Investigating the role of phosphorylation in the regulation of Diacylglycerol Lipase α/β activity

Student thesis: Doctoral ThesisDoctor of Philosophy


Diacylglycerol Lipase α (DAGLα) and β (DAGLβ) hydrolyse diacylglycerol (DAG) to produce 2-arachidonoylglycerol (2-AG), the main endogenous ligand for the cannabinoid receptors 1 (CB1) and 2 (CB2). Anatomical and pharmacological studies, complimented by knockout models, of these enzymes have implicated them in CB1/2 mediated processes such as axonal growth and guidance during development and neurogenesis and synaptic signalling in the adult. Despite considerable knowledge about the functions of these enzymes, little is known about their regulation.
To start to address this, we used bioinformatics tools to generate a model of the catalytic domain of the DAGLs which revealed a regulatory loop (putative lid) shielding the catalytic site of these enzymes, similar to other lipases like hormone sensitive lipase (HSL). A phospho-map of the DAGLs generated by collating phosphoproteomic data revealed several phosphorylation sites that were well placed to regulate an 'open' and 'closed' confirmation of the lid, thereby regulating substrate access to the active site. We developed a cellular assay monitoring DAGL dependent CB1 activation as well as surrogate substrate assays to directly monitor the catalytic activity of the DAGLs. Assay results revealed that the kinases PKA and PKC enhanced DAGL dependent CB1 signalling. However these kinases did not affect DAGL activity against the surrogate substrates; this might be explained by the surrogate substrates having access to the active site irrespective of the position of the regulatory loop. The catalytic domains of the DAGLs were purified using a baculovirus expression system and in the case of DAGLβ we were able to demonstrate using in vitro phosphorylation and phosphoproteomic studies that PKA and PKC can directly phosphorylate the regulatory loop of this enzyme. A phospho-specific antibody against one of these sites (S570) was generated and validated and conditions were identified that could up and down regulate phosphorylation at this site in cells.
In summary, the first structure/function model for the DAGLs is reported here together with considerable evidence for direct phosphorylation of the catalytic domain being a key regulatory mechanism.
Date of Award2013
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorPatrick Doherty (Supervisor) & Gareth Williams (Supervisor)

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