Expression of the phospholipid-dependent Escherichia coli sn-1,2-diacylglycerol kinase in COS cells perturbs cellular lipid composition.
The Escherichia coli sn-1,2-diacylglycerol (DAG) kinase has been successfully expressed in COS cells. The E. coli dgkA locus which contains the coding sequences for DAG kinase was subcloned into an eukaryotic expression vector, pMT2. COS cells transfected with the vector pMT2dgk expressed the DAG kinase as shown by Western analysis. Immunofluorescence studies revealed that the E. coli DAG kinase was prominently but not exclusively located in the endoplasmic reticulum. In addition, mixed micellar assays in beta-octyl glucoside revealed that membranes prepared from pMT2dgk-transfected COS cells contained over a 1500-fold increase in DAG kinase activity: 107 nmol/min/mg compared with only 0.067 nmol/min/mg for controls. DAG kinase activity from the E. coli enzyme was distinguished from endogenous COS cell activity based on differences in thermolability and the ability of the E. coli enzyme to use ceramide as a substrate. No ceramide kinase activity was detected in control COS cells, so the activity detected in pMT2dgk transfectants must have resulted from the expressed E. coli DAG kinase. The Km values for DAG kinase derived from E. coli and COS cells were nearly identical. Finally, transfected COS cells were labeled with [32P]Pi to investigate possible perturbations in lipid composition induced by the action of the E. coli DAG kinase. Ceramide (generated by the action of sphingomyelinase) was also used to clearly implicate the E. coli enzyme. Levels of ceramide phosphate increased more than 150-fold in pMT2dgk-transfected cells relative to controls. The results of these studies show that the E. coli enzyme expressed in COS cells is active and perturbs lipid composition in the intact cell system; the absolute lipid cofactor requirement of E. coli DAG kinase can be satisfied in COS cells.
Ramer
J K
JK
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Bell
R M
RM
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002518:Ceramides / Q000494:pharmacology; D019852:Diacylglycerol Kinase; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D007700:Kinetics; D010743:Phospholipids / Q000378:metabolism* / Q000494:pharmacology; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D002518:Ceramides; D010743:Phospholipids; D010770:Phosphotransferases; D019852:Diacylglycerol Kinase