Physiological implications of the contrasting modulation of the activities of the epsilon- and zeta-isoforms of diacylglycerol kinase

We have shown that the requirement of the epsilon-isoform of diacylglycerol kinase for diacylglycerols containing arachidonic acid is specific for this substrate and cannot be replaced by the presence of an arachidonoyl group in other places in the membrane; rather, it has to be present on the substrate itself. In addition, we demonstrate that the increased activity shown toward 1-stearoyl-2-arachidonoylglycerol by the epsilon-isoform of diacylglycerol kinase is not a consequence of altered membrane physical properties but is rather a specific interaction with the arachidonoyl group. We have also compared the modulation of the activity of the epsilon-isoform of diacylglycerol kinase with that of the zeta-isoform with regard to some of the intermediates involved in phosphatidylinositol cycling. One of the products of the hydrolysis of phosphatidylinositol diphosphate is diacylglycerol enriched in arachidonic acid. The activity of the epsilon-isoform is known to be specific for this form of diacylglycerol. We show that in contrast, the activity of the zeta-isoform is lower against 1-stearoyl-2-arachidonoylglycerol compared with dioleoylglycerol. We demonstrate that addition of phosphatidylserine, as well as other anionic phospholipids including L-alpha-phosphatidylinositol 4,5-bisphosphate, strongly inhibits the epsilon-isoform, but these anionic lipids increase the activity of the zeta-isoform. Addition of Ca(2+), which is released from internal stores as a consequence of phosphatidylinositol cycling, promotes the activity of the epsilon-isoform of this enzyme but has little effect on the zeta-isoform. The contrasting conditions required for maximal activity of these two isoforms of diacylglycerol kinase, as well as their different substrate specificity, suggest that they have different physiological roles in signal transduction.     

Evidence that phosphorylase kinase exhibits phosphatidylinositol kinase activity

Phosphorylase kinase phosphorylates the pure phospholipid phosphatidylinositol. Furthermore, it catalyzed phosphatidylinositol 4-phosphate formation using as substrate phosphatidylinositol that is associated with an isolated trypsin-treated Ca2+-transport adenosinetriphosphatase (ATPase) preparation from skeletal muscle sarcoplasmic reticulum. On this basis a fast and easy assay was developed that allows one to follow the phosphatidylinositol kinase activity during a standard phosphorylase kinase preparation. Both activities are enriched in parallel approximately to the same degree. Neither chromatography on DEAE-cellulose nor that on hydroxyapatite in the presence of 1 M KCl separates phosphatidylinositol kinase from phosphorylase kinase. The presence of a lipid kinase, phosphatidylinositol kinase, in phosphorylase kinase is not a general phenomenon; diacylglycerol kinase can be easily separated from phosphorylase kinase. Polyclonal anti-phosphorylase kinase antibodies as well as a monoclonal antibody directed specifically against the alpha subunit of phosphorylase kinase immunoprecipitate both phosphorylase kinase and phosphatidylinositol kinase.     

Rapid dephosphorylation of protein kinase C substrates by protein kinase A activators results from inhibition of diacylglycerol release

The biochemical events encompassing the dephosphorylation of protein kinase C substrates by protein kinase A activators have been investigated in a neurotumor cell line, NCB-20. Treatment of [32P]orthophosphate-labeled cells with protein kinase A activators (e.g. forskolin, dibutyryl cAMP, prostaglandin E1) resulted in an inhibition of protein kinase C activity due to a failure of the protein kinase C complex to translocate into the membrane. Phospholipase C activity, as measured by the synchronous release of diacylglycerol and inositol phosphates (inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1-phosphate) in response to bradykinin, was inhibited up to 50% following exposure to protein kinase A activators. At the same time, phospholipase C-specific inositol phospholipid substrates (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) were found to accumulate in NCB-20 cells following treatment with protein kinase A activators. This suggests that phospholipase C may be altered through protein kinase A-mediated protein phosphorylation. Second messenger generation (inositol phosphates, diacylglycerol, and Ca2+) is therefore inhibited through cyclic AMP-mediated shutdown of the inositol lipid cycle at the level of phospholipase C.     

Synthesis of polyphosphoinositides in vertebrate photoreceptor membranes

Rod outer segments isolated from bovine retinas incorporated 32P into phospholipids after incubation with [gamma-32P]ATP in a Mg2+-containing medium. Only phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidate were labelled. The incorporation of label into lipids was detected as early as 20 s after the start of incubation and the products were stable for at least 10 min. The reactions were time, protein and ATP-concentration dependent. Entire rod outer segments showed higher diacylglycerol kinase and lower phosphatidylinositol and phosphatidylinositol 4-phosphate kinase activities than the disc membranes obtained from them. Exogenously added phosphatidylinositol (up to 1 mM) in the presence of Triton X-100 increased phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labelling in rod outer segments (8- and 6-fold, respectively). Triton X-100 at a concentration of 0.4% stimulated phosphorylation of endogenous phosphoinositides. Diacylglycerol kinase activity was largely suppressed by the detergent, but this effect was partially reversed by addition of phosphatidylinositol. It is suggested that the rod outer segments contain phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase bound to disc membranes, as well as an active diacylglycerol kinase occurring either as a soluble or a peripherally bound protein in disc membranes.     

Characterization of phosphatidylinositol kinase activity associated with the insulin receptor

Various lipids were tested as substrates for the insulin receptor kinase using either receptor partially purified from rat hepatoma cells by wheat-germ-agglutinin-Sepharose chromatography or receptor purified from human placenta by insulin-Sepharose affinity chromatography. Phosphatidylinositol was phosphorylated to phosphatidylinositol 4-phosphate by the partially purified insulin receptor. In contrast, phosphatidylinositol 4-phosphate and diacylglycerol were not phosphorylated. In some, but not all preparations of partially purified insulin receptor, the phosphatidylinositol kinase activity was stimulated by insulin (mean effect 33%). Phosphatidylinositol kinase activity was retained in insulin receptor purified to homogeneity. Insulin regulation of the phosphatidylinositol kinase was lost in the purified receptor; however, dithiothreitol stimulated both autophosphorylation of the purified receptor and phosphatidylinositol kinase activity in parallel about threefold. (Glu80Tyr20)n, a polymeric substrate specific to tyrosine kinases, inhibited the phosphatidylinositol kinase activity of the purified receptor by greater than 90% and inhibited receptor autophosphorylation by 67%. Immunoprecipitation by specific anti-receptor antibodies depleted by greater than 90% the phosphatidylinositol kinase activity in the supernatant of the purified receptor and the phosphatidylinositol kinase activity was recovered in the precipitate in parallel with receptor autophosphorylation activity. These characteristics of the phosphatidylinositol kinase activity of the purified insulin receptor and its metal ion preference paralleled those of the receptor tyrosine kinase activity and differed from bulk phosphatidylinositol kinase activity in cell extracts, which was not significantly inhibited by (Glu80Tyr20)n, stimulated by dithiothreitol or depleted by immunoprecipitation with anti-(insulin receptor) antibody. These results suggest that the insulin receptor is associated with a phosphatidylinositol kinase activity; however, this activity is not well regulated by insulin. This kinase appears to be distinct from the major phosphatidylinositol kinase(s) of cells. Its relationship to insulin action needs further study.     

Activation of calcium and phospholipid-dependent protein kinase by epidermal growth factor (EGF) in A431 cells: attenuation by 12-0-tetradecanoylphorbol-13-acetate (TPA)

A calcium and phospholipid-dependent protein kinase (protein kinase C) was detected in the crude soluble extracts of A431 human epidermoid carcinoma cells. The enzyme required calcium, phosphatidylserine or phosphatidylinositol, and diacylglycerol (DG) for maximal activation. Protein kinase C phosphorylated both endogenous cytosolic proteins and various histones. Addition of epidermal growth factor (EGF) to A431 cultures resulted in a 2 to 3-fold stimulation of protein kinase activity. 12-0-tetradecanoylphorbol-13-acetate (TPA) in concert with EGF attenuated the EGF-induced enhanced phosphorylation of endogenous proteins. It is conceivable that DG, derived from phosphatidylinositol turnover, acts as a natural activator of protein kinase C activity.     

Phosphoinositide kinase, diacylglycerol kinase, and phospholipase C activities associated to the cytoskeleton: effect of epidermal growth factor

In this paper we demonstrate that cytoskeletons isolated from A431 cells have associated with them high activities of several kinases involved in inositol lipid metabolism, such as phosphatidylinositol kinase, phosphatidylinositol phosphate kinase, and diacylglycerol kinase. In addition also phospholipase C activity was detected on isolated cytoskeletons. Controlled extraction of the cytoskeletons followed by in vitro polymerization of actin demonstrated an association of the kinases to the actin filament system consisting of actin and a number of actin-binding proteins. The cytoskeleton-associated lipid kinase activities were significantly increased upon treatment of intact cells with EGF. These data suggest that the association of the phosphoinositide kinases, diacylglycerol kinase, phospholipase C, and also the EGF receptor to the cytoskeleton may play a role in the efficient signal transduction induced by EGF, by providing a matrix for the various components involved in signal transduction.     

Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

Human neutrophils stimulated with a phorbol ester (phorbol 12-myristrate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 min. In contrast, 4-alpha-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.     

Effect of transforming growth factor-alpha on inositol phospholipid metabolism in human epidermoid carcinoma cells

Transforming growth factor-alpha (TGF-alpha) stimulates (in a dose-dependent manner) the incorporation of [32P]Pi into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) in the human epidermoid carcinoma cell line (A431). The effect of TGF-alpha on the incorporation was found to be similar to that of EGF. On the other hand, a striking difference in the activation of diacylglycerol (DG) kinase activity was seen between TGF-alpha and EGF. At least 100 times more TGF-alpha was required to achieve maximal stimulation of DG kinase activity relative to EGF. These results suggest that the activation of DG kinase by TGF-alpha may involve a mechanism independent from or subsequent to activation of the EGF receptor.     

Phospholipase C activation and diacylglycerol kinase inactivation lead to an increase in diacylglycerol content in spontaneously hypertensive rat

Activities of three kinases, phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol (DG) kinases, and phospholipase C were measured in erythrocyte ghosts from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). PI kinase activity was significantly higher in SHR than WKY but there was no significant difference in PIP kinase activity between SHR and WKY. The activity of phospholipase C, which hydrolyzes PIP2, was also increased in SHR. However, DG kinase activity was, on the contrary, decreased in SHR. These results suggest that there is a tendency to accumulate DG in SHR. Indeed, DG content in erythrocytes of SHR increased 1.7-fold compared to that of WKY. Such DG accumulation may cause the sustained activation of protein kinase C in SHR, since DG is a physiological activator for protein kinase C.     

Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70 kDa protein.

Plasma membranes were isolated from carrot (Daucus carota L.) cells grown in suspension culture and treated with phospholipase A2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinase or phosphatidylinositol monophosphate kinase activity released from the membranes after the phospholipase A2 treatment. Treating the plasma membranes with phospholipase C or D did not release PI kinase activity. The phospholipase A2-released PI kinase was activated over 2-fold by a heat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the Vmax but does not affect the Km of the phospholipase A2-released PI kinase.     

A membrane-bound diacylglycerol kinase that selectively phosphorylates arachidonoyl-diacylglycerol. Distinction from cytosolic diacylglycerol kinase and comparison with the membrane-bound enzyme from Escherichia coli

The membrane-bound diacylglycerol kinase from Swiss 3T3 cells (M-DG kinase) was characterized with a mixed micellar assay system, and compared with the cytosolic diacylglycerol kinase from 3T3 cells and with the membrane-bound diacylglycerol kinase from Escherichia coli. M-DG kinase selectively phosphorylated arachidonoyl-diacylglycerols, at a rate 2- to 8-fold higher than that for other naturally occurring long-chain diacylglycerols. In contrast, the cytosolic 3T3 enzyme exhibited little or no selectivity among long-chain diacylglycerols but had higher activity with more soluble substrates such as 1,2-didecanoylglycerol. Comparison of the properties of M-DG kinase with those of the bacterial membrane-bound enzyme revealed that selectivity for arachidonoyl-diacylglycerol was unique to the mammalian enzyme. All three kinases were activated by phosphatidylserine, but activation did not alter the arachidonoyl selectivity of M-DG kinase. Phosphatidylserine activated M-DG kinase by increasing Vm and decreasing the apparent Km for diacylglycerol. High concentrations of diacylglycerol reduced the Ka for phosphatidylserine, but did not abolish the phosphatidylserine requirement for maximum activity. Examination of the thermal lability of M-DG kinase revealed that this enzyme was rapidly and selectively inactivated by preincubation with its preferred substrate. This novel effect may have obscured previous attempts to discern substrate selectivity. Taken together, the results provide evidence that M-DG kinase is an arachidonoyl-diacylglycerol kinase that may participate in the formation of arachidonoyl-enriched species of phosphatidylinositol.     

Phosphatidylinositol kinase activities in normal and Rous sarcoma virus-transformed cells.

The phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol kinase activities in the plasma membrane-rich fraction of chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus increased when the cells were shifted from the nonpermissive temperature, 41 degrees C, to the permissive temperature, 35 degrees C. Temperature shift from 35 to 41 degrees C decreased the lipid kinase activities in the membrane vesicles. These changes accompanied the changes observed in pp60v-src protein kinase activity. Thermal inactivation at 41 degrees C did not appreciably reduce PI and PIP kinase activities in membrane vesicles prepared from uninfected or Rous sarcoma virus-transformed cells, whereas pp60v-src protein kinase activity in the membrane vesicles was rapidly inactivated under the same conditions. These data suggest that pp60v-src may indirectly enhance PI and PIP phosphorylation but not directly contribute to this pathway.     

Enhancement of inositol phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts

The inositol phospholipid metabolism is one of the main pathways of signal transduction in cells. We measured the activities of its key enzymes in v-Ha-ras-transformed 208F rat fibroblasts. In the ras-transformed clones, incorporation of [32P]Pi into intermediates of the inositol phospholipid metabolism was stimulated. The activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in the transformed clones were about 35-50% more than in untransformed cells, indicating increased inositol phospholipid metabolism. However, the activity of diacylglycerol kinase in their membrane fraction was 25-35% less than that of untransformed cells, although the total diacylglycerol kinase activity did not change. The imbalance of these kinases could constitute one of the main reasons leading to the increased level of inositol phosphates and the accumulation of diacylglycerol to 2-2.2 times that in control 208F cells. Phosphatidylinositol-4,5-bisphosphate-phospholipase C activity did not change on the transformation when assayed under various conditions. The increased level of diacylglycerol caused intracellular translocation, activation, and down-regulation of protein kinase C changes which may be one of the essential events in transformation by the v-Ha-ras gene.     

Regulation of diacylglycerol kinase reaction in Swiss 3T3 cells. Increased phosphorylation of endogenous diacylglycerol and decreased phosphorylation of didecanoylglycerol in response to platelet-derived growth factor

We studied the influence of platelet-derived growth factor (PDGF) on diacylglycerol phosphorylation in Swiss 3T3 cells. Rates of incorporation of 32P into phosphatidic acid (PA) and phosphatidylinositol (PtdIns) were determined in prelabeled cells into which sn-1,2-didecanoylglycerol (diC10) had been introduced. PDGF stimulated the formation of [32P]PA and -PtdIns from endogenous substrates but decreased the formation of [32P]PA10 and -PtdIns10. Direct measurements of diacylglycerol phosphorylation in lysates of quiescent and stimulated cells showed that PDGF stimulated the phosphorylation of endogenous diacylglycerol 2-fold in parallel with diacylglycerol accumulation but decreased by 50% the phosphorylation of diC10. Total diacylglycerol kinase activity, measured in a mixed micellar assay, was not changed by PDGF treatment. The maximum activity of diacylglycerol kinase exceeded that needed to phosphorylate all of the endogenous diacylglycerol, suggesting that the PDGF-dependent increase in diacylglycerol mass would account for the increase in PA formation. The increased mass of diacylglycerol also could explain the inhibition of diC10 phosphorylation, via substrate competition. The predominant species of endogenous diacylglycerol was 1-stearoyl-2-arachidonoyl-glycerol (18:0/20:4 diacylglycerol). In mixed micelles, the rate of phosphorylation of 18:0/20:4 diacylglycerol was 8-fold higher than that of diC10, and the 18:0/20:4 species competed with diC10 for phosphorylation. Studies showed that a membrane-bound enzyme accounted for the PDGF effect on PA formation; there was no evidence for translocation of cytosolic enzyme to the membrane. The results support these conclusions: 1) PDGF stimulates the phosphorylation of cellular diacylglycerol by promoting a transient accumulation of this lipid. 2) The stimulated phosphorylation is catalyzed by a diacylglycerol kinase that preferentially phosphorylates 18:0/20:4 diacylglycerol over diC10. 3) The diacylglycerol kinase responsible for the PDGF effect is membrane-bound.     

Phosphoinositide reorganization in human erythrocyte membrane upon cholesterol depletion.

The effect of cholesterol depletion on the activity of phosphatidylinositol/phosphatidylinositol 4-phosphate and diacylglycerol kinases and polyphosphoinositide phosphodiesterase has been studied in isolated membranes of human normal and cholesterol-depleted erythrocytes. Polyphosphoinositide synthesis (phosphatidylinositol/phosphatidylinositol 4-phosphate kinase activities) were found to depend on the permeability and sidedness characteristics of the membrane vesicles, which could limit the accessibility of ATP for the enzymes. When measured under proper conditions, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate synthesis were decreased in cholesterol-depleted membranes as compared with control membranes. The same level of synthesis could be obtained in both membranes by the addition of phosphatidylinositol (and Triton X-100) or of phosphatidylinositol 4-phosphate. Phosphatidic acid synthesis (diacylglycerol kinase activity) was also decreased in cholesterol-depleted membranes as compared with control membranes when measured in the presence of Ca2+. Addition of diolein (and Triton X-100) caused a large increase in phosphatidic acid synthesis which reached approximately the same level in both membranes. This showed that the apparent inhibition of polyphosphoinositide and phosphatidic acid synthesis was not due to a loss or to an inactivation of the kinases. Ca2+-activated polyphosphoinositide phosphodiesterase promoted the hydrolysis of 65-70% of the polyphosphoinositides in control and of only 45-55% in cholesterol-depleted membranes without changing the Ca2+ concentration for half-maximum hydrolysis (1 microM). Upon addition of sodium oleate, the extent of polyphosphoinositide hydrolysis became identical in both membranes, indicating again that there was no loss nor inactivation of the polyphosphoinositide phosphodiesterase in the cholesterol-depleted membranes. Since the concentration of the polyphosphoinositides was not changed by cholesterol depletion [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200], the reduction in both their synthesis and degradation observed here could be attributed to a reorganization of the phosphoinositides in membrane domains where they were not accessible to the kinases and phosphodiesterase. The reduction in phosphatidic acid synthesis was likely caused by a reduction in the total amount of the substrate diacylglycerol in cholesterol-depleted membranes as already shown [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200].     

Identification of two cytosolic diacylglycerol kinase isoforms in rat brain, and in NIH-3T3 and ras-transformed fibroblasts.

Two major species of diacylglycerol kinase (type I and type II) were separated from brain cytosol and from NIH-3T3 or ras-transformed 3T3 cells by heparin-agarose chromatography. Multiple species of diacylglycerol kinase were also detected by non-denaturing isoelectric focusing. The two peaks of activity were of similar size, both co-eluted at approximately 95 kDa from a Superose f.p.l.c. column. Type II enzyme (pI 8.0) was more active when substrate was presented in a deoxycholate/phosphatidylserine undefined environment, as opposed to an octyl glucoside/phosphatidylserine micellar environment. Type II activity was also enhanced by the presence of phosphatidylcholine as cofactor. Type I enzyme (pI 4.0) was more active in the presence of either phosphatidylserine or phosphatidylinositol. Type I and II enzymes had different ATP affinities. Both enzymes showed a preference for diacylglycerol substrates with saturated acyl chains of 10-12 carbon atoms. The cytosolic enzyme activity was able to bind to diacylglycerol-enriched membranes in NIH-3T3 fibroblasts, and this translocation was unaffected in ras-transformed 3T3 cells. These results demonstrate the presence of multiple diacylglycerol kinases in brain cytosol and NIH-3T3 and ras-transformed 3T3 cells. The enzymes differ in cofactor, ATP and substrate requirements. These results can explain some of the contradictions between previous studies of cytosolic diacylglycerol kinase activity, and suggest the presence of a family of such kinases that are differentially regulated by phospholipid cofactors.     

Diacylglycerol kinase defect in a Drosophila retinal degeneration mutant rdgA

The Drosophila visual mutant rdgA is known to show age-dependent retinal degeneration with defective diacylglycerol (DG) kinase activity. In this study we examined DG kinase activity of several visual mutants and found that only rdgA mutant eyes showed the lack of DG kinase activity in a gene dosage-dependent manner. The enzyme activity is already absent at the time of eclosion from pupal case when the degeneration is not yet apparent. To examine whether rdgA gene dosage effect holds for other enzymes related to the phosphatidylinositol turnover, phospholipase C was analyzed which did not show any gene dosage effect. Therefore, it is strongly suggested that rdgA gene correlates closely with DG kinase activity, and the defect of DG kinase activity is a primary cause of retinal degeneration in rdgA mutant.     

Protein kinase C from small intestine epithelial cells

Protein kinase C activity has been identified in cytosolic and membrane fractions from rat and rabbit small intestine epithelial cells. The cytosolic fraction comprised about the 75% of total activity. Protein kinase C activity was resolved from other protein kinase activities by ion exchange chromatography. Phosphatidylserine or phosphatidylinositol were required for protein kinase C to be active. In addition, the activity was enhanced by the presence of a diacylglycerol. Diolein and dimyristin were the most effective (13-14 fold activation). In the presence of phosphatidylserine and diolein, the Ka for activation by Ca2+ was 10(-7)M. The phorbol ester TPA substituted for diacylglycerol in activating protein kinase C. Brush border and basolateral membranes contained protein kinase C activity, although the specific activity of the basal lateral membranes was four-fold higher than the specific activity of the brush border membranes. The presence of PKC in small intestine epithelial cells might have important implications in the Ca2+ mediated control of ionic transport in this tissue.     

Diacylglycerol kinases

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol to form phosphatidic acid. In most cases, members of this large family of enzymes appear to bind and regulate proteins activated by either diacylglycerol or phosphatidic acid. Proteins that appear to be regulated, in part, by DGKs include protein kinase Cs, RasGRPs, and phosphatidylinositol kinases. By modulating the activity of these proteins, DGKs potentially affect a number of biological events including-but likely not limited to-cell growth, neuronal transmission, and cytoskeleton remodeling.