Lipid modulation of the activity of diacylglycerol kinase alpha- and zeta-isoforms: activation by phosphatidylethanolamine and cholesterol.
Diacylglycerol kinase (DGK) isoforms alpha and zeta were extracted from transfected cells that overexpressed these enzymes. We determined the lipid dependence of the binding of these isoforms to liposomes. The modulation by lipid of the rate of phosphorylation of diacylglycerol by these enzymes was also measured. Incorporation of phosphatidylethanolamine into the liposomes resulted in an increased partitioning of both isoforms of DGK to the membrane as well as an increased catalytic rate. We demonstrate that the increased catalytic rate is a consequence of both increased portioning of the enzyme to the membrane and increased catalytic activity of the membrane-bound form. DGKalpha, a calcium-dependent isoform, can be activated in a calcium-independent fashion in the presence of phosphatidylethanolamine. Similar effects are observed with cholesterol. In contrast, sphingomyelin inhibits the activity of both isoforms of DGK. Our results demonstrate that the translocation to membranes and activity of DGKalpha and DGKzeta are modulated by the composition and properties of the membrane. The enzymes are activated by the presence of lipids that promote the formation of inverted phases. However, the promotion of negative curvature is not the sole factor contributing to the lipid effects on enzyme binding and activity. A truncated form of DGKalphalacking both the E-F hand and the recoverin homology domain is constitutively active and is not further activated by any of the lipids tested or by calcium. However, a truncated form lacking only the recoverin homology domain is partially activated by either calcium or certain lipids.
Fanani
Maria Laura
ML
Department of Biochemistry and Biomedical Sciences, McMaster University Health Science Center, Hamilton, Ontario L8N 3Z5, Canada.
Topham
Matthew K
MK
Walsh
James P
JP
Epand
Richard M
RM
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000737:chemistry*; D019852:Diacylglycerol Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020832:EF Hand Motifs / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008081:Liposomes; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D014162:Transfection
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D002135:Calcium-Binding Proteins; D007527:Isoenzymes; D008081:Liposomes; D010714:Phosphatidylethanolamines; C483858:phosphatidylethanolamine; D002784:Cholesterol; D019852:Diacylglycerol Kinase; D002118:Calcium