Membrane interactions of the hydrophobic segment of diacylglycerol kinase epsilon

Diacylglycerol kinase epsilon (DGKε) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKε of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH₂. Flanking Lys residues mimic the natural setting of this peptide in DGKε, while facilitating peptide synthesis and characterization. Circular dichroism and fluorescence spectroscopic analysis demonstrated that the peptide has increased helical content and significant blue shifts in the presence of anionic - but not zwitterionic - bilayer membranes. When labeled with fluorophores that can undergo fluorescence resonance energy transfer, the peptide was found to dimerize - a result also observed from migration rates on SDS-PAGE gels under both reducing and non-reducing disulfide bridge conditions. The peptide was shown to preferentially interact with cholesterol in lipid films comprised of homogeneous mixtures of cholesterol and phosphatidylcholine, yet the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. The peptide was also able to inhibit the activity of DGKε protein in vitro. Our overall findings suggest that the peptide ultimately cannot leave the bulk water for attachment/insertion into the outer leaflet of an erythrocyte-like bilayer, yet its core sequence is sufficiently hydrophobic to insert into membrane core regions when membrane attachment is promoted by electrostatic attraction to anionic lipid head groups of the inner leaflet of an erythrocyte-like bilayer.     

Role of the hydrophobic segment of diacylglycerol kinase epsilon

Diacylglycerol kinase epsilon (DGKepsilon) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids. We have evaluated the contributions of this segment to the membrane interactions and functions of this protein. To test the role of the hydrophobic segment, we have compared the properties of DGKepsilon with those of a truncated form of the protein (DGKDeltaepsilon) lacking the 40 N-terminal amino acids, which includes the hydrophobic segment. The proteins were expressed in COS-7 cells from a gene for human DGKepsilon or from a gene for a truncated form (DGKDeltaepsilon), both of which had a FLAG tag at the amino terminus. Full-length FLAG-DGKepsilon and truncated FLAG-DGKDeltaepsilon were both more specific for 1-stearoyl-2-arachidonoyl-sn-glycerol than for 1,2-dioleoyl-sn-glycerol. 1-Stearoyl-2-linoleoyl-sn-glycerol exhibited intermediate specificity for both forms of the enzyme. The results show that the truncated form of the enzyme maintains substrate specificity for lipids with an arachidonoyl moiety present at the sn-2 position. The truncation increases the catalytic rate constant for all three substrates and may suggest a role in the negative regulation of this enzyme. A full-length DGKepsilon with a C-terminal His tag exhibited substrate specificity similar to that of the other two forms of the enzyme, indicating that the nature and position of the epitope tag did not strongly affect this property. Using an ultracentrifugation floatation assay, we showed that at neutral pH DGKDeltaepsilon is extracted with 1.5 M KCl while DGKepsilon remains essentially fully membrane bound. The full-length protein had a weak tendency to oligomerize in the presence of weak detergents. DGKepsilon was monomeric on SDS-PAGE but exhibited partial dimerization with low concentrations of perfluorooctanoic acid. The major conclusions of this work are that the hydrophobic domain of DGKepsilon does not contribute to substrate specificity but plays a role in permanently sequestering the enzyme to a membrane.     

Determination of the topology of the hydrophobic segment of mammalian diacylglycerol kinase epsilon in a cell membrane and its relationship to predictions from modeling

The epsilon isoform of diacylglycerol kinase (DGKepsilon) is unique among mammalian DGKs in having a segment of hydrophobic amino acids comprising approximately residues 20 to 41. Several algorithms predict this segment to be a transmembrane (TM) helix. Using PepLook, we have performed an in silico analysis of the conformational preference of the segment in a hydrophobic environment comprising residues 18 to 42 of DGKepsilon. We find that there are two distinct groups of stable conformations, one corresponding to a straight helix that would traverse the membrane and the second corresponding to a bent helix that would enter and leave the same side of the membrane. Furthermore, the calculations predict that substituting the Pro32 residue in the hydrophobic segment with an Ala will cause the hydrophobic segment to favor a TM orientation. We have expressed the P32A mutant of DGKepsilon, with a FLAG tag (an N-terminal 3xFLAG epitope tag) at the amino terminus, in COS-7 cells. We find that this mutation causes a large reduction in both k(cat) and K(m) while maintaining k(cat)/K(m) constant. Specificity of the P32A mutant for substrates with polyunsaturated acyl chains is retained. The P32A mutant also has higher affinity for membranes since it is more difficult to extract from the membrane with high salt concentration or high pH compared with the wild-type DGKepsilon. We also evaluated the topology of the proteins with confocal immunofluorescence microscopy using NIH 3T3 cells. We find that the FLAG tag at the amino terminus of the wild-type enzyme is not reactive with antibodies unless the cell membrane is permeabilized with detergent. We also demonstrate that at least a fraction of the wild-type DGKepsilon is present in the plasma membrane and that comparable amounts of the wild-type and P32A mutant proteins are in the plasma membrane fraction. This indicates that in these cells the hydrophobic segment of the wild-type DGKepsilon is not TM but takes up a bent conformation. In contrast, the FLAG tag at the amino terminus of the P32A mutant is exposed to antibody both before and after membrane permeabilization. This modeling approach thus provides an explanation, not provided by simple predictive algorithms, for the observed topology of this protein in cell membranes. The work also demonstrates that the wild-type DGKepsilon is a monotopic protein.     

Membrane topology of Escherichia coli diacylglycerol kinase.

The topology of Escherichia coli diacylglycerol kinase (DAGK) within the cytoplasmic membrane was elucidated by a combined approach involving both multiple aligned sequence analysis and fusion protein experiments. Hydropathy plots of the five prokaryotic DAGK sequences available were uniform in their prediction of three transmembrane segments. The hydropathy predictions were experimentally tested genetically by fusing C-terminal deletion derivatives of DAGK to beta-lactamase and beta-galactosidase. Following expression, the enzymatic activities of the chimeric proteins were measured and used to determine the cellular location of the fusion junction. These studies confirmed the hydropathy predictions for DAGK with respect to the number and approximate sequence locations of the transmembrane segments. Further analysis of the aligned DAGK sequences detected probable alpha-helical N-terminal capping motifs and two amphipathic alpha-helices within the enzyme. The combined fusion and sequence data indicate that DAGK is a polytopic integral membrane protein with three transmembrane segments with the N terminus of the protein in the cytoplasm, the C terminus in the periplasmic space, and two amphipathic helices near the cytoplasmic surface.     

Inhibition of lipid signaling enzyme diacylglycerol kinase epsilon attenuates mutant huntingtin toxicity

Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine expansion in the protein huntingtin (Htt). Striatal and cortical neuronal loss are prominent features of this disease. No disease-modifying treatments have been discovered for HD. To identify new therapeutic targets in HD, we screened a kinase inhibitor library for molecules that block mutant Htt cellular toxicity in a mouse HD striatal cell model, Hdh(111Q/111Q) cells. We found that diacylglycerol kinase (DGK) inhibitor II (R59949) decreased caspase-3/7 activity after serum withdrawal in striatal Hdh(111Q/111Q) cells. In addition, R59949 decreased the accumulation of a 513-amino acid N-terminal Htt fragment processed by caspase-3 and blocked alterations in lipid metabolism during serum withdrawal. To identify the diacylglycerol kinase mediating this effect, we knocked down all four DGK isoforms expressed in the brain (β, γ, ε, and ζ) using siRNA. Only the knockdown of the family member, DGKε, blocked striatal Hdh(111Q/111Q)-mediated toxicity. We also investigated the significance of these findings in vivo. First, we found that reduced function of the Drosophila DGKε homolog significantly improves Htt-induced motor dysfunction in a fly model of HD. In addition, we find that the levels of DGKε are increased in the striatum of R6/2 HD transgenic mice when compared with littermate controls. Together, these findings indicate that increased levels of kinase DGKε contribute to HD pathogenesis and suggest that reducing its levels or activity is a potential therapy for HD.     

Role of hydrophobic interactions in yeast phosphoglycerate kinase stability

Cold denaturation of yeast phosphoglycerate kinase (yPGK) was investigated by a combination of far UV circular dichroism (CD), steady-state and time-resolved fluorescence, and small angle X-ray scattering. It was shown that cold denaturation of yPGK cannot be accounted for by a simple two-state process and that an intermediate state can be stabilized under mild denaturing conditions. Comparison between far UV CD and fluorescence shows that in this state the protein displays a fluorescence signal corresponding mainly to exposed tryptophans, whereas its CD signal is only partially modified. Comparison with spectroscopic data obtained from a mutant missing the last 12 amino-acids (yPGK delta404) suggests that lowering the temperature mainly results in a destabilization of hydrophobic interactions between the two domains. Small angle X-ray scattering measurements give further information about this stabilized intermediate. At 4 degrees C and in the presence of 0.45 M Gdn-HCl, the main species corresponds to a protein as compact as native yPGK, whereas a significant proportion of ellipticity has been lost. Although various techniques have shown the existence of residual structures in denatured proteins, this is one example of a compact denatured state devoid of its main content in alpha helices.     

Characterization of the human diacylglycerol kinase epsilon gene and its assessment as a candidate for inherited retinitis pigmentosa

Human diacylglycerol kinase epsilon (hDGK epsilon) displays high selectivity for arachidonate-containing substrates and may be essential in the termination of signals transmitted through arachidonoyl-diacylglycerol and/or the synthesis of phospholipids with defined fatty acid composition. We herein report the genomic structure, chromosomal mapping, and mutation screening of hDGK epsilon gene. hDGK epsilon gene contains at least 12 exons spanning approximately 30 kb of genomic sequence and was mapped to chromosome 17q22 by fluorescence in situ hybridization. A search for disease gene linkage revealed that a locus for autosomal dominant retinitis pigmentosa (adRP) known as RP17 resided in that region, and Northern blot analysis showed that hDGK epsilon was expressed in human retina. The hDGK epsilon gene was then localized to one of the YAC clones containing a STS marker for the RP17 locus by YAC contig mapping. Direct sequencing following PCR amplification of two affected DNA samples from that type of adRP patients, however, did not reveal any mutation in hDGK epsilon exons.     

A transmembrane segment mimic derived from Escherichia coli diacylglycerol kinase inhibits protein activity

The function of membrane proteins is inextricably linked to the proper packing and assembly of their independently helical transmembrane (TM) segments. Here we examined whether an externally added TM peptide analogue could specifically inhibit the function of the membrane protein from which it is derived by competing for native TM helix packing sites, thereby producing a non-functional peptide-protein complex. This hypothesis was tested using Lys-tagged peptides synthesized with sequences corresponding to the three TM segments of the homotrimeric Escherichia coli diacylglycerol kinase (DGK). The peptide corresponding to wild-type DGK TM-2 inhibited the protein's enzymatic activity in a dose-dependent manner through formation of an inactive pseudo-complex, whereas peptides derived from TM-1 and TM-3 were benign toward DGK structure/function. Also, substitution of a conserved residue (Glu-69) within the TM-2 peptide abolished these effects, demonstrating the strict sequence requirements for TM-2-mediated association. This strategy, coupled with the practical advantages of the water solubility of Lys-tagged TM peptides, may constitute an attractive approach for the design of therapeutic membrane protein modulators even in the absence of a high resolution structure.     

The hydrophobic segment of Arabidopsis thaliana cluster I diacylglycerol kinases is sufficient to target the proteins to cell membranes

Diacylglycerol kinases (DGKs) catalyze the phosphorylation of diacylglycerol into phosphatidic acid. To fulfill their role in many signalling processes, DGKs must be located at, or in, membranes. Most mammalian DGKs are cytosolic and are recruited to membranes upon stimulation, except for epsilon type DGKs that are permanently membrane-associated through a hydrophobic segment. Nothing is known about the mechanism(s) involved in the membrane localization of plant DGKs. By fusion to fluorescent proteins, we show that two DGKs from cluster I in Arabidopsis thaliana possess amino-terminal hydrophobic segments that are sufficient to address them to endoplasmic reticulum membranes.     

Diacylglycerol kinase epsilon, but not zeta, selectively removes polyunsaturated diacylglycerol, inducing altered protein kinase C distribution in vivo

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, epsilon and zeta. In vitro, the former is specific for polyunsaturated structures, whereas the latter shows no apparent selectivity. Overexpression of DAGKepsilon 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 PKCalpha and epsilon from the membrane to the cytosol. Overexpression of DAGKzeta caused a general reduction in DAG levels but had little effect on PKC distribution. These results for the first time show that DAGKepsilon specifically phosphorylates polyunsaturated DAG in 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 DAGKepsilon being a physiological terminator of DAG second messenger signaling.     

Membrane translocation of protein kinase Ctheta during T lymphocyte activation requires phospholipase C-gamma-generated diacylglycerol

Protein kinase C (PKC) is the only PKC isoform recruited to the immunological synapse after T cell receptor stimulation, suggesting that its activation mechanism differs from that of the other isoforms. Previous studies have suggested that this selective PKC recruitment may operate via a Vav-regulated, cytoskeletal-dependent mechanism, independent of the classical phospholipase C/diacylglycerol pathway. Here, we demonstrate that, together with tyrosine phosphorylation of PKC in the regulatory domain, binding of phospholipase C-dependent diacylglycerol is required for PKC recruitment to the T cell synapse. In addition, we demonstrate that diacylglycerol kinase alpha-dependent diacylglycerol phosphorylation provides the negative signal required for PKC inactivation, ensuring fine control of the T cell activation response.     

The role of hydrophobic interactions in the phospholipid-dependent activation of protein kinase C.

The effects of hydrophobic interaction on the activation of Ca2+-stimulated phospholipid-dependent protein kinase (protein kinase C), isolated from mouse brain, by phosphatidylserine (PS) and diacylglycerol (DAG) or phorbol 12-myristate 13-acetate were studied. To maintain bilayer structure during assay conditions, phosphatidylcholine was added to the PS vesicles. The vesicular structure of all types of PS was confirmed by freeze-fracture electron microscopy. The PS-dependent activation of purified protein kinase C from mouse brain is affected by the fatty acid composition of PS: an inverse relationship between the unsaturation index of PS (isolated from bovine heart, bovine spinal cord or bovine brain) and the ability to activate protein kinase C was demonstrated. In highly saturated PS lipid dispersions, only slight additional activation of protein kinase C by DAG was found, in contrast with highly unsaturated PS lipid dispersion, where DAG increased protein kinase C activity by 2-3-fold at optimal PS concentrations. We quantified the formation of the protein kinase C-Ca2+-PS-phorbol ester complex by using [3H]phorbol 12,13-dibutyrate [( 3H]PDBu). The efficiency of complex-formation, determined as the amount of [3H]PDBu bound, is not affected by variations in the hydrophobic part of PS. These results indicate a role of the hydrophobic part of the activating phospholipid in the activation mechanism of protein kinase C and in the action of cofactors.     

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.     

Membrane interactions of the sodium channel S4 segment and its fluorescently-labeled analogues.

A 24-amino acid peptide corresponding to the S4 segment of the sodium channel was synthesized. In order to perform fluorescence energy transfer measurements and to monitor the interaction of the peptide with lipid vesicles, the peptide was selectively labeled with fluorescence probes at either its N- or C-terminal amino acids. The fluorescent emission spectra of 7-nitrobenz-2-oxa-1,3-diazol-4- yl-(NBD-)labeled analogues displayed blue shifts upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. The results revealed that both the N- and C-terminus of the S4 segment are located within the lipid bilayer. Titration of solutions containing NBD-labeled peptides with SUV was used to generate binding isotherms, from which surface partition constants, in the range of 10(4) M-1, were derived. The shape of the binding isotherms as well as fluorescence energy transfer measurements suggest that aggregation of peptide monomers within the membrane readily occurs in acidic but not in zwitterionic vesicles. Furthermore, the results provide good correlation between the incidence of aggregation in PC/PS vesicles and the ability of the peptides to permeate the vesicle's membrane. However, a transmembrane diffusion potential had no detectable effect on the location of the peptide within the lipid bilayer or on its aggregation state.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Association of diacylglycerol kinase zeta with protein kinase C alpha: spatial regulation of diacylglycerol signaling

Activation of PKC depends on the availability of DAG, a signaling lipid that is tightly and dynamically regulated. DAG kinase (DGK) terminates DAG signaling by converting it to phosphatidic acid. Here, we demonstrate that DGKzeta inhibits PKCalpha activity and that DGK activity is required for this inhibition. We also show that DGKzeta directly interacts with PKCalpha in a signaling complex and that the binding site in DGKzeta is located within the catalytic domain. Because PKCalpha can phosphorylate the myristoylated alanine-rich C-kinase substrate (MARCKS) motif of DGKzeta, we tested whether this modification could affect their interaction. Phosphorylation of this motif significantly attenuated coimmunoprecipitation of DGKzeta and PKCalpha and abolished their colocalization in cells, indicating that it negatively regulates binding. Expression of a phosphorylation-mimicking DGKzeta mutant that was unable to bind PKCalpha did not inhibit PKCalpha activity. Together, our results suggest that DGKzeta spatially regulates PKCalpha activity by attenuating local accumulation of signaling DAG. This regulation is impaired by PKCalpha-mediated DGKzeta phosphorylation.     

An endogenous regulator of diacylglycerol kinase

During the initial steps of the subcellular fractionation of rat brain homogenate, we recovered more than 100% of diacylglycerol kinase activity. The unusually high yields prompted us to examine the possibility that we had removed an endogenous inhibitor from diacylglycerol kinase during those steps. Our study revealed the existence of a potent inhibitor of diacylglycerol kinase in the crude synaptosomal-mitochondrial fraction (P2 pellet). The inhibitory substance was water soluble upon organic solvent extraction. The inhibitory activity of the substance was retained after extensive dialysis, suggesting the macromolecular nature of this compound. This substance may represent an important physiological regulator of diacylglycerol kinase.     

Membrane Potential of the Root-base segment

By cutting segments of different lengths from 1-week-old barleyroots and interposing the remainder as a membrane in concentrationcells, it was found that the root-base segment of 2—3cm is similar to a homogeneous membrane with constant transportnumbers for ions. The transport number for potassium was foundto be 0.52 indicating that the root-base segment possesses electricfields of low intensities with little effect on the mobilitiesof the ions. The effect of pH on the potentials and the pointof zero charge of the root-base segment show close similaritiesto earlier results obtained with whole roots.