John de Widt

PURIFICATION, CDNA-CLONING AND EXPRESSION OF HUMAN DIACYLGLYCEROL KINASE

Diacylglycerol (DG) kinase attenuates the level of the second messenger DG in signal transduction, and therefore possibly modulates protein kinase C (PKC). DG kinase was purified to homogeneity from human white blood cells, showing an M 1 of 86 kDa as determined by SDS‐PAGE and gel filtration. Two amino acid sequences of tryptic peptides from DG kinase were determined and degenerate oligonucleotides were prepared and used in the polymerase chain reaction. An amplified DNA fragment was subsequently used to clone the full‐length human DG kinase cDNA. This sequence is the human homolog of porcine DG kinase cDNA sequence reported recently [1]. The sequence contains a double EF‐ hand structure typical for Ca2+ binding proteins. DG kinase further contains a double cysteine repeat that is present in all PKC isoforms, where it constitutes the phorbol ester (and most likely diacylglycerol) binding site. Therefore we speculate that the double cysteine repeat in DG kinase is involved in DG binding. DG kinase is transcribed as a single mRNA of 3.2 kb, that is highly expressed in T‐lymphocytes. The human DG kinase cDNA when transfected in mammalian cells (COS‐7) results in a 6–7‐fold increase of DG kinase activity.

DIACYLGLYCEROL KINASE THETA BINDS TO AND IS NEGATIVELY REGULATED BY ACTIVE RHOA

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. To date, very little is known about the regulation of DGK activity. We have previously identified the DGKθ isotype, which is predominantly expressed in brain (Houssa, B., Schaap, D., van der Wal, J., Goto, K., Kondo, H., Yamakawa, A., Shibata, M., Takenawa, T., and Van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 10422–10428). We now report that DGKθ binds specifically to activated RhoA in transfected COS cells as well as in nontransfected neuronal N1E-115 cells. Binding is abolished by a point mutation (Y34N) in the effector loop of RhoA. DGKθ does not bind to inactive RhoA, nor to the other Rho-family GTPases, Rac or Cdc42. Like active RhoA, DGKθ localizes to the plasma membrane. Strikingly, the binding of activated RhoA to DGKθ completely inhibits DGK catalytic activity. Our results suggest that DGKθ is a downstream effector of RhoA and that its activity is negatively regulated by RhoA. Through accumulation of newly produced diacylglycerol, RhoA-mediated inhibition of DGKθ may lead to enhanced PKC activity in response to external stimuli.

Translocation of Diacylglycerol Kinase θ from Cytosol to Plasma Membrane in Response to Activation of G Protein-coupled Receptors and Protein Kinase C

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. We previously identified DGKθ as one of nine mammalian DGK isoforms and reported on its regulation by interaction with RhoA and by translocation to the plasma membrane in response to noradrenaline. Here, we have investigated how the localization of DGKθ, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGKθ translocation from the cytoplasm to the plasma membrane within 2–6 min. This translocation, independent of DGK activity, was preceded by protein kinase C (PKC) translocation and was blocked by PKC inhibitors. Conversely, activation of PKC by 12-O-tetradecanoylphorbol-13-acetate induced DGKθ translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGKθ translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGKθ abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGKθ recruitment to the membrane. We show that DGKθ interacts selectively with and is phosphorylated by PKCϵ and -η and that peptide agonist-induced selective activation of PKCϵ directly leads to DGKθ translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGKθ, which may be important in the negative regulation of PKCϵ and/or PKCη activity.

Involvement of Phosphatidylcholine-specific Phospholipase C in Platelet-derived Growth Factor-induced Activation of the Mitogen-activated Protein Kinase Pathway in Rat-1 Fibroblasts

The role of phosphatidylcholine (PC) hydrolysis in activation of the mitogen-activated protein kinase (MAPK) pathway by platelet-derived growth factor (PDGF) was studied in Rat-1 fibroblasts. PDGF induced the transient formation of phosphatidic acid, choline, diacylglycerol (DG), and phosphocholine, the respective products of phospholipase D (PLD) and phospholipase C (PC-PLC) activity, with peak levels at 5-10 min. PLD-catalyzed transphosphatidylation (with n-butyl alcohol) diminished DG formation at 5 min but not at later stages of PDGF stimulation. Phorbol ester-induced down-regulation of protein kinase C (PKC) completely blocked PLD activation but not the formation of DG and phosphocholine at 10 min of PDGF stimulation. Collectively, these data indicate that PDGF activates both PLD and PC-PLC. In contrast, epidermal growth factor did not activate PC-PLC in these cells, and it activated PLD only weakly. DG formation by itself, through Bacillus cereus PC-PLC treatment of cells, was sufficient to mimic PDGF in activation of MAPK independent of phorbol ester-sensitive PKC. Since PKC down-regulation blocked PDGF-induced PLD but not MAPK activation, we conclude that PLD is not involved in MAPK signaling. In contrast, MAPK activation by exogenous (bacterial) PLD was not affected by PKC down-regulation, indicating that signals evoked by exogenous PLD differ from endogenous PLD. D609 (2-10 μg/ml), an inhibitor of PC-PLC, blocked PDGF- but not epidermal growth factor-induced MAPK activation. However, D609 should be used with caution since it also affects PLD activity. The results suggest that PC-PLC rather than PLD plays a critical role in the PDGF-activated MAPK pathway.

The Retinoblastoma Family Proteins Bind to and Activate Diacylglycerol Kinaseζ

The retinoblastoma protein (pRB) is a tumor suppressor and key regulator of the cell cycle. We have previously shown that pRB interacts with phosphatidylinositol-4-phosphate 5-kinases, lipid kinases that can regulate phosphatidylinositol 4,5-bisphosphate levels in the nucleus. Here, we investigated pRB binding to another lipid kinase in the phosphoinositide cycle, diacylglycerol kinase (DGK) that phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. We found that DGKζ, but not DGKα or DGKθ, interacts with pRB in vitro and in vivo. Binding of DGKζ to pRB is dependent on the phosphorylation status of pRB, since only hypophosphorylated pRB interacts with DGKζ. DGKζ also binds to the pRB-related pocket proteins p107 and p130 in vitro and in cells. Although DGKζ did not affect the ability of pRB to regulate E2F-mediated transcription, we found that pRB, p107, and p130 potently stimulate DGKζ activity in vitro. Finally, overexpression of DGKζ in pRB-null fibroblasts reconstitutes a cell cycle arrest induced by γ-irradiation. These results suggest that DGKζ may act in vivo as a downstream effector of pRB to regulate nuclear levels of diacylglycerol and phosphatidic acid.

A metabolite of an antineoplastic ether phospholipid may inhibit transmembrane signalling via protein kinase C.

In our search for the mechanisms by which the drug 1-O-alkyl-2-O-methylglycero-3-phosphocholine (AMG-PC) inhibits tumor growth and metastasis, we have detected a metabolite, 1-O-alkyl-2-O-methylglycerol (AMG), in membranes of MO4 mouse fibrosarcoma cells grown in the presence of the drug. Synthetic AMG inhibited the activation of highly purified human protein kinase C by diacylglycerol in the presence of phosphatidylserine. Furthermore, AMG also inhibited the receptor-specific binding of3H-phorbol-12,13-dibutyrate to human HL-60 promyeloid leukemia cells in a dose-dependent fashion. AMG-PC was not effective or much less so in these assays. We suggest that interaction of the metabolite AMG with protein kinase C may inhibit stimulus-response coupling in tumor cells and may thus potentially contribute to the mechanism by which AMG-PC exerts its anticancer activities.

Metabolic conversion of the biologically active phospholipid, lysophosphatidic acid, in fibroblasts

Lysophosphatidic acid (3-sn-lysophosphatidic acid; LPA) can activate cells similar to hormones and growth factors. We have considered the question whether metabolic conversion of LPA taken up by the cell could be of any importance in this activation. Addition of [14C-glycerol]LPA to quiescent Rat-1 fibroblasts resulted in rapid formation of [14C]monoacylglycerol (MG), closely followed by accumulation of [14C]triacylglycerol. Only very little [14C]diacylglycerol and [14C]phosphatidic acid was formed (approx. 100-fold less than MG). MG, when added exogenously to cells, lacks detectable biological activity. The results suggest that LPA itself, rather than one of its metabolites is the biologically active principle.

Alterations in biosynthesis and homeostatis of cholesterol and in lipoprotein patterns in mice bearing a transplanted lymphoid tumor

The membrane fluidity of murine lymphoid GRSL tumor cells has been shown to depend on their site of growth in the host. Tumor cells located in ascites, in contrast to those in the enlarged spleen, show a very high plasma membrane fluidity, mainly due to a decreased level of cellular (membrane) cholesterol. Yet, the rate of cholesterol biosynthesis in the tumor cells as estimated by the activity of HMG-CoA reductase and the incorporation of [14C]acetate into cholesterol was extremely high when compared to various lymphoid cells in normal control mice. Also the rate of biosynthesis and the cholesterol content in liver and spleen of tumor-bearing mice were substantially higher than in the organs of control mice. Blood plasma cholesterol, however, was decreased in tumor-bearing mice as a result of altered lipoprotein patterns. Outgrowth of the tumor was accompanied by a strong reduction in plasma high-density lipoproteins. Low-density lipoproteins became transiently increased, but eventually all lipoproteins, and consequently the plasma cholesterol content decreased to very low levels, especially so in the ascites plasma. The low transfer of [14C]cholesteryl ester-labeled lipoproteins between blood and ascites plasma after either intravenous or intraperitoneal injection suggested a hampered flow between the two compartments. Also apparent differences in cholesteryl ester fatty acid composition between lipoproteins of the blood and ascites plasma indicated the lack of a rapid equilibration between the two compartments. The results suggest that the limited availability of lipoproteins as an additional source of cholesterol to the rapidly growing ascites cells promotes their high membrane fluidity.

Effect of dietary lipids on plasma lipoproteins and fluidity of lymphoid cell membranes in normal and leukemic mice

Mice of the GR/A strain were fed four different isocaloric semipurified diets, enriched in either (1) saturated fatty acids (palm oil), or (2) polyunsaturated fatty acids (corn oil), or (3) palm oil plus cholesterol, or (4) a fat-poor diet containing only a minimal amount of essential fatty acids. We have studied the effects of these dietary lipids on the density profile and composition of the plasma lipoproteins and on the lipid composition and fluidity of (purified) lymphoid cell membranes in healthy mice and in mice bearing a transplanted lymphoid leukemia (GRSL). Tumor development in these mice occurred in the spleen and in ascites. While the fatty acid composition of the VLDL-triacylglycerols still strongly resembled the dietary lipids, the effects of the diets decreased in the order VLDL-triacylglycerols greater than HDL-phospholipids greater than plasma membrane phospholipids. Diet-induced differences in the latter fraction were virtually confined to the content of oleic acid and linoleic acid, and they were too small to affect the membrane fluidity, as measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. Healthy mice were almost irresponsive to dietary cholesterol, but in the tumor bearers, where lipoprotein metabolism has been shown to be disturbed, the cholesterol diet caused a substantial increase in the low- and very-low density regions of both blood and ascites plasma lipoproteins. The cholesterol-rich diet also increased the cholesterol/phospholipid molar ratio and lipid structural order (decreased fluidity) in GRSL ascites cell membranes, but not in the splenic GRSL cell membranes. We conclude that the composition of plasma lipoproteins and cell membrane lipids in GR/A mice is subject to exquisite homeostatic control. However, in these low-responders to dietary lipids the development of an ascites tumor may lead to increased responsiveness to dietary cholesterol. The elevated level of membrane cholesterol thus obtained in GRSL ascites cells did not affect the expression of various cell surface antigens or tumor cell growth.

Accumulation of HDL-like lipoproteins in the plasma low-density fractions of tumor-bearing mice

Outgrowth of the transplanted GRSL lymphoma in GR mice yielded several-fold increased blood plasma levels of low- and very-low-density lipoproteins, while high-density lipoproteins were strongly reduced. Changes in cholesteryl ester fatty acid profiles indicated an accumulation of HDL-like particles rather than LDL in the low-density fractions. By intravenous injection of [14C]cholesteryl ester-labeled HDL into tumor-bearing mice, conversion of HDL into lipoproteins of low density was demonstrated.