Role of the hydrophobic segment of diacylglycerol kinase epsilon.
Diacylglycerol kinase epsilon (DGKepsilon) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids. We have evaluated the contributions of this segment to the membrane interactions and functions of this protein. To test the role of the hydrophobic segment, we have compared the properties of DGKepsilon with those of a truncated form of the protein (DGKDeltaepsilon) lacking the 40 N-terminal amino acids, which includes the hydrophobic segment. The proteins were expressed in COS-7 cells from a gene for human DGKepsilon or from a gene for a truncated form (DGKDeltaepsilon), both of which had a FLAG tag at the amino terminus. Full-length FLAG-DGKepsilon and truncated FLAG-DGKDeltaepsilon were both more specific for 1-stearoyl-2-arachidonoyl-sn-glycerol than for 1,2-dioleoyl-sn-glycerol. 1-Stearoyl-2-linoleoyl-sn-glycerol exhibited intermediate specificity for both forms of the enzyme. The results show that the truncated form of the enzyme maintains substrate specificity for lipids with an arachidonoyl moiety present at the sn-2 position. The truncation increases the catalytic rate constant for all three substrates and may suggest a role in the negative regulation of this enzyme. A full-length DGKepsilon with a C-terminal His tag exhibited substrate specificity similar to that of the other two forms of the enzyme, indicating that the nature and position of the epitope tag did not strongly affect this property. Using an ultracentrifugation floatation assay, we showed that at neutral pH DGKDeltaepsilon is extracted with 1.5 M KCl while DGKepsilon remains essentially fully membrane bound. The full-length protein had a weak tendency to oligomerize in the presence of weak detergents. DGKepsilon was monomeric on SDS-PAGE but exhibited partial dimerization with low concentrations of perfluorooctanoic acid. The major conclusions of this work are that the hydrophobic domain of DGKepsilon does not contribute to substrate specificity but plays a role in permanently sequestering the enzyme to a membrane.
Dicu
Armela O
AO
Department of Biochemistry and Biomedical Sciences, McMaster University Health Science Center, Hamilton, Ontario L8N 3Z5, Canada.
Topham
Matthew K
MK
Ottaway
Lindsay
L
Epand
Richard M
RM
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002522:Chlorocebus aethiops; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D007700:Kinetics; D009842:Oligopeptides; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D007527:Isoenzymes; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D000255:Adenosine Triphosphate; C085706:FLAG peptide; D019852:Diacylglycerol Kinase