Differential regulation of phosphoinositide and phosphatidylcholine hydrolysis by protein kinase C-beta 1 overexpression. Effects on stimulation by alpha-thrombin, guanosine 5'-O-(thiotriphosphate), and calcium.
Rat 6 fibroblasts that stably overexpress cDNA for the beta 1 isozyme of protein kinase C (PKC3 cells) were used to determine the effect of protein kinase C (PKC) overexpression on hormonal stimulation of phospholipid hydrolysis. In control Rat 6 cells, inositol trisphosphate levels (InsP3) were increased 9-fold in 15 s in response to 10 nM alpha-thrombin, compared with only a 2-fold increase in PKC3 cells. PKC overexpression also inhibited thrombin-stimulated production of 1,2-diacylglycerol, the other product of phosphatidylinositol 4,5-bisphosphate hydrolysis, by 73% at 15 s. In permeabilized cells, PKC overexpression greatly reduced guanosine thiotriphosphate-stimulated InsP3 accumulation, but did not affect InsP3 stimulation by increased free calcium concentration. These data suggest that desensitization of thrombin-stimulated phosphoinositide-phospholipase C is enhanced by PKC-beta 1 overexpression and may involve modulation of G-protein/phospholipase C coupling. In contrast, thrombin was 4.5-fold more effective in stimulation of phosphatidylcholine-phospholipase D activity in PKC3 cells than in control cells, as determined by phosphatidylethanol formation. In permeabilized cells, guanosine thiotriphosphate also stimulated phospholipase D activity more effectively in PKC3 cells than in control cells, suggesting that upregulation of phospholipase D activity by PKC overexpression occurs distal to the thrombin receptor. These results suggest that PKC may act as a switch to up-regulate phosphatidylcholine-phospholipase D and down-regulate phosphoinositide-phospholipase C stimulations.
Pachter
J A
JA
Molecular Pharmacology Section, Schering-Plough Research, Bloomfield, New Jersey 07003.
Pai
J K
JK
Mayer-Ezell
R
R
Petrin
J M
JM
Dobek
E
E
Bishop
W R
WR
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D004075:Diglycerides / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013917:Thrombin / Q000494:pharmacology*
D004075:Diglycerides; D007527:Isoenzymes; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D010739:Phospholipase D; D013917:Thrombin; D002118:Calcium