Regulation of DAGL activity by kinases and phosphatases; implications for on-demand endocannabinoid signalling.

As the population ages, the incidence of psychiatric disorders, neurodegenerative diseases, and pain will increase. In terms of therapeutic intervention, this is self-evidently an area that requires the identification of novel pathways and targets. The CB1 and CB2 cannabinoid receptors are being investigated as potential therapeutic targets, however there are also likely to be considerable therapeutic opportunities based on modulating the synthesis of the most abundant endocannabinoid in the adult brain, 2-arachidonlyglycerol (2-AG). The molecule is generated from diacylglycerol by the sn1-specific DAG lipases (DAGL alpha and DAGL beta) that we recently cloned (1). We have recently established that these enzymes regulate the level of both 2-AG and arachidonic acid throughout the body, and that they are responsible for endocannabinoid mediated synaptic plasticity and neurogenesis in the adult brain (2). To fully exploit the DAGLs as a targets, we need to understand how their activity is regulated. In this context we have identified a number of conserved serine (S) and threonine (T) containing motifs within the catalytic domains of the enzymes. We have also generated a unique cell line that allows us to readily measure DAGL activity in cells. This has involved modification of a commercially available CB1-TANGO cell line. This line contains a chimeric CB1 receptor with a protease sensitive site - in brief, when the CB1 receptor is activated a transcription factor is released and this drives expression of a beta-lactamase reporter gene (for full details see (3)). We have used a lentivirus approach to stably express human DAGL alpha in these cells, and identified conditions where DAGL is inactive in the cells, and conditions where it is active (e.g. following stimulation with calcium). Activity can be monitored over several hours and is reflected in a DAGL/CB1 dependent increase in expression of the beta lactamase reporter. We have used a panel of selective antagonists in pharmacological screen to identify several kinases that are required for DAGL alpha activation by calcium (including PKA, PKC and Cdk5), and used bioinformatics to identify the sites within the catalytic domains that these kinases are most likely to phosphorylate. The project will build upon and extent the pharmacological approach to identify the kinases and phosphatases that regulate DAGL activity. siRNA will be used to further test the importance of candidate kinases/phosphatases by knocking them down. We will use proteomics to directly map the sites that are phosphorylated on DAGL by the candidate kinases in cells, as well as following treatment of purified DAGL protein with the candidate kinases. We will screen a number of commercially available antibodies that recognise 'generic' phosphorylated epitopes in order to identify tools that directly report on the activation status of DAGL. We will also raise phospo-specific antibodies to the key activation sites. Finally, we will mutate key phosphorylation sites and determine the effects of this on enzyme activity following expression of the constructs in the CB1-TANGO cells. 1. Bisogno, T., Howell, F., Williams, G., Minassi, A., Cascio, M. G., Ligresti, A., Matias, I., Schiano-Moriello, A., Paul, P., Williams, E. J., Gangadharan, U., Hobbs, C., Di Marzo, V., and Doherty, P. (2003) J Cell Biol 163, 463-468 2. Gao, Y., Vasilyev, D. V., Goncalves, M. B., Howell, F. V., Hobbs, C., Reisenberg, M., Shen, R., Zhang, M. Y., Strassle, B. W., Lu, P., Mark, L., Piesla, M. J., Deng, K., Kouranova, E. V., Ring, R. H., Whiteside, G. T., Bates, B., Walsh, F. S., Williams, G., Pangalos, M. N., Samad, T. A., and Doherty, P. (2010) J Neurosci 30, 2017-2024 3. van der Lee, M. M., Blomenrohr, M., van der Doelen, A. A., Wat, J. W., Smits, N., Hanson, B. J., van Koppen, C. J., and Zaman, G. J. (2009) J Biomol Screen