Structure-activity relationship of diacylglycerol kinase theta

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid (PA). Among the nine mammalian isotypes identified, DGKtheta is the only one with three cysteine-rich domains (CRDs) (instead of two) in its N-terminal regulatory region. We previously reported that DGKtheta binds to and is negatively regulated by active RhoA. We now report that RhoA strongly binds to the C-terminal catalytic domain, which would explain its inhibition of DGK activity. To help finding a physiological function of DGKtheta, we further determined its activity in vitro as a function of 15 different truncations and point mutations in the primary structure. Most of these alterations, located throughout the protein, inactivated the enzyme, suggesting that catalytic activity depends on all of its conserved domains. The most C-terminal CRD is elongated with a stretch of 15 amino acids that is highly conserved among DGK isotypes. Mutation analysis revealed a number of residues in this region that were essential for enzyme activity. We suggest that this CRD extension plays an essential role in the correct folding of the protein and/or in substrate presentation to the catalytic region of the protein.     

Endocannabinoids and diacylglycerol kinase activity

Mammalian diacylglycerol kinases are a family of enzymes that catalyze the phosphorylation of diacylglycerol to produce phosphatidic acid. The extent of interaction of these enzymes with monoacylglycerols is the focus of the present study. Because of the structural relationship between mono- and diacylglycerols, one might expect the monoacylglycerols to be either substrates or inhibitors of diacylglycerol kinases. This would have some consequence to lipid metabolism. One of the lipid metabolites that would be affected is 2-arachidonoyl glycerol, which is an endogenous ligand for the CB1 cannabinoid receptor. We determined if the monoglycerides 2-arachidonoyl glycerol or 2-oleoyl glycerol affected diacylglycerol kinase activity. We found that 2-arachidonoyl glycerol is a very poor substrate for either the epsilon or the zeta isoforms of diacylglycerol kinases. Moreover, 2-arachidonoyl glycerol is an inhibitor for both of these diacylglycerol kinase isoforms. 2-oleoyl glycerol is also a poor substrate for these two isoforms of diacylglycerol kinases. As an inhibitor, 2-oleoyl glycerol inhibits diacylglycerol kinase ε less than does 2-arachidonoyl glycerol, while for diacylglycerol kinase ζ, these two monoglycerides have similar inhibitory potency. These results have implications for the known role of diacylglycerol kinase ε in neuronal function and in epilepsy since the action of this enzyme will remove 1-stearoyl-2-arachidonoylglycerol, the precursor of the endocannabinoid 2-arachidonoyl glycerol.     

Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization

Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.     

Inhibition of rat diacylglycerol kinase activity by synthetic L-lysine containing polypeptides

We investigated whether or not synthetic polymers of amino acids affected the activity of rat diacylglycerol kinase. Among all the polypeptides of amino acids tested, only the polymers containing significant amounts of basic amino acid residues inhibited diacylglycerol kinase activity. Poly-L-lysine was a better inhibitor than poly-L-arginine. Both the density of positive charge and the nature of spacing amino acids of the inhibitory peptides were determining factors in interacting with the enzyme. Various polyamines were ineffective. Our observation indicated that the activity of diacylglycerol kinase might be controlled by a site which binds to a polycation.     

Modulation of human nuclear receptor LRH-1 activity by phospholipids and SHP

The human nuclear receptor liver receptor homolog 1 (hLRH-1) plays an important role in the development of breast carcinomas. This orphan receptor is efficiently downregulated by the unusual co-repressor SHP and has been thought to be ligand-independent. We present the crystal structure at a resolution of 1.9 A of the ligand-binding domain of hLRH-1 in complex with the NR box 1 motif of human SHP, which we find contacts the AF-2 region of hLRH-1 using selective structural motifs. Electron density indicates phospholipid bound within the ligand-binding pocket, which we confirm using mass spectrometry of solvent-extracted samples. We further show that pocket mutations reduce phospholipid binding and receptor activity in vivo. Our results indicate that hLRH-1's control of gene expression is mediated by phospholipid binding, and establish hLRH-1 as a novel target for compounds designed to slow breast cancer development.     

Modulation of creatine kinase activity by ruthenium complexes

Creatine kinase is a crucial enzyme for brain, heart and skeletal muscle energy homeostasis, and a decrease of its activity has been associated with cell death. Many biological properties have been attributed to ruthenium complexes. In this context, this work was performed in order to evaluate creatine kinase activity from rat brain, heart and skeletal muscle (quadriceps) after administration of ruthenium complexes, trans-[RuCl(2)(nic)(4)] (nic=3-pyridinecarboxylic acid) 180.7 micromol/kg (complex I), trans-[RuCl(2)(i-nic)(4)] (i-nic=4-pyridinecarboxylic acid) 13.6 micromol/kg (complex II), trans-[RuCl(2)(dinic)(4)] (dinic=3,5-pyridinedicarboxylic acid) 180.7 micromol/kg (complex III) and trans-[RuCl(2)(i-dinic)(4)] (i-dinic=3,4-pyridinedicarboxylic acid) 180.7 micromol/kg (complex IV). Our results showed that complex I caused inhibition of creatine kinase activity in hippocampus, striatum, cerebral cortex, heart and skeletal muscle. Besides, complex II did not affect the enzyme activity. complexes III and IV increased creatine kinase activity in hippocampus, striatum, cerebral cortex and heart, but not in skeletal muscle. Besides, none of the complexes in vitro altered creatine kinase activity, suggesting that enzymatic activity is indirectly affected by complexes I, III and IV. It is believed that diminution of creatine kinase in brain of rats caused by complex I may be related to results from other study reporting memory impairment caused by the same complex. Further research is necessary in order to elucidate the effects of ruthenium complexes in other important metabolic enzymes.     

Modulation of adenylate cyclase activity of fibroblasts by free fatty acids and phospholipids

The effect of certain lipids on adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from fibroblasts in culture has been investigated. The unsaturated fatty acids, as well as lysolecithin, were found to act as potent inhibitors of fibroblast adenylate cyclase activity. Increasing the degree of unsaturation increases the extent of inhibition noted at a given fatty acid concentration. The inhibitory effect of the unsaturated fatty acids or lysolecithin is not selective for a specific function of the adenylate cyclase system since basal, and hormone- or fluoride-stimulated cyclase activities are inhibited to the same extent. The fatty acid-inactivated state of fibroblast adenylate cyclase is not readily reversed for enzyme activity is not restored when arachidonate-treated membranes are washed with Tris buffer containing 10 mm EDTA, 0.15 mm albumin, or 0.15 m KCl. Previous studies have shown that the adenylate cyclase system from Moloney sarcoma virus-transformed NRK (MNRK) cells is not stimulated by the addition of GTP or hormones. Of interest is the present finding that the addition of unsaturated fatty acids, or lysolecithin, over a narrow concentration range (0.1 – 0.2 mm) leads to partial restoration of GTP activation of MNRK cyclase activity. Hormonal responsiveness to l-epinephrine or prostaglandin E₁ is not restored to the MNRK enzyme with fatty acid or lysolecithin treatment.     

Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation

FXYD10 is a 74 amino acid small protein which regulates the activity of shark Na,K-ATPase. The lipid dependence of this regulatory interaction of FXYD10 with shark Na,K-ATPase was investigated using reconstitution into DOPC/cholesterol liposomes with or without the replacement of 20 mol % DOPC with anionic phospholipids. Specifically, the effects of the cytoplasmic domain of FXYD10, which contains the phosphorylation sites for protein kinases, on the kinetics of the Na,K-ATPase reaction were investigated by a comparison of the reconstituted native enzyme and the enzyme where 23 C-terminal amino acids of FXYD10 had been cleaved by mild, controlled trypsin treatment. Several kinetic properties of the Na,K-ATPase reaction cycle as well as the FXYD-regulation of Na,K-ATPase activity were found to be affected by acidic phospholipids like PI, PS, and PG. This takes into consideration the Na+ and K+ activation, the K+-deocclusion reaction, and the poise of the E1/E2 conformational equilibrium, whereas the ATP activation was unchanged. Anionic phospholipids increased the intermolecular cross-linking between the FXYD10 C-terminus (Cys74) and the Cys254 in the Na,K-ATPase A-domain. However, neither in the presence nor in the absence of anionic phospholipids did protein kinase phosphorylation of native FXYD10, which relieves the inhibition, affect such cross-linking. Together, this seems to indicate that phosphorylation involves only modest structural rearrangements between the cytoplasmic domain of FXYD10 and the Na,K-ATPase A-domain.     

Modulation by phospholipids of the activity of monoamine oxidase purified from pig liver

Monoamine oxidase was purified from pig liver mitochondria to homogeneity. The enzyme sample contained a large amount of phospholipids. Depletion of lipids from the enzyme sample resulted in a decrease in its activity, while activity was restored by the binding of the lipid-depleted enzyme to phosphatidylcholine, phosphatidylethanolamine, or mitochondrial lipids. Upon binding the lipid-depleted enzyme to the mixture of phosphatidylcholine and phosphatidylethanolamine (molar ratio, 1 : 1), the enzymatic activity toward serotonin was elevated over that of the purified enzyme, but not toward benzylamine, suggesting a change in substrate specificity. Upon lipid depletion, inhibition by deprenyl became weaker, while that by clorgyline became stronger. This alteration was reversed by the binding to lipids. By the binding of the lipid-depleted enzyme to some lipids such as the mixture of phosphatidylcholine and phosphatidylethanolamine (molar ratio, 1 : 1), inhibition by clorgyline became even weaker than for the original enzyme sample.     

Translocation of diacylglycerol kinase theta 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 DGK 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 DGK, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGK 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 DGK translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGK translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGK abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGK recruitment to the membrane. We show that DGK interacts selectively with and is phosphorylated by PKCepsilon and -eta and that peptide agonist-induced selective activation of PKCepsilon directly leads to DGK translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGK, which may be important in the negative regulation of PKCepsilon and/or PKCeta activity.     

Phosphorylation of pig brain diacylglycerol kinase by endogenous protein kinase

Pig brain diacylglycerol kinase did not catalyze autophosphorylation. However, the kinase was phosphorylated on serine, when immunoprecipitated from the partially purified enzyme preparation preincubated with Mg2+ and [gamma-32P]ATP. The action of the endogenous protein kinase phosphorylating diacylglycerol kinase was independent of cyclic nucleotides and Ca2+, and became maximum at pH 5.5. Although the extent of enzyme phosphorylation was limited (maximally about 0.25 mol Pi incorporated per mol kinase), the results show that diacylglycerol kinase can be a phosphoprotein.     

Modulation of the catalytic activity of pyridoxal kinase by metallothionein

Pyridoxal kinase displays high catalytic activity in the presence of metallothionein. The apoprotein of metallothionein as well as the peptide LYS-CYS-THR-CYS-CYS-ALA exert a strong inhibitory effect upon pyridoxal kinase by sequestering free Zn ions. Several steps intervene in the process of pyridoxal kinase activation, i.e. binding of Zn ions by ATP and interaction of Zn-ATP with the enzyme; but direct interaction between metallothionein and pyridoxal kinase (protein association) could not be detected by emission anisotropy measurements. Since the concentration of free Zn++ in mammalian tissues is lower than 10(-9)M, it is postulated that the concentration of metallothionein regulates the catalytic activity of pyridoxal kinase. The mechanism of reconstitution of the metalloenzyme yeast aldolase in the presence of metallothionein was also investigated.     

Regulation of clathrin-dependent endocytosis by diacylglycerol kinase delta: importance of kinase activity and binding to AP2alpha

DGKdelta (diacylglycerol kinase delta), which phosphorylates DAG (diacylglycerol) and converts it into PA (phosphatidic acid), has an important role in signal transduction. In the present study, we have demonstrated the molecular mechanism of DGKdelta-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGKdelta in the regulation of clathrin-dependent endocytosis was previously proposed following genome-wide RNAi (RNA interference) screening. Clathrin-coated pits are mainly formed by clathrin and AP-2 (adaptor protein 2) complex. These proteins assemble a polyhedral lattice at the membrane and gather several endocytic accessory proteins. As the intracellular localization of DGKdelta2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGKdelta2 and its interaction with some endocytosis-regulatory proteins. DGKdelta2 contained the DXF-type binding motifs, and DGKdelta2 bound to AP2alpha, a subunit of the AP-2 complex. DGKdelta2 interacted with the platform subdomain in the AP2alpha ear domain via F369DTFRIL and D746PF sequences in the catalytic domain of DGKdelta2. For further insight into the role for DGKdelta2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptake-expressing wild-type or mutant DGKdelta2 under knockdown of endogenous DGKdelta. Mutants lacking binding ability to AP2alpha as well as kinase-negative mutants could not compensate for the uptake of transferrin inhibited by siRNA (small interfering RNA) treatment, whereas overexpression of wild-type DGKdelta2 completely recovered the transferrin uptake. These results demonstrate that binding between DGKdelta2 and AP2alpha is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.     

Phosphorylation of diacylglycerol kinase in vitro by protein kinase C.

We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as Mr-80,000 proteins, were highly purified from pig thymus cytosol. Protein kinase C phosphorylated diacylglycerol kinase (up to 1 mol of 32P/mol of enzyme) much more actively than did cyclic AMP-dependent protein kinase. Phosphorylated and non-phosphorylated diacylglycerol kinase showed a similar pI, approx. 6.8. Diacylglycerol kinase phosphorylated by either protein kinase C or cyclic AMP-dependent protein kinase was almost exclusively associated with phosphatidylserine membranes. In contrast, soluble kinase consisted of the non-phosphorylated form. The catalytic properties of the lipid kinase were not much affected by phosphorylation, although phosphorylation-linked binding with phosphatidylserine vesicles resulted in stabilization of the enzyme activity.     

Role of the kinase activation loop on protein kinase C theta activity and intracellular localisation

Multiple protein kinase C (PKC) theta species, identified in an erythroleukaemia cell line, have been characterised in terms of their molecular properties and intracellular distribution. PKCthetas localised in the detergent-soluble cell fraction have an Mr of 76 kDa (theta-76) and contain Thr538 or pThr538 in the kinase activation loop. In contrast, PKCthetas localised in the Golgi complex have an Mr of 85 kDa (theta-85) and, although unphosphorylated at Thr538, are catalytically active. Strikingly, only theta-76 species which are unphosphorylated at Thr538 can undergo autocatalytic conversion to theta-85. Moreover, a Thr538-->Ala PKCtheta mutant is constitutively localised in the Golgi complex, confirming that changes in the phosphorylation state of this residue play a pivotal role in the overall control of catalytic properties and localisation of this kinase.     

Modulation of gastrointestinal wound repair and inflammation by phospholipids

The mucosal surface of the digestive tract is a critical barrier between a broad spectrum of noxious and immunogenic substances present in the gastrointestinal lumen and the underlying mucosal immune system. Its preservation following various forms of injury or physiological damage is essential to prevent the invasion of harmful luminal factors into the host, which subsequently may lead to inflammation, uncontrolled immune response, and a disequilibrium of the homeostasis of the host. The preservation of this barrier following injuries is regulated by a broad spectrum of structurally distinct regulatory molecules, including phospholipids. Phospholipids play a pivotal role in the modulation of intestinal inflammation. They have been demonstrated to both promote and inhibit inflammation, and their overall impact in an individual setting seems to be dependent on several factors, including the level of immune cell activation and the presence of other mediators. Modulation of lipid mediators through administration of lysophosphatidic acid (LPA) or lisofylline (LSF), inhibitors of phospholipase A2 (PLA2) biosynthesis or monoclonal antibodies against thromboxane (TBX) or platelet-activating factor (PAF) as a therapeutic approach have been used in several models of inflammation; however, beneficial effects were not always convincing and further studies are warranted.     

Modulation of N-type calcium channel activity by G-proteins and protein kinase C

N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming alpha(1B) subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-protein-mediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-gamma-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-gamma-S-induced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-protein-mediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-protein-mediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potential-dependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-beta-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.     

Utilization of diacylglycerol in phospholipid bilayers by pig brain diacylglycerol kinase and Rhizopus arrhizus lipase

Diacylglycerol kinase purified from pig brain cytosol could use sonication-dispersed diacylglycerol in the presence of its activator, phosphatidylcholine vesicles. However, the kinase failed to significantly use diacylglycerol cosonicated with phosphatidylcholine. Similarly, the kinase could not use diacylglycerol generated in microsomes by the back reaction of diacylglycerol choline phosphotransferase, though phospholipase C treatment of microsomes yielded effective substrate for the kinase. In order to elucidate the mechanism of these discrepant findings, we studied the activity of the purified kinase and Rhizopus arrhizus lipase utilizing dioleoylglycerol incorporated into various phospholipid vesicles. The inaccessibility of diacylglycerol contained in phospholipid vesicles was observed similarly for the two different enzymes. We considered that the apparent enzymic latency of diacylglycerol could be best accounted for by an extremely limited solubility of diacylglycerol in the outer leaflet of phospholipid bilayers. The experimental bases for this interpretation are: 1) diacylglycerol cosonicated with dihexanoyl phosphatidylcholine was exceptionally effective as substrate for the kinase; 2) the enzyme activities with cosonicated and separately sonicated lipids became similar when bile salts were present; 3) both enzymes could use diacylglycerol generated on phosphatidylcholine vesicles by a limited phospholipase C hydrolysis; and 4) phosphatidylcholine diacylglycerol vesicles at widely different molar ratios (from 1:0.014 to 1:0.2) were similarly ineffective as substrate for both enzymes.