Epidermal growth factor stimulates distinct diradylglycerol species generation in Swiss 3T3 fibroblasts: evidence for a potential phosphatidylcholine-specific phospholipase C-catalysed pathway.
Stimulation of 3T3 fibroblasts with epidermal growth factor (EGF) results in an increase in 1,2-diacylglycerol (DAG) mass which is maximal at 25 s, declining at 1 min and returning to basal levels by 30 min. No changes in alkylacylglycerol or alkenylacylglycerol were detected. Three species account for most of this mass increase: 18:0/20:5,n-3, 18:0/20:4,n-6 and 18:0/20:3,n-9. These species are characteristic of the phosphoinositides; however, previous work failed to detect any EGF-stimulated rise in inositol phosphates in these cells [Cook and Wakelam (1992) Biochem. J. 285, 247-253]. This ruled out phosphoinositide hydrolysis by phospholipase C, but raised the possibility of phospholipase D/phosphatidate phosphohydrolase-catalysed hydrolysis of phosphatidylinositol. The inclusion of butanol in the incubation medium failed to block the diacylglycerol changes, indicating that the phospholipase D pathway is not involved and that DAG must be derived from another source, probably via phospholipase C-catalysed hydrolysis of a phosphatidylcholine pool that is particularly rich in these species. The tyrosine kinase inhibitor ST-271 almost abolished the elevation in 18:0/20:5,n-3, 18:0/20:4, n-6 and 18:0/20:3,n-9 at 25 s, but only reduced the rise in total DAG mass by about 50%. The protein kinase C (PKC) inhibitor Ro-31-8220 increased DAG levels at all time points but had no effect on the species profiles. This provides additional evidence for PKC-mediated regulation of cell-surface EGF receptors, since the inhibition of PKC would increase the availability and/or ligand binding affinity of receptors at the plasma membrane and hence increase and prolong the response to EGF.
Pettitt
T R
TR
Institute of Biochemistry, University of Glasgow, Scotland, U.K.
Zaqqa
M
M
Wakelam
M J
MJ
D016475:3T3 Cells; D000818:Animals; D002384:Catalysis; D004075:Diglycerides / Q000096:biosynthesis*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D010738:Type C Phospholipases / Q000378:metabolism*
C011439:1,2-diacylglycerol; D004075:Diglycerides; D007211:Indoles; D010713:Phosphatidylcholines; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D010739:Phospholipase D; C064758:Ro 31-8220