An intact zinc finger motif of the C1B domain is critical for stability and activity of diacylglycerol kinase-?

Diacylglycerol kinase-e (DGKe) 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 DGKe activity is crucial for the turnover of these signaling lipids. Malfunction of DGKe contributes to several patho-physiological 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 DGKe, as some aHUS-linked mutations affect this ill-defined part of the kinase. For this, we introduce a novel fluorescent assay for deter-mination of DGKe 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-DGKe, also endogenous DGKe or overexpressed mouse DGKe-Myc in cell lysates, homogenates, and kinase im-munoprecipitates. 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 DGKe-Myc activity and stability. Substi-tution of any of these amino acids with Ala or Trp in DGKe-Myc abolished its activity and led to its proteasomal degradation, possibly assisted by Hsp70/90/40 chaperones. Inhibition of the 26S 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 DGKe-linked aHUS, as the Cys164Trp substitution in mouse DGKe corresponds to the Cys167Trp one in human DGKe found in some aHUS patients.

Automated summary

This study analyzed the role of the zinc finger motif in the C1B domain of DGKe. A novel fluorescent assay was introduced to determine DGKe activity, and it was found that four amino acids in the C1B domain are crucial for the activity and stability of DGKe-Myc.