Stimulation of phosphatidylcholine breakdown by thrombin and carbachol but not by tyrosine kinase receptor ligands in cells transfected with M1 muscarinic receptors. Rapid desensitization of phosphocholine-specific (PC) phospholipase D but sustained activity of PC-phospholipase C.
In order to evaluate the possible contribution of phospholipase D (PLD) stimulation to the mitogenic response, a screening of a variety of different compounds, some of which are known to be potent mitogens, was performed using the well characterized Chinese hamster lung fibroblast (CCL39) cell line. In wild type CCL39 cells, or derivatives expressing high levels of either the human M1 muscarinic receptor (Hm1) or the human epidermal growth factor (EGF) receptor (39M1-81 and 39ER22 clones, respectively), thrombin, a potent mitogen for all three cell types, elicited the rapid activation of PLD (t1/2 activation, 30 s). Carbachol-mediated activation of the Hm1 receptor in the 39M1-81 clone, which is not a mitogenic signal, produced a similarly rapid although greater activation of PLD. Addition of EGF to the 39ER22 clone was able to provoke both a mitogenic response and stimulate PLD, albeit a comparatively small effect. In each case, the stimulation of PLD correlated closely with the ability to stimulate inositol phospholipid breakdown and was entirely dependent on the activation of protein kinase C. Moreover, the ability of both thrombin and carbachol to stimulate PLD was found to be rapidly desensitized, with a similar time course of desensitization (t1/2 desensitization, 90 s). It has recently been reported that an increase in phospholipase C (PLC)-mediated phosphocholine (PC) hydrolysis by either addition of agonist or by extracellular addition of PC-specific PLC enzyme constitutes a mitogenic signal. In this regard, in addition to stimulation of PLD, thrombin and carbachol were both able to stimulate the activity of a phosphocholine-specific phospholipase C (PC-PLC), which did not appear to desensitize within the time course employed. By contrast, EGF was unable to elicit the stimulation of PC-PLC. Ligands such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), which bind to and activate receptors with intrinsic tyrosine kinase activity, are potent mitogens for CCL39 cells but were unable to stimulate either PLD or PC-PLC activity. Furthermore, exogenous addition of purified PC-PLC enzyme, although able to induce a strong and lasting hydrolysis of PC, was unable to produce a mitogenic signal on its own. On the basis of these results, we conclude that the activation of both PLD and PC-PLC is neither sufficient nor required to produce a mitogenic response.
McKenzie
F R
FR
Centre de Biochimie, UMR. 134, Nice, France.
Seuwen
K
K
Pouysségur
J
J
D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D002794:Choline / Q000302:isolation & purification / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004075:Diglycerides / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010713:Phosphatidylcholines / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010767:Phosphorylcholine / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D004075:Diglycerides; D006133:Growth Substances; D008024:Ligands; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D011956:Receptors, Cell Surface; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D010767:Phosphorylcholine; D015240:Phorbol 12,13-Dibutyrate; D002217:Carbachol; D011505:Protein-Tyrosine Kinases; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D010739:Phospholipase D; D013917:Thrombin; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002794:Choline