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.
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