Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon.
In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor.
Schaap
D
D
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.
van der Wal
J
J
van Blitterswijk
W J
WJ
van der Bend
R L
RL
Ploegh
H L
HL
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D019852:Diacylglycerol Kinase; D004815:Epidermal Growth Factor / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D010449:Peptide Mapping; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D010770:Phosphotransferases / Q000276:immunology / Q000378:metabolism*; D019000:Phosphotyrosine; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D005576:Colforsin; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D010770:Phosphotransferases; D011494:Protein Kinases; D019852:Diacylglycerol Kinase; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D015056:1-Methyl-3-isobutylxanthine