5 and p35 in nociceptive neurons, and discovered that this expression is modulated during peripheral inflammation. We also discovered that induced inflammation results in increased calpain activity in sensory neurons, which activates cleavage of p35 to p25 and subsequently increases Cdk5 kinase activity. The p35-/- mice, which exhibit significantly decreased Cdk5 activity, showed delayed responses towards painful thermal stimulation compared to their wild-type controls. In contrast, mice overexpressing p35 with elevated levels of Cdk5 activity were more sensitive to painful thermal stimuli than controls. We analyzed TRPV1 for potential phosphorylation by Cdk5 and found that Cdk5 can directly phosphorylate TRPV1 at threonine 407, and this in turn modulates agonist-induced calcium influx. We also found that inhibiting Cdk5 activity resulted in attenuation of capsaicin-induced calcium influx in cultured DRG neurons, and this attenuation was reversible. These observations suggest that Cdk5-mediated phosphorylation of TRPV1 is important for capsaicin-mediated calcium influx through this receptor. Since Cdk5-/- mice die perinatally, we generated primary nociceptor-specific Cdk5 conditional knockout (COKO) mice to identify the precise role of Cdk5 in primary afferent pain signaling. In the basal state, the conditional knockout mice had significant hypoalgesia, confirming the direct role of normal Cdk5 activity in primary afferents. Collectively, our findings show a novel molecular mechanism for the functional regulation of TRPV1 by Cdk5 and suggest that Cdk5/p35 may be a target for development of analgesic drugs. The aim of our current study in FY08 was to identify the proinflammatory molecules that regulate Cdk5/p35 activity in response to inflammation. We constructed a vector that contains the mouse p35 promoter driving luciferase expression. We transiently transfected this vector in PC12 cells to test the effect of several cytokines on p35 transcriptional activity and Cdk5 activity. Our results indicate that tumor necrosis factor-alpha (TNF-alpha) activates p35 promoter activity in a dose- and time-dependent manner and concomitantly upregulates Cdk5 activity. Because TNF-alpha is known to activate ERK1/2, p38 MAPK, JNK, and NF-kB signaling pathways, we examined their involvement in the activation of p35 promoter activity. MEK inhibitor, which inhibits ERK activation, decreased p35 promoter activity, while the inhibitors of p38 MAPK, JNK, and NF-kB increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The mRNA and protein levels of early gene response 1 (Egr-1), a transcription factor, were increased by TNF-alpha treatment, and this increase was dependent on ERK signaling. In a mouse model of inflammation-induced pain in which carrageenan injection into the hind paw causes hypersensitivity to heat stimuli, TNF-alpha mRNA was increased at the site of injection. These findings suggest that TNF-alpha-mediated regulation of Cdk5 activity plays an important role in inflammation-induced pain signaling. Phosphoproteomic analysis of Cdk5 targets: The human genome encodes over 500 different protein kinases, 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 performed in different laboratories 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 phosphorylation status and total protein levels of 258 different proteins by simple Western blotting analyses of Cdk5-/- and WT wild-type brains. The antibodies used in this analysis are 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 6 different groups. The first group contained 25 different proteins involved in apoptosis, the second consisted of 77 different other kinases, the third consisted of 39 different substrates for these kinases, the fourth consisted of 43 different proteins involved in the cell cycle, the fifth contained 37 different proteins involved in numerous neurobiological functions, and the sixth consisted of 37 different proteins controlling different kinase pathways. This phosphoproteomic screening gave us a broad overview of Cdk5 targets involved in these biochemical pathways and cellular processes. We will develop strategies to help us identify the role of Cdk5 in tooth pain and develop potential analgesics. Work on other potentially important questions involving Cdk5 have been shifted to ongoing major collaborations with a number of leading laboratories in neurobiology. This shift will benefit us in our increasingly predominant program on Cdk5 and pain signaling.