Title: Diacylglycerol and Phosphatidate Generated by Phospholipases C and D, Respectively, Have Distinct Fatty Acid Compositions and Functions
Abstract: Stimulation of cells with certain agonists often activates both phospholipases C and D. These generate diacylglycerol and phosphatidate, respectively, although the two lipids are also apparently interconvertable through the actions of phosphatidate phosphohydrolase and diacylglycerol kinase. Diacylglycerol activates protein kinase C while one role for phosphatidate is the activation of actin stress fiber formation. Therefore, if the two lipids are interconvertable, it is theoretically possible that an uncontrolled signaling loop could arise. To address this issue structural analysis of diacylglycerol, phosphatidate, and phosphatidylbutanol (formed in the presence of butan-1-ol) from both Swiss 3T3 and porcine aortic endothelial cells was performed. This demonstrated that phospholipase C activation generates primarily polyunsaturated species while phospholipase D activation generates saturated/monounsaturated species. In the endothelial cells, where phospholipase D was activated by lysophosphatidic acid independently of phospholipase C, there was no activation of protein kinase C. Thus we propose that only polyunsaturated diacylglycerols and saturated/monounsaturated phosphatidates function as intracellular messengers and that their interconversion products are inactive. Stimulation of cells with certain agonists often activates both phospholipases C and D. These generate diacylglycerol and phosphatidate, respectively, although the two lipids are also apparently interconvertable through the actions of phosphatidate phosphohydrolase and diacylglycerol kinase. Diacylglycerol activates protein kinase C while one role for phosphatidate is the activation of actin stress fiber formation. Therefore, if the two lipids are interconvertable, it is theoretically possible that an uncontrolled signaling loop could arise. To address this issue structural analysis of diacylglycerol, phosphatidate, and phosphatidylbutanol (formed in the presence of butan-1-ol) from both Swiss 3T3 and porcine aortic endothelial cells was performed. This demonstrated that phospholipase C activation generates primarily polyunsaturated species while phospholipase D activation generates saturated/monounsaturated species. In the endothelial cells, where phospholipase D was activated by lysophosphatidic acid independently of phospholipase C, there was no activation of protein kinase C. Thus we propose that only polyunsaturated diacylglycerols and saturated/monounsaturated phosphatidates function as intracellular messengers and that their interconversion products are inactive. Stimulation of cells by particular agonists which occupy either heterotrimeric G-protein-coupled receptors or those with an intrinsic tyrosine kinase activity induce an increase in the mass of diradylglycerols (collectively diacylglycerol, alkyl, acylglycerol and alkenyl, acylglycerol; DRG), 1The abbreviations used are: DRG, diradylglycerol; DAG, diacylglycerol; PLC, phospholipase C; PLD, phospholipase D; PKC, protein kinase C; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidate; PBut, phosphatidylbutanol; PAE, porcine aortic endothelial; LPA, lysophosphatidic acid; HPLC, high performance liquid chromatography. 1The abbreviations used are: DRG, diradylglycerol; DAG, diacylglycerol; PLC, phospholipase C; PLD, phospholipase D; PKC, protein kinase C; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidate; PBut, phosphatidylbutanol; PAE, porcine aortic endothelial; LPA, lysophosphatidic acid; HPLC, high performance liquid chromatography. in particularsn-1,2-diacylglycerol (DAG), the physiological activator of protein kinase C (PKC) (1Divecha N. Irvine R.F. Cell. 1995; 80: 269-278Abstract Full Text PDF PubMed Scopus (588) Google Scholar). DAG is produced, together with inositol 1,4,5-trisphosphate which stimulates the elevation of intracellular free calcium concentration, by phospholipase C (PLC)-catalyzed phosphatidylinositol 4,5-bisphosphate hydrolysis. Agonist stimulation of this pathway is rapidly desensitized, DAG generation has been demonstrated to be rapid, but transient, declining toward basal levels within 1–2 min (2Wright T.M. Rangan L.A. Shin H.S. Raben D.M. J. Biol. Chem. 1988; 263: 9374-9380Abstract Full Text PDF PubMed Google Scholar, 3Cook S.J. Palmer S. Plevin R. Wakelam M.J.O. Biochem. J. 1990; 265: 617-620Crossref PubMed Scopus (39) Google Scholar). However, there is frequently a second sustained phase of DAG generation. This phase has been associated with an increase in the activation of phospholipase D (PLD)-catalyzed phosphatidylcholine (PC) hydrolysis, producing phosphatidate (PA) which can be converted to DAG by the action of phosphatidate phosphohydrolase. It has also been proposed that DAG can be derived from other pathways, e.g. through a PC-PLC pathway, although the evidence for stimulation of this pathway in mammalian cells remains mostly circumstantial (4Cook S.J. Wakelam M.J.O. Rev. Physiol. Biochem. Pharmacol. 1992; 119: 14-45Google Scholar, 5Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (916) Google Scholar).Cells contain multiple species of DAG, however, a limited subset of these change following stimulation. Comparison of the acyl chain DAG structures with those of the cellular phospholipids indicated that the initial phase of DAG increase was predominantly from inositol phospholipids, while the sustained phase, which was accompanied by an increase in choline release, was probably produced from PC (6Pessin M.S. Raben D.M. J. Biol. Chem. 1989; 264: 8729-8738Abstract Full Text PDF PubMed Google Scholar, 7Pettitt T.R. Wakelam M.J.O. Biochem. J. 1993; 289: 487-495Crossref PubMed Scopus (27) Google Scholar, 8Pettitt T.R. Zaqqa M. Wakelam M.J.O. Biochem. J. 1994; 298: 655-660Crossref PubMed Scopus (34) Google Scholar, 9Pessin M.S. Baldassare J.J. Raben D.M. J. Biol. Chem. 1990; 265: 7959-7966Abstract Full Text PDF PubMed Google Scholar). The initial phase of DAG generation was made up of specific polyunsaturated DAG species, in particular 18:0/20:3n-9, 18:0/20:4n-6, and 18:0/20:5n-3, while the second phase was predominantly represented by more saturated species (7Pettitt T.R. Wakelam M.J.O. Biochem. J. 1993; 289: 487-495Crossref PubMed Scopus (27) Google Scholar).The role of the PLD pathway remains incompletely defined. We have recently demonstrated that PA, generated by the activation of PLD, can stimulate rho-mediated actin stress fiber formation in porcine aortic endothelial (PAE) cells (10Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar). This would suggest that while PLD activation clearly results in an increase in DAG, it is not certain that this lipid plays a signaling role. Rather, it may be that the initial product, PA, functions as a messenger and that it is converted to DAG to attenuate the signal. In vitro studies have demonstrated that essentially all DAG species can activate PKC, however, the in vivo evidence for activation by PLD-derived DAG remains mixed. One report has demonstrated no activation of PKC in IIC9 fibroblasts stimulated with a concentration of thrombin which did not activate inositol lipid hydrolysis but did activate PLD (11Leach K.L. Ruff V.A. Wright T.M. Pessin M.S. Raben D.M. J. Biol. Chem. 1991; 266: 3215-3221Abstract Full Text PDF PubMed Google Scholar), while another report found activation of PKCε under the same conditions (12Ha K.-S. Exton J.H. J. Biol. Chem. 1993; 268: 10534-10539Abstract Full Text PDF PubMed Google Scholar). A number of signaling roles have been proposed for PA including activation of a kinase (13Khan W.A. Blobe G.C. Richards A.L. Hannun Y.A. J. Biol. Chem. 1994; 269: 9729-9735Abstract Full Text PDF PubMed Google Scholar), membrane fusion (14Wakelam M.J.O. Curr. Top. Membr. Transp. 1988; 32: 87-112Crossref Scopus (25) Google Scholar), and actin stress fiber formation (10Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar). Since DAG is converted to PA in cells by DAG kinase activity and PA is metabolized to DAG by phosphatidate phosphohydrolase activity, if both PA and DAG are second messengers an uncontrolled signaling cycle could result. Therefore we have re-examined the role of the PLD pathway in regulating PKC and have analyzed the acyl chain structure of DAG and PA in stimulated cells. We demonstrate here that the DAG and thus PA derived from PLC activation is predominantly polyunsaturated while the lipids generated by PLD activation are saturated or monounsaturated. In addition PLD-derived DAG does not appear to regulate PKC activity in stimulated PAE cells.DISCUSSIONAgonist-stimulated PLD activity has been proposed to provide the source of sustained DAG generation in cells leading to the sustained activation of protein kinase C. However, it is becoming increasingly apparent that the PA product of PLD activation itself functions as an intracellular messenger. The potential for interconversion of PA and DAG in cells thus raises the possibility of uncontrolled signaling in a normal cell and makes it difficult to reconcile the possibility that both lipids can indeed function as messenger molecules. The results presented in this report provide a molecular basis for separate signaling functions of DAG and PA.We and others have previously reported that in stimulated cells the rapidly generated DAG is predominantly polyunsaturated and apparently derived from inositol phospholipids (6Pessin M.S. Raben D.M. J. Biol. Chem. 1989; 264: 8729-8738Abstract Full Text PDF PubMed Google Scholar, 7Pettitt T.R. Wakelam M.J.O. Biochem. J. 1993; 289: 487-495Crossref PubMed Scopus (27) Google Scholar, 8Pettitt T.R. Zaqqa M. Wakelam M.J.O. Biochem. J. 1994; 298: 655-660Crossref PubMed Scopus (34) Google Scholar, 9Pessin M.S. Baldassare J.J. Raben D.M. J. Biol. Chem. 1990; 265: 7959-7966Abstract Full Text PDF PubMed Google Scholar, 18Lee C. Fisher S.K. Agranoff B.W. Hajra A.K. J. Biol. Chem. 1991; 266: 22837-22846Abstract Full Text PDF PubMed Google Scholar). We now show that the acyl chain structure of the PA in stimulated Swiss 3T3 and PAE cells is predominantly saturated or monounsaturated, in particular 16:0, 18:0, and 18:1n-9. Conversion of this PA to DAG by the action of phosphatidate phosphohydrolase thus produces a saturated/monounsaturated rather than a polyunsaturated species. We have only been able to detect extremely small quantities of polyunsaturated acyl groups in PA, e.g. 20:4n-6 in the presence or absence of butan-1-ol. This presumably reflects the rapid utilization of these species by PA-cytidyl transferase, forming cytidyl monophosphate-PA for the resynthesis of inositol phospholipids, a possibility supported by the observed reduction in polyunsaturated PA mass in stimulated Swiss 3T3 cells. Attempts to trap cytidyl monophosphate-PA, by inositol depletion and LiCl treatment as described by other groups (e.g. Ref. 20Rodriguez R. Imai A. Gershengorn M.C. Mol. Endocrinol. 1987; 1: 802-806Crossref PubMed Scopus (11) Google Scholar) were largely unsuccessful, with no significant accumulation of this lipid (data not shown). Others have also found that this trapping technique did not to work with all cell types (19Drummond A.H. Raeburn C.A. Biochem. J. 1984; 224: 129-135Crossref PubMed Scopus (94) Google Scholar, 20Rodriguez R. Imai A. Gershengorn M.C. Mol. Endocrinol. 1987; 1: 802-806Crossref PubMed Scopus (11) Google Scholar, 21Monaco M.E. Adelson J.R. Biochem. J. 1991; 279: 337-341Crossref PubMed Scopus (20) Google Scholar). Gas chromatography-mass spectrometry analysis of cytidyl monophosphate-PA showed predominantly 16:0, 18:0, and 18:1n-6 fatty acids with no detectable 20:3n-9, 20:4n-6, or 20:5n-3 at any time point, suggesting that molecular species containing these fatty acids are selectively metabolized more rapidly than other species.Analysis of the acyl structure of the PBut formed in cells stimulated in the presence of 30 mm butan-1-ol demonstrated that the saturated/monounsaturated PA was produced by PLD activation, rather than by PLC-catalyzed phospholipid hydrolysis followed by DAG kinase-catalyzed phosphorylation of the generated DAG. The reduction in stimulated DAG generation in the presence of the alcohol defined the fraction generated by PLD activation. Thus the results in Fig. 1 and Table I demonstrate that PLD activation is responsible for sustained DAG generation in both Swiss 3T3 and PAE cells. It was considered possible that the “alcohol trap” of generated PA was incomplete, however, in the LPA-stimulated PAE cells, where PLD is the only agonist-stimulated phospholipase, butan-1-ol, but not butan-2-ol completely prevented DAG generation (Fig. 1).It has been proposed that PLD-derived DAG can stimulate PKC activity in chronically stimulated cells (see Ref. 5Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (916) Google Scholar, for review). This implies that the acyl chain structure of the DAG is not relevant to the ability of a species to function as an activator. The PAE cell provided a useful experimental model to test this hypothesis since LPA stimulation only increased saturated/monounsaturated DAGs which were produced as a result of PLD activation. In the PAE cells all the PKCα and -δ and a significant proportion of the PKCε and -ζ were found in the membrane fraction under basal conditions. This membrane localization was probably a consequence of the high level of 18:0/20:4n-6 and 18:0/20:3n-9 DAGs (Fig. 2) found in the resting cells. This provides support for a specific role for the polyunsaturated DAGs in activating PKC. Fig. 5 clearly shows that the increase in the saturated/monounsaturated DAG species in LPA-stimulated PAE cells was unable to induce the translocation of PKCε to a membrane fraction. PKCε is a calcium independent isoform, thus it would be expected to be translocated by an increase in DAG mass; as a control inclusion of PMA clearly induced complete translocation. This lack of translocation reflected the inability of LPA to stimulate PKC activity in the PAE cells (Fig. 6). Thus the DAG species produced as a result of PLD activation do not appear to be regulators of PKC, at least in this cell line. Therefore, while they can activate in vitro, we suggest that saturated/monounsaturated DAG species do not regulate PKC activity in an intact normal cell.Previous reports (11Leach K.L. Ruff V.A. Wright T.M. Pessin M.S. Raben D.M. J. Biol. Chem. 1991; 266: 3215-3221Abstract Full Text PDF PubMed Google Scholar) have suggested that PKCα translocation is a consequence of phosphatidylinositol 4,5-bisphosphate, rather than PC hydrolysis since an increase in both DAG and [Ca2+] are required. It was previously proposed that the DAG derived from PC hydrolysis did not activate PKC (11Leach K.L. Ruff V.A. Wright T.M. Pessin M.S. Raben D.M. J. Biol. Chem. 1991; 266: 3215-3221Abstract Full Text PDF PubMed Google Scholar). However, this study only examined PKCα and a later study in the same cell line suggested that the DAG derived from PC hydrolysis could stimulate PKCε translocation (12Ha K.-S. Exton J.H. J. Biol. Chem. 1993; 268: 10534-10539Abstract Full Text PDF PubMed Google Scholar), the results reported here differ from that report. A possible explanation for the differences in results may be that in the work reported here we have been able to clearly demonstrate that all of the increased DAG in the stimulated PAE cells is indeed PLD derived.An alternative explanation for the lack of stimulation of PKC by PLD-derived DAG is that the phospholipase has been activated in a compartment devoid of PKC. Subcellular fractionation studies have provided evidence for PLD activity in plasma membranes, Golgi membranes, endoplasmic reticulum, and the nuclear membrane (22Ktistakis N.T. Brown H.A. Sternweis P.C. Roth M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4952-4956Crossref PubMed Scopus (181) Google Scholar, 23Whatmore J. Morgan C.P. Cunningham E. Collison K.S. Willison K.R. Cockcroft S. Biochem. J. 1996; 320: 785-794Crossref PubMed Scopus (66) Google Scholar, 24Balboa M.A. Insel P.A. J. Biol. Chem. 1995; 270: 29843-29847Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 25Provost J.J. Fudge J. Israelit S. Siddiqi A.R. Exton J.H. Biochem. J. 1996; 319: 285-291Crossref PubMed Scopus (72) Google Scholar, 26Baldassare J.J. Jarpe M.B. Alferes L. Raben D.M. J. Biol. Chem. 1997; 272: 4911-4914Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Thus it is unlikely that PLD-derived DAG would be formed in a membrane devoid of PKC, particularly since PKC isoenzymes appear to be able to translocate to most membranes in the cell including those where PLD activity has been detected. Additional support for our proposal that PLD-derived DAG does not activate PKC is the observation that incubation of HL-60 cells with Streptomyces chromofuscus PLD had no effect upon PKC redistribution, while incubation withBacillus cereus phosphatidylinositol-specific phospholipase C induced cytosol to membrane translocation (27Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In keeping with this result, we were only able to observe sustained activation of PKCδ and -ε in Swiss 3T3 cells where polyunsaturated DAG species were elevated (28Olivier A.R. Hansra G. Pettitt T.R. Wakelam M.J.O. Parker P.J. Biochem. J. 1996; 318: 425-519Crossref PubMed Scopus (16) Google Scholar).Thus we propose that the DAG derived from PLD hydrolysis is not involved in signaling, rather it is a metabolite utilized in the resynthesis of phospholipids. This also suggests that the monounsaturated/saturated PA species themselves play a signaling role in cells. Indeed, in our recent demonstration of PA-stimulated actin stress fiber formation in PAE cells, the stimulant was the dioleoyl structure (10Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar). In order for PA to function as a signal a target molecule must exist and thus proof of PA's signaling function is dependent upon the identification of specific PA targets. It is, however, implicit in the proposed function for PA, that the polyunsaturated PA species do not bind to and activate such target proteins under physiological conditions and thus do not function as signaling molecules. In conclusion we suggest that, while both PLD and PLC pathways directly generate second messengers, it is only the polyunsaturated DAGs and the saturated/monounsaturated PAs which serve as signals under physiological conditions. Stimulation of cells by particular agonists which occupy either heterotrimeric G-protein-coupled receptors or those with an intrinsic tyrosine kinase activity induce an increase in the mass of diradylglycerols (collectively diacylglycerol, alkyl, acylglycerol and alkenyl, acylglycerol; DRG), 1The abbreviations used are: DRG, diradylglycerol; DAG, diacylglycerol; PLC, phospholipase C; PLD, phospholipase D; PKC, protein kinase C; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidate; PBut, phosphatidylbutanol; PAE, porcine aortic endothelial; LPA, lysophosphatidic acid; HPLC, high performance liquid chromatography. 1The abbreviations used are: DRG, diradylglycerol; DAG, diacylglycerol; PLC, phospholipase C; PLD, phospholipase D; PKC, protein kinase C; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidate; PBut, phosphatidylbutanol; PAE, porcine aortic endothelial; LPA, lysophosphatidic acid; HPLC, high performance liquid chromatography. in particularsn-1,2-diacylglycerol (DAG), the physiological activator of protein kinase C (PKC) (1Divecha N. Irvine R.F. Cell. 1995; 80: 269-278Abstract Full Text PDF PubMed Scopus (588) Google Scholar). DAG is produced, together with inositol 1,4,5-trisphosphate which stimulates the elevation of intracellular free calcium concentration, by phospholipase C (PLC)-catalyzed phosphatidylinositol 4,5-bisphosphate hydrolysis. Agonist stimulation of this pathway is rapidly desensitized, DAG generation has been demonstrated to be rapid, but transient, declining toward basal levels within 1–2 min (2Wright T.M. Rangan L.A. Shin H.S. Raben D.M. J. Biol. Chem. 1988; 263: 9374-9380Abstract Full Text PDF PubMed Google Scholar, 3Cook S.J. Palmer S. Plevin R. Wakelam M.J.O. Biochem. J. 1990; 265: 617-620Crossref PubMed Scopus (39) Google Scholar). However, there is frequently a second sustained phase of DAG generation. This phase has been associated with an increase in the activation of phospholipase D (PLD)-catalyzed phosphatidylcholine (PC) hydrolysis, producing phosphatidate (PA) which can be converted to DAG by the action of phosphatidate phosphohydrolase. It has also been proposed that DAG can be derived from other pathways, e.g. through a PC-PLC pathway, although the evidence for stimulation of this pathway in mammalian cells remains mostly circumstantial (4Cook S.J. Wakelam M.J.O. Rev. Physiol. Biochem. Pharmacol. 1992; 119: 14-45Google Scholar, 5Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (916) Google Scholar). Cells contain multiple species of DAG, however, a limited subset of these change following stimulation. Comparison of the acyl chain DAG structures with those of the cellular phospholipids indicated that the initial phase of DAG increase was predominantly from inositol phospholipids, while the sustained phase, which was accompanied by an increase in choline release, was probably produced from PC (6Pessin M.S. Raben D.M. J. Biol. Chem. 1989; 264: 8729-8738Abstract Full Text PDF PubMed Google Scholar, 7Pettitt T.R. Wakelam M.J.O. Biochem. J. 1993; 289: 487-495Crossref PubMed Scopus (27) Google Scholar, 8Pettitt T.R. Zaqqa M. Wakelam M.J.O. Biochem. J. 1994; 298: 655-660Crossref PubMed Scopus (34) Google Scholar, 9Pessin M.S. Baldassare J.J. Raben D.M. J. Biol. Chem. 1990; 265: 7959-7966Abstract Full Text PDF PubMed Google Scholar). The initial phase of DAG generation was made up of specific polyunsaturated DAG species, in particular 18:0/20:3n-9, 18:0/20:4n-6, and 18:0/20:5n-3, while the second phase was predominantly represented by more saturated species (7Pettitt T.R. Wakelam M.J.O. Biochem. J. 1993; 289: 487-495Crossref PubMed Scopus (27) Google Scholar). The role of the PLD pathway remains incompletely defined. We have recently demonstrated that PA, generated by the activation of PLD, can stimulate rho-mediated actin stress fiber formation in porcine aortic endothelial (PAE) cells (10Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar). This would suggest that while PLD activation clearly results in an increase in DAG, it is not certain that this lipid plays a signaling role. Rather, it may be that the initial product, PA, functions as a messenger and that it is converted to DAG to attenuate the signal. In vitro studies have demonstrated that essentially all DAG species can activate PKC, however, the in vivo evidence for activation by PLD-derived DAG remains mixed. One report has demonstrated no activation of PKC in IIC9 fibroblasts stimulated with a concentration of thrombin which did not activate inositol lipid hydrolysis but did activate PLD (11Leach K.L. Ruff V.A. Wright T.M. Pessin M.S. Raben D.M. J. Biol. Chem. 1991; 266: 3215-3221Abstract Full Text PDF PubMed Google Scholar), while another report found activation of PKCε under the same conditions (12Ha K.-S. Exton J.H. J. Biol. Chem. 1993; 268: 10534-10539Abstract Full Text PDF PubMed Google Scholar). A number of signaling roles have been proposed for PA including activation of a kinase (13Khan W.A. Blobe G.C. Richards A.L. Hannun Y.A. J. Biol. Chem. 1994; 269: 9729-9735Abstract Full Text PDF PubMed Google Scholar), membrane fusion (14Wakelam M.J.O. Curr. Top. Membr. Transp. 1988; 32: 87-112Crossref Scopus (25) Google Scholar), and actin stress fiber formation (10Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar). Since DAG is converted to PA in cells by DAG kinase activity and PA is metabolized to DAG by phosphatidate phosphohydrolase activity, if both PA and DAG are second messengers an uncontrolled signaling cycle could result. Therefore we have re-examined the role of the PLD pathway in regulating PKC and have analyzed the acyl chain structure of DAG and PA in stimulated cells. We demonstrate here that the DAG and thus PA derived from PLC activation is predominantly polyunsaturated while the lipids generated by PLD activation are saturated or monounsaturated. In addition PLD-derived DAG does not appear to regulate PKC activity in stimulated PAE cells. DISCUSSIONAgonist-stimulated PLD activity has been proposed to provide the source of sustained DAG generation in cells leading to the sustained activation of protein kinase C. However, it is becoming increasingly apparent that the PA product of PLD activation itself functions as an intracellular messenger. The potential for interconversion of PA and DAG in cells thus raises the possibility of uncontrolled signaling in a normal cell and makes it difficult to reconcile the possibility that both lipids can indeed function as messenger molecules. The results presented in this report provide a molecular basis for separate signaling functions of DAG and PA.We and others have previously reported that in stimulated cells the rapidly generated DAG is predominantly polyunsaturated and apparently derived from inositol phospholipids (6Pessin M.S. Raben D.M. J. Biol. Chem. 1989; 264: 8729-8738Abstract Full Text PDF PubMed Google Scholar, 7Pettitt T.R. Wakelam M.J.O. Biochem. J. 1993; 289: 487-495Crossref PubMed Scopus (27) Google Scholar, 8Pettitt T.R. Zaqqa M. Wakelam M.J.O. Biochem. J. 1994; 298: 655-660Crossref PubMed Scopus (34) Google Scholar, 9Pessin M.S. Baldassare J.J. Raben D.M. J. Biol. Chem. 1990; 265: 7959-7966Abstract Full Text PDF PubMed Google Scholar, 18Lee C. Fisher S.K. Agranoff B.W. Hajra A.K. J. Biol. Chem. 1991; 266: 22837-22846Abstract Full Text PDF PubMed Google Scholar). We now show that the acyl chain structure of the PA in stimulated Swiss 3T3 and PAE cells is predominantly saturated or monounsaturated, in particular 16:0, 18:0, and 18:1n-9. Conversion of this PA to DAG by the action of phosphatidate phosphohydrolase thus produces a saturated/monounsaturated rather than a polyunsaturated species. We have only been able to detect extremely small quantities of polyunsaturated acyl groups in PA, e.g. 20:4n-6 in the presence or absence of butan-1-ol. This presumably reflects the rapid utilization of these species by PA-cytidyl transferase, forming cytidyl monophosphate-PA for the resynthesis of inositol phospholipids, a possibility supported by the observed reduction in polyunsaturated PA mass in stimulated Swiss 3T3 cells. Attempts to trap cytidyl monophosphate-PA, by inositol depletion and LiCl treatment as described by other groups (e.g. Ref. 20Rodriguez R. Imai A. Gershengorn M.C. Mol. Endocrinol. 1987; 1: 802-806Crossref PubMed Scopus (11) Google Scholar) were largely unsuccessful, with no significant accumulation of this lipid (data not shown). Others have also found that this trapping technique did not to work with all cell types (19Drummond A.H. Raeburn C.A. Biochem. J. 1984; 224: 129-135Crossref PubMed Scopus (94) Google Scholar, 20Rodriguez R. Imai A. Gershengorn M.C. Mol. Endocrinol. 1987; 1: 802-806Crossref PubMed Scopus (11) Google Scholar, 21Monaco M.E. Adelson J.R. Biochem. J. 1991; 279: 337-341Crossref PubMed Scopus (20) Google Scholar). Gas chromatography-mass spectrometry analysis of cytidyl monophosphate-PA showed predominantly 16:0, 18:0, and 18:1n-6 fatty acids with no detectable 20:3n-9, 20:4n-6, or 20:5n-3 at any time point, suggesting that molecular species containing these fatty acids are selectively metabolized more rapidly than other species.Analysis of the acyl structure of the PBut formed in cells stimulated in the presence of 30 mm butan-1-ol demonstrated that the saturated/monounsaturated PA was produced by PLD activation, rather than by PLC-catalyzed phospholipid hydrolysis followed by DAG kinase-catalyzed phosphorylation of the generated DAG. The reduction in stimulated DAG generation in the presence of the alcohol defined the fraction generated by PLD activation. Thus the results in Fig. 1 and Table I demonstrate that PLD activation is responsible for sustained DAG generation in both Swiss 3T3 and PAE cells. It was considered possible that the “alcohol trap” of generated PA was incomplete, however, in the LPA-stimulated PAE cells, where PLD is the only agonist-stimulated phospholipase, butan-1-ol, but not butan-2-ol completely prevented DAG generation (Fig. 1).It has been proposed that PLD-derived DAG can stimulate PKC activity in chronically stimulated cells (see Ref. 5Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (916) Google Scholar, for review). This implies that the acyl chain structure of the DAG is not relevant to the ability of a species to function as an activator. The PAE cell provided a useful experimental model to test this hypothesis since LPA stimulation only increased saturated/monounsaturated DAGs which were produced as a result of PLD activation. In the PAE cells all the PKCα and -δ and a significant proportion of the PKCε and -ζ were found in the membrane fraction under basal conditions. This membrane localization was probably a consequence of the high level of 18:0/20:4n-6 and 18:0/20:3n-9 DAGs (Fig. 2) found in the resting cells. This provides support for a specific role for the polyunsaturated DAGs in activating PKC. Fig. 5 clearly shows that the increase in the saturated/monounsaturated DAG species in LPA-stimulated PAE cells was unable to induce the translocation of PKCε to a membrane fraction. PKCε is a calcium independent isoform, thus it would be expected to be translocated by an increase in DAG mass; as a control inclusion of PMA clearly induced complete translocation. This lack of translocation reflected the inability of LPA to stimulate PKC activity in the PAE cells (Fig. 6). Thus the DAG species produced as a result of PLD activation do not appear to be regulators of PKC, at least in this cell line. Therefore, while they can activate in vitro, we suggest that saturated/monounsaturated DAG species do not regulate PKC activity in an intact normal cell.Previous reports (11Leach K.L. Ruff V.A. Wright T.M. Pessin M.S. Raben D.M. J. Biol. Chem. 1991; 266: 3215-3221Abstract Full Text PDF PubMed Google Scholar) have suggested that PKCα translocation is a consequence of phosphatidylinositol 4,5-bisphosphate, rather than PC hydrolysis since an increase in both DAG and [Ca2+] are required. It was previously proposed that the DAG derived from PC hydrolysis did not activate PKC (11Leach K.L. Ruff V.A. Wright T.M. Pessin M.S. Raben D.M. J. Biol. Chem. 1991; 266: 3215-3221Abstract Full Text PDF PubMed Google Scholar). However, this study only examined PKCα and a later study in the same cell line suggested that the DAG derived from PC hydrolysis could stimulate PKCε translocation (12Ha K.-S. Exton J.H. J. Biol. Chem. 1993; 268: 10534-10539Abstract Full Text PDF PubMed Google Scholar), the results reported here differ from that report. A possible explanation for the differences in results may be that in the work reported here we have been able to clearly demonstrate that all of the increased DAG in the stimulated PAE cells is indeed PLD derived.An alternative explanation for the lack of stimulation of PKC by PLD-derived DAG is that the phospholipase has been activated in a compartment devoid of PKC. Subcellular fractionation studies have provided evidence for PLD activity in plasma membranes, Golgi membranes, endoplasmic reticulum, and the nuclear membrane (22Ktistakis N.T. Brown H.A. Sternweis P.C. Roth M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4952-4956Crossref PubMed Scopus (181) Google Scholar, 23Whatmore J. Morgan C.P. Cunningham E. Collison K.S. Willison K.R. Cockcroft S. Biochem. J. 1996; 320: 785-794Crossref PubMed Scopus (66) Google Scholar, 24Balboa M.A. Insel P.A. J. Biol. Chem. 1995; 270: 29843-29847Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 25Provost J.J. Fudge J. Israelit S. Siddiqi A.R. Exton J.H. Biochem. J. 1996; 319: 285-291Crossref PubMed Scopus (72) Google Scholar, 26Baldassare J.J. Jarpe M.B. Alferes L. Raben D.M. J. Biol. Chem. 1997; 272: 4911-4914Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Thus it is unlikely that PLD-derived DAG would be formed in a membrane devoid of PKC, particularly since PKC isoenzymes appear to be able to translocate to most membranes in the cell including those where PLD activity has been detected. Additional support for our proposal that PLD-derived DAG does not activate PKC is the observation that incubation of HL-60 cells with Streptomyces chromofuscus PLD had no effect upon PKC redistribution, while incubation withBacillus cereus phosphatidylinositol-specific phospholipase C induced cytosol to membrane translocation (27Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In keeping with this result, we were only able to observe sustained activation of PKCδ and -ε in Swiss 3T3 cells where polyunsaturated DAG species were elevated (28Olivier A.R. Hansra G. Pettitt T.R. Wakelam M.J.O. Parker P.J. Biochem. J. 1996; 318: 425-519Crossref PubMed Scopus (16) Google Scholar).Thus we propose that the DAG derived from PLD hydrolysis is not involved in signaling, rather it is a metabolite utilized in the resynthesis of phospholipids. This also suggests that the monounsaturated/saturated PA species themselves play a signaling role in cells. Indeed, in our recent demonstration of PA-stimulated actin stress fiber formation in PAE cells, the stimulant was the dioleoyl structure (10Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar). In order for PA to function as a signal a target molecule must exist and thus proof of PA's signaling function is dependent upon the identification of specific PA targets. It is, however, implicit in the proposed function for PA, that the polyunsaturated PA species do not bind to and activate such target proteins under physiological conditions and thus do not function as signaling molecules. In conclusion we suggest that, while both PLD and PLC pathways directly generate second messengers, it is only the polyunsaturated DAGs and the saturated/monounsaturated PAs which serve as signals under physiological conditions. Agonist-stimulated PLD activity has been proposed to provide the source of sustained DAG generation in cells leading to the sustained activation of protein kinase C. However, it is becoming increasingly apparent that the PA product of PLD activation itself functions as an intracellular messenger. The potential for interconversion of PA and DAG in cells thus raises the possibility of uncontrolled signaling in a normal cell and makes it difficult to reconcile the possibility that both lipids can indeed function as messenger molecules. The results presented in this report provide a molecular basis for separate signaling functions of DAG and PA. We and others have previously reported that in stimulated cells the rapidly generated DAG is predominantly polyunsaturated and apparently derived from inositol phospholipids (6Pessin M.S. Raben D.M. J. Biol. Chem. 1989; 264: 8729-8738Abstract Full Text PDF PubMed Google Scholar, 7Pettitt T.R. Wakelam M.J.O. Biochem. J. 1993; 289: 487-495Crossref PubMed Scopus (27) Google Scholar, 8Pettitt T.R. Zaqqa M. Wakelam M.J.O. Biochem. J. 1994; 298: 655-660Crossref PubMed Scopus (34) Google Scholar, 9Pessin M.S. Baldassare J.J. Raben D.M. J. Biol. Chem. 1990; 265: 7959-7966Abstract Full Text PDF PubMed Google Scholar, 18Lee C. Fisher S.K. Agranoff B.W. Hajra A.K. J. Biol. Chem. 1991; 266: 22837-22846Abstract Full Text PDF PubMed Google Scholar). We now show that the acyl chain structure of the PA in stimulated Swiss 3T3 and PAE cells is predominantly saturated or monounsaturated, in particular 16:0, 18:0, and 18:1n-9. Conversion of this PA to DAG by the action of phosphatidate phosphohydrolase thus produces a saturated/monounsaturated rather than a polyunsaturated species. We have only been able to detect extremely small quantities of polyunsaturated acyl groups in PA, e.g. 20:4n-6 in the presence or absence of butan-1-ol. This presumably reflects the rapid utilization of these species by PA-cytidyl transferase, forming cytidyl monophosphate-PA for the resynthesis of inositol phospholipids, a possibility supported by the observed reduction in polyunsaturated PA mass in stimulated Swiss 3T3 cells. Attempts to trap cytidyl monophosphate-PA, by inositol depletion and LiCl treatment as described by other groups (e.g. Ref. 20Rodriguez R. Imai A. Gershengorn M.C. Mol. Endocrinol. 1987; 1: 802-806Crossref PubMed Scopus (11) Google Scholar) were largely unsuccessful, with no significant accumulation of this lipid (data not shown). Others have also found that this trapping technique did not to work with all cell types (19Drummond A.H. Raeburn C.A. Biochem. J. 1984; 224: 129-135Crossref PubMed Scopus (94) Google Scholar, 20Rodriguez R. Imai A. Gershengorn M.C. Mol. Endocrinol. 1987; 1: 802-806Crossref PubMed Scopus (11) Google Scholar, 21Monaco M.E. Adelson J.R. Biochem. J. 1991; 279: 337-341Crossref PubMed Scopus (20) Google Scholar). Gas chromatography-mass spectrometry analysis of cytidyl monophosphate-PA showed predominantly 16:0, 18:0, and 18:1n-6 fatty acids with no detectable 20:3n-9, 20:4n-6, or 20:5n-3 at any time point, suggesting that molecular species containing these fatty acids are selectively metabolized more rapidly than other species. Analysis of the acyl structure of the PBut formed in cells stimulated in the presence of 30 mm butan-1-ol demonstrated that the saturated/monounsaturated PA was produced by PLD activation, rather than by PLC-catalyzed phospholipid hydrolysis followed by DAG kinase-catalyzed phosphorylation of the generated DAG. The reduction in stimulated DAG generation in the presence of the alcohol defined the fraction generated by PLD activation. Thus the results in Fig. 1 and Table I demonstrate that PLD activation is responsible for sustained DAG generation in both Swiss 3T3 and PAE cells. It was considered possible that the “alcohol trap” of generated PA was incomplete, however, in the LPA-stimulated PAE cells, where PLD is the only agonist-stimulated phospholipase, butan-1-ol, but not butan-2-ol completely prevented DAG generation (Fig. 1). It has been proposed that PLD-derived DAG can stimulate PKC activity in chronically stimulated cells (see Ref. 5Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (916) Google Scholar, for review). This implies that the acyl chain structure of the DAG is not relevant to the ability of a species to function as an activator. The PAE cell provided a useful experimental model to test this hypothesis since LPA stimulation only increased saturated/monounsaturated DAGs which were produced as a result of PLD activation. In the PAE cells all the PKCα and -δ and a significant proportion of the PKCε and -ζ were found in the membrane fraction under basal conditions. This membrane localization was probably a consequence of the high level of 18:0/20:4n-6 and 18:0/20:3n-9 DAGs (Fig. 2) found in the resting cells. This provides support for a specific role for the polyunsaturated DAGs in activating PKC. Fig. 5 clearly shows that the increase in the saturated/monounsaturated DAG species in LPA-stimulated PAE cells was unable to induce the translocation of PKCε to a membrane fraction. PKCε is a calcium independent isoform, thus it would be expected to be translocated by an increase in DAG mass; as a control inclusion of PMA clearly induced complete translocation. This lack of translocation reflected the inability of LPA to stimulate PKC activity in the PAE cells (Fig. 6). Thus the DAG species produced as a result of PLD activation do not appear to be regulators of PKC, at least in this cell line. Therefore, while they can activate in vitro, we suggest that saturated/monounsaturated DAG species do not regulate PKC activity in an intact normal cell. Previous reports (11Leach K.L. Ruff V.A. Wright T.M. Pessin M.S. Raben D.M. J. Biol. Chem. 1991; 266: 3215-3221Abstract Full Text PDF PubMed Google Scholar) have suggested that PKCα translocation is a consequence of phosphatidylinositol 4,5-bisphosphate, rather than PC hydrolysis since an increase in both DAG and [Ca2+] are required. It was previously proposed that the DAG derived from PC hydrolysis did not activate PKC (11Leach K.L. Ruff V.A. Wright T.M. Pessin M.S. Raben D.M. J. Biol. Chem. 1991; 266: 3215-3221Abstract Full Text PDF PubMed Google Scholar). However, this study only examined PKCα and a later study in the same cell line suggested that the DAG derived from PC hydrolysis could stimulate PKCε translocation (12Ha K.-S. Exton J.H. J. Biol. Chem. 1993; 268: 10534-10539Abstract Full Text PDF PubMed Google Scholar), the results reported here differ from that report. A possible explanation for the differences in results may be that in the work reported here we have been able to clearly demonstrate that all of the increased DAG in the stimulated PAE cells is indeed PLD derived. An alternative explanation for the lack of stimulation of PKC by PLD-derived DAG is that the phospholipase has been activated in a compartment devoid of PKC. Subcellular fractionation studies have provided evidence for PLD activity in plasma membranes, Golgi membranes, endoplasmic reticulum, and the nuclear membrane (22Ktistakis N.T. Brown H.A. Sternweis P.C. Roth M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4952-4956Crossref PubMed Scopus (181) Google Scholar, 23Whatmore J. Morgan C.P. Cunningham E. Collison K.S. Willison K.R. Cockcroft S. Biochem. J. 1996; 320: 785-794Crossref PubMed Scopus (66) Google Scholar, 24Balboa M.A. Insel P.A. J. Biol. Chem. 1995; 270: 29843-29847Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 25Provost J.J. Fudge J. Israelit S. Siddiqi A.R. Exton J.H. Biochem. J. 1996; 319: 285-291Crossref PubMed Scopus (72) Google Scholar, 26Baldassare J.J. Jarpe M.B. Alferes L. Raben D.M. J. Biol. Chem. 1997; 272: 4911-4914Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Thus it is unlikely that PLD-derived DAG would be formed in a membrane devoid of PKC, particularly since PKC isoenzymes appear to be able to translocate to most membranes in the cell including those where PLD activity has been detected. Additional support for our proposal that PLD-derived DAG does not activate PKC is the observation that incubation of HL-60 cells with Streptomyces chromofuscus PLD had no effect upon PKC redistribution, while incubation withBacillus cereus phosphatidylinositol-specific phospholipase C induced cytosol to membrane translocation (27Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In keeping with this result, we were only able to observe sustained activation of PKCδ and -ε in Swiss 3T3 cells where polyunsaturated DAG species were elevated (28Olivier A.R. Hansra G. Pettitt T.R. Wakelam M.J.O. Parker P.J. Biochem. J. 1996; 318: 425-519Crossref PubMed Scopus (16) Google Scholar). Thus we propose that the DAG derived from PLD hydrolysis is not involved in signaling, rather it is a metabolite utilized in the resynthesis of phospholipids. This also suggests that the monounsaturated/saturated PA species themselves play a signaling role in cells. Indeed, in our recent demonstration of PA-stimulated actin stress fiber formation in PAE cells, the stimulant was the dioleoyl structure (10Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar). In order for PA to function as a signal a target molecule must exist and thus proof of PA's signaling function is dependent upon the identification of specific PA targets. It is, however, implicit in the proposed function for PA, that the polyunsaturated PA species do not bind to and activate such target proteins under physiological conditions and thus do not function as signaling molecules. In conclusion we suggest that, while both PLD and PLC pathways directly generate second messengers, it is only the polyunsaturated DAGs and the saturated/monounsaturated PAs which serve as signals under physiological conditions.