Available online 13 September 2022, 106295

A new fluorescent DGKε activity assay using NBD-SAG as a substrate is developed

The assay allows activity determination of purified DGKε and in cell lysates

DGKε activity and stability are affected by four amino acids of a zinc finger motif

Cys167Trp substitution in the zinc finger motif of DGKε is found in aHus patients

Disruption of the zinc finger motif of C1B domain is deleterious to DGKε

Diacylglycerol kinase-ε (DGKε) phosphorylates DAG to phosphatidic acid with unique specificity toward 18:0/20:4 DAG (SAG). SAG is a typical backbone of phosphatidylinositol and its derivatives, therefore DGKε activity is crucial for the turnover of these signaling lipids. Malfunction of DGKε contributes to several pathophysiological conditions, including atypical hemolytic uremic syndrome (aHUS) linked with DGKE mutations. In the present study we analyzed the role of a zinc finger motif of the C1B domain of DGKε, as some aHUS-linked mutations affect this ill-defined part of the kinase. For this, we introduce a novel fluorescent assay for determination of DGKε activity which relies on the use of NBD-SAG in mixed micelles as a substrate, followed by TLC separation of NBD-phosphatidic acid formed. The assay reliably determines the activity of purified human GST-DGKε, also endogenous DGKε or overexpressed mouse DGKε-Myc in cell lysates, homogenates, and kinase immunoprecipitates. Using the above assay we found that four amino acids, Cys135, Cys138, His161 and Cys164, forming the zinc finger motif in the C1B domain are required for the DGKε-Myc activity and stability. Substitution of any of these amino acids with Ala or Trp in DGKε-Myc abolished its activity and led to its proteasomal degradation, possibly assisted by Hsp70/90/40 chaperones. Inhibition of the 26 S proteasome prevented the degradation but the mutated proteins were inactive. The present data on the deleterious effect of the zinc finger motif disruption contribute to the understanding of the DGKε-linked aHUS, as the Cys164Trp substitution in mouse DGKε corresponds to the Cys167Trp one in human DGKε found in some aHUS patients.

atypical hemolytic uremic syndrome-

cysteine-rich conserved homology-1

1-NBD-stearoyl-2-arachidonoyl-sn-glycerol

1-palmitoyl-2-NBD-dodecanoyl-sn-glycerol

1-palmitoyl-2-NBD-dodecanoyl-sn-glycero-3-phosphocholine

1-NBD-stearoyl-2-arachidonoyl-sn-glycero-3-phosphate

1-palmitoyl-2-NBD-dodecanoyl-sn-glycero-3-phosphate

phosphatidylcholine-specific phospholipase C

1-palmitoyl-2-dodecanoyl-sn-glycerol or 16:0/12:0-DAG

phosphorylated derivative of PI

1,2-diacyl-sn-glycero-phosphoserine

1-stearoyl-2-arachidonoyl-sn-glycerol or 18:0/20:4-DAG

Tris(2-carboxyethyl)phosphine hydrochloride

thin layer chromatography

atypical hemolytic uremic syndrome

diacylglycerol kinase

kinase activity assay

phosphatidic acid

zinc finger motif

© 2022 The Author(s). Published by Elsevier Ltd.