Diacylglycerol Kinase ε, but Not ζ, Selectively Removes Polyunsaturated Diacylglycerol, Inducing Altered Protein Kinase C Distribution in Vivo

Abstract

Porcine aortic endothelial cells have previously been shown to contain particularly high basal levels of polyunsaturated diacylglycerol (DAG) together with a very high degree of membrane-associated protein kinase C (PKC), which is largely insensitive to further activation (Pettitt, T. R., Martin, A., Horton, T., Liossis, C., Lord, J. M., and Wakelam, M. J. O. (1997) J. Biol. Chem. 272, 17354–17359). To investigate the possibility that the high polyunsaturated DAG levels were constitutively activating PKC, we transfected porcine aortic endothelial cells with two different forms of human diacylglycerol kinase, ε and ζ. In vitro, the former is specific for polyunsaturated structures, whereas the latter shows no apparent selectivity. Overexpression of DAGKε specifically reduced the level of polyunsaturated DAG in the transfected cells while having little effect on the more saturated structures. It also caused the redistribution of PKCα and ε from the membrane to the cytosol. Overexpression of DAGKζ caused a general reduction in DAG levels but had little effect on PKC distribution. These results for the first time show that DAGKε specifically phosphorylates polyunsaturated DAGin vivo and that in so doing it regulates PKC localization and activity. This provides support for the proposal that it is the polyunsaturated DAGs that function as messengers and convincing evidence for DAGKε being a physiological terminator of DAG second messenger signaling. Porcine aortic endothelial cells have previously been shown to contain particularly high basal levels of polyunsaturated diacylglycerol (DAG) together with a very high degree of membrane-associated protein kinase C (PKC), which is largely insensitive to further activation (Pettitt, T. R., Martin, A., Horton, T., Liossis, C., Lord, J. M., and Wakelam, M. J. O. (1997) J. Biol. Chem. 272, 17354–17359). To investigate the possibility that the high polyunsaturated DAG levels were constitutively activating PKC, we transfected porcine aortic endothelial cells with two different forms of human diacylglycerol kinase, ε and ζ. In vitro, the former is specific for polyunsaturated structures, whereas the latter shows no apparent selectivity. Overexpression of DAGKε specifically reduced the level of polyunsaturated DAG in the transfected cells while having little effect on the more saturated structures. It also caused the redistribution of PKCα and ε from the membrane to the cytosol. Overexpression of DAGKζ caused a general reduction in DAG levels but had little effect on PKC distribution. These results for the first time show that DAGKε specifically phosphorylates polyunsaturated DAGin vivo and that in so doing it regulates PKC localization and activity. This provides support for the proposal that it is the polyunsaturated DAGs that function as messengers and convincing evidence for DAGKε being a physiological terminator of DAG second messenger signaling. phospholipase C sn-1,2-diacylglycerol phosphatidic acid 5-bisphosphate lysophosphatidic acid diacylglycerol kinase protein kinase C porcine aortic endothelial (cells) 12-O-tetradecanoylphorbol-13-acetate high performance liquid chromatography Agonist stimulation of cells often induces the rapid and transient phospholipase C (PLC)1-catalyzed generation of polyunsaturated sn-1,2-diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP2). This is generally closely followed by a second, sustained phase of diradylglycerol generation lasting for up to 60 min, characterized by more saturated species, primarily DAGs but also potentially includingsn-1-O-alkyl-2-acylglycerols andsn-1-O-alkenyl-2-acylglycerols. These second-phase diradylglycerols are often derived through a phospholipase D/phosphatidic acid phosphohydrolase pathway utilizing phosphatidylcholine as substrate (1Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (389) Google Scholar). Sustained mitogenic stimulation also induces polyunsaturated DAG generation at much longer times, where it correlates with progression hrough the cell cycle (2Olivier A.R. Hansra G. Pettitt T.R. Wakelam M.J.O. Parker P.J. Biochem. J. 1996; 318: 519-525Crossref PubMed Scopus (16) Google Scholar). The best-characterized roles of these DAGs are as lipid second messengers, activating both the classical and novel PKC families (3Newton A.C. Johnson J.E. Biochim. Biophys. Acta. 1998; 1376: 155-172Crossref PubMed Scopus (245) Google Scholar,4Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1366) Google Scholar). However, although all DAG species can activate PKC to some extentin vitro, we have previously proposed that the polyunsaturated forms are the true messengers under physiological conditions, whereas the more saturated DAGs have little or no activity (5Hodgkin M.H. Pettitt T.R. Martin A. Michell R.H. Pemberton A.J. Wakelam M.J.O. Trends Biochem. Sci. 1998; 23: 200-204Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 6Wakelam M.J.O. Biochim. Biophys. Acta. 1998; 1436: 117-126Crossref PubMed Scopus (160) Google Scholar). An intracellular messenger needs a mechanism of generation that can be rapidly activated in response to the appropriate stimulus. However, it also requires an equally rapid and specific mechanism of removal to terminate the signal. A major route for the removal of DAG is through phosphorylation catalyzed by diacylglycerol kinase (DAGK) to form phosphatidic acid (PA) (7Sakane F. Kanoh H. Int. J. Biochem. Cell Biol. 1997; 29: 1139-1143Crossref PubMed Scopus (81) Google Scholar). This enzyme exists as at least eight different isoforms that, on the basis of their structure, have been separated into five classes. Each shows a slightly different tissue distribution. Only one, DAGKε, a type III enzyme found at high levels in testis but also present in other tissues has been shown to exhibit differential selectivity in vitro, where it preferentially phosphorylates polyunsaturated DAG, particularlysn-1-acyl-2-arachidonyl glycerol (8Tang W. Bunting M. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1996; 271: 10237-10241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). These observations suggest that DAGKε could be responsible for terminating the polyunsaturated DAG intracellular messenger signal; however, this has not been shown in vivo. Although PA is a putative messenger itself and is required for progression from G1 into S phase in interleukin-2-stimulated mouse CTLL-2 cells (9Flores I. Casaseca T. Martinez-A C. Kanoh H. Merida I. J. Biol. Chem. 1996; 271: 10334-10340Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), polyunsaturated PA formed by DAGKε is probably inactive as an intracellular signal (5Hodgkin M.H. Pettitt T.R. Martin A. Michell R.H. Pemberton A.J. Wakelam M.J.O. Trends Biochem. Sci. 1998; 23: 200-204Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) as it is rapidly utilized to reform phosphoinositide. To determine if distinct DAGK isoforms have distinct in vivosubstrates, we transfected porcine aortic endothelial (PAE) cells with human DAGKε and DAGKζ, then examined the effect on DAG composition and PKC distribution. Cells transfected with DAGKε have significantly reduced polyunsaturated DAG as compared with the very high levels seen in the parental cells (10Pettitt T.R. Martin A. Horton T. Liossis C. Lord J.M. Wakelam M.J.O. J. Biol. Chem. 1997; 272: 17354-17359Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), and there is a concomitant redistribution of PKC from the membrane to the cytosol. Transfection with DAGKζ, a form with no known species selectivity (7Sakane F. Kanoh H. Int. J. Biochem. Cell Biol. 1997; 29: 1139-1143Crossref PubMed Scopus (81) Google Scholar, 11Bunting M. Tang W. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1996; 271: 10230-10236Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), did not alter the DAG species profile and had little effect on PKC distribution. All solvents were of Analytical or HPLC grade from Fisher. Lipid standards were purchased from Avanti Polar Lipids Inc., Alabaster, AL. Other chemicals were from Sigma-Aldrich. Cell culture reagents were purchased from Life Technologies, Inc. The cDNA for DAGKε was cloned into pcDNA1/Neo (Invitrogen), whereas that for DAGKζ was cloned into pcDNA3.1/Zeo(+) (Invitrogen); they were generously provided by Drs. M. K. Topham and S. M. Prescott, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT. DAGK antibodies raised in rabbit as described previously (11Bunting M. Tang W. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1996; 271: 10230-10236Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) were also supplied by Drs. Topham and Prescott. Mouse monoclonal PKCα and ε antibodies were obtained from Transduction Laboratories. PAE cells were cultured in Ham's F-12 nutrient mixture containing Glutamax, 10% fetal calf serum, penicillin, and streptomycin and used upon reaching 70–80% confluency. Most cells were serum-starved for 24 h before use. Transfection was by electroporation. Geneticin was (100 μg/ml) used to select clones expressing human DAGKε, whereas zeocin (100 μg/ml) was used to select clones expressing human DAGKζ. Cells were washed twice with phosphate-buffered saline then incubated for 15 min in Ham's F-12 containing 20 mm HEPES, pH 7.4, and 0.1% bovine serum albumin (fraction V; Sigma) at 37 °C before stimulation with 10 μm sn-1-18 :1n-9,2-lysophosphatidic acid (LPA) where described. Incubations were terminated by aspiration of the media followed by the addition of ice-cold methanol and 1 μg of 1,2-12:0/12.0 DAG internal standard. Lipids were extracted, and diradylglycerols were derivatized and analyzed by HPLC as described previously (12Pettitt T.R. Wakelam M.J.O. Biochem. J. 1993; 289: 487-495Crossref PubMed Scopus (27) Google Scholar, 13Pettitt T.R. Zaqqa M. Wakelam M.J.O. Biochem. J. 1994; 298: 655-660Crossref PubMed Scopus (34) Google Scholar). Data are expressed as means ± S.D., wheren = 3–6. Cells were labeled with myo-[2-3H]inositol (0.5 μCi/ml) in inositol-free medium for 48 h and stimulated in the presence of 20 mm LiCl with vehicle or LPA (10 μm), and the inositol phosphates were analyzed as described previously (14Wakelam M.J.O. Murphy G.J. Hruby V.J. Houslay M.D. Nature. 1986; 323: 68-71Crossref PubMed Scopus (274) Google Scholar). PAE cells stimulated with LPA (10 μm) for 20 s and 5 min where described were washed in ice-cold phosphate-buffered saline and harvested in 20 mm HEPES, pH 7.4, containing 1 mm phenylmethylsulfonyl fluoride and 20 μg/ml leupeptin. The samples were frozen at −70 °C overnight, thawed, and sonicated for 20 s in a bath sonicator, and membranes and cytosol were separated by centrifugation (300,000 ×g, 20 min). The membranes were washed once. The cytosolic proteins were concentrated by precipitation with 12.5% trichloroacetic acid (4 °C, 1 h) and centrifugation (13,000 ×g, 10 min), and the pellet was washed with 200 μl of ice-cold acetone. The cytosolic and membrane proteins were solubilized by boiling in 75 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 4 m urea, 5% β-mercaptoethanol, bromphenol blue for 5 min, then cell fraction equivalents were separated by electrophoresis on 7.5% SDS-polyacrylamide minigels. After transfer onto polyvinylidene difluoride membranes (Immobilon-P; Millipore), the PKC isozymes were probed using mouse monoclonal antibodies to PKCα (1 in 2500 dilution) and PKCε (1 in 2500) and detected using enhanced chemiluminescence. DAGK isozymes were probed with rabbit polyclonal antibodies to DAGKε (1 in 2000) and DAGKζ (1 in 1000). Cells grown in 10-cm dishes were washed twice with phosphate-buffered saline, lysed for 15 min at 4 °C with 1 ml of ice-cold hypertonic lysis buffer (20 mm Tris, pH 7.6, 250 mm NaCl, 3 mmEDTA, 3 mm EGTA, 1 mm sodium orthovanadate, 1 mm dithiothreitol, 0.5% Nonidet P-40, protease inhibitor mixture; (Roche Molecular Biochemicals), scraped into Eppendorf tubes, sonicated for 5 min in a sonicating bath cooled with ice, centrifuged (13000 × g, 5 min), and the cleared supernatant was transferred to clean tubes. Each sample was mixed for 1 h at 4 °C with 5 μl of mouse monoclonal PKC antibody followed by a further 30 min with 10 μl of goat anti-mouse IgG antibody (Sigma). Each sample was then split into two equal fractions and mixed with 50 μl of 20% Protein G-Sepharose beads (Amersham for 1 h at 4 After centrifugation (13,000 × g, 5 min), the pellet was washed with μl of kinase buffer mm HEPES, pH mm NaCl, 75 mm 20 10 mm mm 1 mm sodium orthovanadate, 1 mm and then in 20 μl of kinase To the was 5 μl of PKC substrate type or 1 Sigma) in kinase 10 μl of lipid μm μm mixed DAG, 0.5% in kinase and 5 μl of kinase buffer 5 μm in kinase buffer for the 10 μl of μm containing of was to the After (1 30 the was with 20 μl of 5% The substrate was separated by electrophoresis on SDS-polyacrylamide for whereas with the sample was onto washed 4 × 10 min with ml of 0.5% acid and washed with substrate was detected and using a Molecular of DAGKε and DAGKζ in parental PAE cells and clones was examined by Although the parental cells both the clones enhanced levels with in DAGKε and in DAGKζ of cytosolic and membrane fractions that these were to the membrane fraction both in and cells not The of the at and in the DAGKζ but be of the The parental cells the DAGK clones with of and h on cell different culture for DAGKε and DAGKζ The of both the parental and transfected PAE cells was largely insensitive to serum for 48 h from with a 20% in The DAGK clones have a to into The degree of clones but to with the level of DAGK expressing more enzyme The DAGKζ clones this at confluency. The DAGKε clones were more into and whereas the parental cells a This with the parental and DAGKε clones to in the of and in different of cell culture serum and these the DAG PA by PAE cells contain high levels of polyunsaturated DAG under conditions, and these largely insensitive to further stimulation (10Pettitt T.R. Martin A. Horton T. Liossis C. Lord J.M. Wakelam M.J.O. J. Biol. Chem. 1997; 272: 17354-17359Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). DAGKε had the in vivo vitro, of enzyme be to these lipid were derivatized with and separated by HPLC to the different DAG 3 shows a of DAG from DAGKε and the parental both grown in 10% species levels are reduced in the glycerol and glycerol are with an Other DAGKε clones results with reduced polyunsaturated DAG levels but little in more saturated species, as in I. Overexpression of DAGKε reduced DAG by ± compared with ± This is to the of polyunsaturated structures, particularly and which together for cells in the parental cells but cells in the DAGKε for 24 h had little effect on the DAG but did by ± to ± cells in DAGKε The DAGKζ clones very little in the DAG species to the parental cells not However, the basal DAG levels were to the parental cells grown in 10% serum ± cells compared with ± or ± cells compared with ± composition of parental and PAE cells grown in 10% = = = = species in which are significantly reduced in PAE cells species in which are significantly reduced in PAE cells species in which are significantly reduced in PAE cells species in which are significantly reduced in PAE cells species in which are significantly reduced in PAE cells Molecular species in which are significantly reduced in PAE cells in a the reduced basal polyunsaturated DAG levels in the DAGKε clones had the for activation of as by rapid polyunsaturated DAG and inositol LPA stimulation for 1 min, or 5 min caused no inositol generation and had no effect on polyunsaturated DAG levels not However, 5 min, LPA did a in more saturated species, particularly which from cells to These were probably through the phospholipase D/phosphatidic acid phosphohydrolase pathway we previously that the of these in PAE cells (10Pettitt T.R. Martin A. Horton T. Liossis C. Lord J.M. Wakelam M.J.O. J. Biol. Chem. 1997; 272: 17354-17359Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). the that LPA is to an activity in these if basal polyunsaturated DAG levels are have shown previously that all the PKCα and of the PKCε was with the membrane fraction in PAE cells (10Pettitt T.R. Martin A. Horton T. Liossis C. Lord J.M. Wakelam M.J.O. J. Biol. Chem. 1997; 272: 17354-17359Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). that this was to the high basal levels of polyunsaturated DAG, in constitutively activating the enzyme and it onto the by the polyunsaturated DAG more PKC be found in the cytosol. we compared the PKC in the DAGKε clones with that in the parental both cultured for the 24 h under or 10% serum cultured in conditions, the clones with their reduced levels of polyunsaturated DAG, but not the parental PKC redistribution to the cytosol PKCα was membrane in the compared with in the whereas for PKCε the membrane was compared with the parental cells and the clones cultured in 10% serum, all the PKCα was on the whereas for PKCε was on the parental cells show the PKC as that seen grown in 10% with LPA for 20 s and 5 min had no effect on PKC not PKCα in the DAGK clones was to that seen in the parental however, PKCε was in the DAGKε clones and probably also the DAGKζ although there was some The DAGKζ clones a PKC to that in the parental cells under both serum of DAGK on PKC distribution. and membrane fractions were from cells grown for the 24 h in or 10% serum conditions, separated on 7.5% SDS-polyacrylamide and analyzed by To investigate if DAGK PKC kinase PKCα and ε were from and their to or a PKC substrate to from were examined in the presence or of the Although PKCα activity was generally in the parental and DAGK under basal and basal activity was in the DAGKζ clones and in the parental cells Although not the also a in PKCε particularly in the DAGKζ is to activate all in vitro, the activity seen in the presence of is to with PKC in the in C and the basal PKC activity is expressed as a of activity This an to the PKC specific under DAGK had no effect on basal PKCα which at In PKCε activity was particularly in the DAGKζ where the protein kinase was activated under basal DAGK is a enzyme with roles in and cell signaling. Most to have the in of novel The the first that DAGK can DAG levels in In the results show the of DAG species levels by a DAGKε, but not by DAGKζ. This that the substrate by in (8Tang W. Bunting M. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1996; 271: 10237-10241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 11Bunting M. Tang W. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1996; 271: 10230-10236Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) in vivo. The removal of polyunsaturated DAG by DAGKε is by of the of PKCα and This provides support for the that polyunsaturated DAG, derived through a phospholipase C is the true form of this whereas the more saturated species derived through a phospholipase D/phosphatidic acid phosphohydrolase pathway are largely DAGKε clones grown in 10% serum a PKC to the parental however, grown for 24 h in the PKC to the cytosol in the of serum reduced the DAG cells as compared with but had no effect upon the DAG species the reduction in polyunsaturated DAG be to PKC In DAGKε and together cells under as compared with cells grown in 10% In the parental although DAG a cells as compared with the polyunsaturated DAG at cells that seen in the DAGKε clones grown with 10% In these parental cells the PKC under to that seen in 10% serum, with enzyme at the membrane the of PKC redistribution is the of polyunsaturated DAG in a cell to more saturated However, it also be that different to the differential effect of the two DAGK forms on PKC distribution. DAGKζ and the are to different then DAGK of DAG levels not the PKC found in the In be to have a effect on PKC The activity for PKCα A and support the that although different of PKC be at the membrane under basal conditions, to the cell needs a level of kinase activity it in for the parental and The activity in the presence of that the cell have levels of In the for PKCε suggest that there is more enzyme in the DAGK clones This with the where more PKCε was detected in the transfected cells 4 and The kinase activity in the under basal these enzyme although the activity to be in the parental particularly for the DAGKζ clones However, the enzyme had to be and then vitro, a of we that the PKC redistribution are a more of PKC activation in although not of kinase activity. In with the in PKCε protein in the DAGK a reduction in the membrane polyunsaturated DAG the of enzyme The of DAGKζ from is but as it slightly this on SDS-polyacrylamide (11Bunting M. Tang W. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1996; 271: 10230-10236Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). cells transfected with the DAGKζ expressed a of and as did human cells (11Bunting M. Tang W. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Biol. Chem. 1996; 271: 10230-10236Abstract Full Text Full Text PDF PubMed Scopus (159) Google however, both parental and transfected PAE cells expressed a of together with of at and be a form has been found in human although to of the form Bunting M. Topham McIntyre T.M. Zimmerman G.A. Prescott S.M. Sci. S. A. 1997; PubMed Scopus Google Scholar). The DAGK protein with the DAGKζ clones as compared with the DAGKε clones that the latter is probably more to the cell present at slightly This is a with or other can major membrane and cell This is to be for in lipid signaling. To membrane and for the substrate for polyunsaturated DAG messenger be rapidly The first catalyzed by the polyunsaturated DAG signal by polyunsaturated This is the substrate for at least form of S. K. A. H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), which is then utilized by phosphatidylinositol for A. S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation then DAGKε J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and S. K. A. H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) are by in vitro, a mechanism to Overexpression of DAGKε alter this enhanced removal of polyunsaturated DAG the PKC which regulates activity T.R. Zaqqa M. Wakelam M.J.O. Biochem. J. 1994; 298: 655-660Crossref PubMed Scopus (34) Google Scholar), potentially to sustained This of and DAGKε as the cell to phosphoinositide. a the cell into an which if DAGK particularly DAGKζ, a in cell at through on membrane caused by the reduction in diradylglycerol However, the that DAGKζ is found at high levels the and has a C kinase substrate that can as a localization Bunting M. Topham McIntyre T.M. Zimmerman G.A. Prescott S.M. Sci. S. A. 1997; PubMed Scopus Google Scholar, K. H. Sci. S. A. 1996; PubMed Scopus Google Scholar, Bunting M. Zimmerman G.A. McIntyre T.M. P.J. Prescott S.M. Nature. 1998; PubMed Scopus Google Scholar) that primarily this of this by PKCα or has been shown to DAGKζ of the and from DAG Bunting M. Zimmerman G.A. McIntyre T.M. P.J. Prescott S.M. Nature. 1998; PubMed Scopus Google Scholar). this by PKC or of DAGKζ in the the cell cycle time Bunting M. Zimmerman G.A. McIntyre T.M. P.J. Prescott S.M. Nature. 1998; PubMed Scopus Google Scholar). A localization with observations that DAGKζ is with the membrane fraction from PAE cell this contain that the cell time was for the DAGKζ with mouse DAGKζ, which shows a with the human found that is both and and correlates with the of and McIntyre T.M. Zimmerman G.A. Prescott S.M. 1998; PubMed Scopus Google Scholar), a for this enzyme in to an of other and of the type Overexpression of DAGKε the of DAGKζ, in the more seen at in the DAGKε In DAGKε, but not DAGKζ, PKCα and ε activation by specifically the levels of DAG by polyunsaturated the of the phosphoinositide. S. M. Prescott and M. K. Topham for the of DAGK cDNA and which this and for their