Study of Arachidonoyl Specificity in Two Enzymes of the PI Cycle

We identified a conserved pattern of residues L-X(3-4)-R-X-(2)-L-X-(4)-G, in which -X-(n)- is n residues of any amino acid, in two enzymes acting on the polyunsaturated fatty acids, diacylglycerol kinase epsilon (DGK epsilon) and phosphatidylinositol-4-phosphate-5-kinase I alpha (PIP5K I alpha). DGK epsilon is the only one of the 10 mammalian isoforms of DGK that exhibits arachidonoyl specificity and is the only isoform with the motif mentioned above. Mutations of the essential residues in this motif result in the loss of arachidonoyl specificity. Furthermore, DGK alpha can be converted to an enzyme having this motif by substituting only one residue. When DGK alpha was mutated so that it gained the motif, the enzyme also gained some specificity for arachidonoyl-containing diacylglycerol. This motif is present also in an isoform of phosphatidylinositol-4-phosphate-5-kinase that we demonstrated had arachidonoyl specificity for its substrate. Single residue mutations within the identified motif of this isoform result in the loss of activity against an arachidonoyl substrate. The importance of acyl chain specificity for the phosphatidic acid activation of phosphatidylinositol-4-phosphate-5-kinase is also shown. We demonstrate that the acyl chain dependence of this phosphatidic acid activation is dependent on the substrate. This is the first demonstration of a motif that endows specificity for an acyl chain in enzymes DGK epsilon and PIP5K I alpha. (C) 2011 Elsevier Ltd. All rights reserved.