Molecular Roles of Cdk5 in Neuronal Functions and Pain Signaling

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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.