Determination of the topology of the hydrophobic segment of mammalian diacylglycerol kinase epsilon in a cell membrane and its relationship to predictions from modeling.

The epsilon isoform of diacylglycerol kinase (DGKepsilon) is unique among mammalian DGKs in having a segment of hydrophobic amino acids comprising approximately residues 20 to 41. Several algorithms predict this segment to be a transmembrane (TM) helix. Using PepLook, we have performed an in silico analysis of the conformational preference of the segment in a hydrophobic environment comprising residues 18 to 42 of DGKepsilon.

Substrate chirality and specificity of diacylglycerol kinases and the multisubstrate lipid kinase.

The alpha, zeta, and epsilon isoforms of diacylglycerol kinase exhibit a high degree of stereospecificity in the phosphorylation of diacylglycerol. In comparison, a multiple lipid kinase, MuLK, shows much less stereospecificity, phosphorylating 1,2-dioleoylglycerol only approximately 2-3 times more rapidly than 2,3-dioleoylglycerol. The alpha and zeta isoforms of diacylglycerol kinase are inhibited by 2,3-dioleoylglycerol, but not the more substrate-selective epsilon isoform.

Membrane interactions 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 as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKepsilon of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH(2). Flanking Lys residues mimic the natural setting of this peptide in DGKepsilon, while facilitating peptide synthesis and characterization.

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.