Molecular Properties of Diacylglycerol Kinase Epsilon

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The proposal is focused on a unique member of the family of mammalian diacylglycerol kinases, i.e. DGK. DGK plays a key role in signal transduction and lipid synthesis. It is the only known member of this group that appears to have specificity for one particular species of substrate, i.e. 1-stearoyl-2-arachidonoyl glycerol. This is the same acyl chain composition as is found in lipid intermediates of the phosphatidylinositol (PI) cycle, the sole pathway for the synthesis of PI and its phosporylated products that play a major role in metabolic regulation and cancer. Interconversion of these phosphorylated forms of PI is highly dependent on the nature of the acyl chains. DGK contributes to the specific acyl chain composition of several lipids involved in cell signaling. The product of the reaction catalyzed by DGK, phosphatidic acid, is also an important signaling agent as well as being a precursor for several other lipids. We have successfully purified catalytically active DGK. This provides an opportunity to understand the molecular structure of this protein. It would be the first mammalian DGK isoform to have its structure determined and would provide a model for the analysis of other isoforms that have some homologous segments and for understanding the functioning of this important family of signaling proteins. We will also study how this protein interacts with membranes and with its substrate. It will be an important example of a peripheral membrane protein that interacts with lipid segments buried in the membrane. In cells DGK functions when bound to a bilayer membrane. Before our success in purifying this enzyme, all activity studies were done in assays using detergent micelles. However, we are now able to use the purified enzyme to assay the activity in liposomes made of bilayer membranes. We found a marked difference between the micellar-based and the liposome-based assays and a dependence of both the activity and specificity on the chemical and physical properties of the lipids used to make the liposomes. This will enable us to gain an understanding of how the functioning of DGK is dependent on the nature of the membrane to which it is bound. This research program will determine how peripheral membrane proteins can bind to membranes and recognize lipid substrates. The example of DGK is of particular importance because of its involvement in determining the acyl chain composition of PI. There is currently no structural information on this protein and no reported studies of its activity on liposomes. Our work will provide important novel structural and functional information. It will contribute to our general understanding of the mechanism of acyl chain recognition by interfacial enzymes and in particular as applied to DGK. This information will provide a more accurate description of an aspect of the regulation of biological systems that eventually will be used for their control.