Protein kinase C alpha phosphorylates and negatively regulates diacylglycerol kinase zeta

Diacylglycerol kinase (DGK) terminates diacylglycerol (DAG) signaling by phosphorylating DAG to produce phosphatidic acid, which also has signaling properties. Thus, precise control of DGK activity is essential for proper signal transduction. We demonstrated previously that a peptide corresponding to the myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation site domain (PSD) in DGK zeta was phosphorylated in vitro by an active fragment of protein kinase C (PKC). In the present study, we tested full-length DGK zeta and found that PKC alpha phosphorylated DGK zeta on serines within the MARCKS PSD in vitro and in vivo. DGK zeta also coimmunoprecipitated with PKC alpha, suggesting that they reside in a regulated signaling complex. We then tested whether phosphorylation affected DAG kinase activity. We found that a mutant (DGK zeta S/D) in which serines within the MARCKS PSD were altered to aspartates (to mimic phosphorylation) had lower activity compared with wild-type DGK zeta or a control mutant (DGK zeta S/N) in which the same serines were changed to asparagines. Furthermore, activation of PKC alpha by phorbol 12-myristate 13-acetate inhibited the activity of wild-type DGK zeta, but not DGK zeta S/D, in human embryonic kidney 293 cells. These results suggest that by phosphorylating the MARCKS PSD, PKC alpha attenuates DGK zeta activity. Supporting this, we found that cells expressing DGK zeta S/D had higher DAG levels and grew more rapidly compared with cells expressing DGK zeta S/N that could not be phosphorylated. Taken together, these results indicate that PKC alpha phosphorylates DGK zeta in cells, and this phosphorylation inhibits its kinase activity to remove cellular DAG, thereby affecting cell growth.     

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.     

Protein kinase C inhibits binding of diacylglycerol kinase-zeta to the retinoblastoma protein

We previously showed that the retinoblastoma protein (pRB), a key regulator of G1 to S-phase transition of the cell cycle, binds to and stimulates diacylglycerol kinase-zeta (DGKzeta) to phosphorylate the lipid second messenger diacylglycerol into phosphatidic acid. pRB binds to the MARCKS phosphorylation-site domain of DGKzeta that can be phosphorylated by protein kinase C (PKC). Here, we report that activation of PKC by phorbol ester inhibits DGKzeta binding to pRB. Ro 31-8220, a specific inhibitor of PKC, alleviated this inhibition of binding. Mimicking of PKC phosphorylation of serine residues (by S/D but not S/N mutations) within the DGKzeta-MARCKS phosphorylation-site domain also prevented DGKzeta binding to pRB, suggesting that PKC phosphorylation of these residues negatively regulates the interaction between DGKzeta and pRB. In PKC overexpression studies, it appeared that activation of particularly the (wild-type) PKCalpha isoform inhibits DGKzeta binding to pRB, whereas dominant-negative PKCalpha neutralized this inhibition. PKCalpha activation thus prevents DGKzeta regulation by pRB, which may have implications for nuclear diacylglycerol and phosphatidic acid levels during the cell cycle.     

Arachidonic acid and diacylglycerol act synergistically to activate protein kinase C in vitro and in vivo

Using a well-defined model membrane bilayer system, incorporation of both lipid second messengers, 1,2-diacylglycerol and arachidonic acid, at submaximal activating concentrations, resulted in a synergistic activation of protein kinase C in a Ca2+/phosphatidylserine-dependent manner as measured by monitoring phosphorylation of phosphoprotein substrates. The arachidonic acid appears to modulate membrane properties both at the hydrocarbon core and the membrane surface increasing the availability of the diacylglycerol which can bind to and subsequently activate the enzyme. Co-application of these two lipid activators to the Hermissenda photoreceptor reduced K+ channel conductance in a synergistic manner via a PKC-dependent pathway. Thus, these in vivo and in vitro studies suggest that the membrane bilayer properties of these PKC lipid activators interact to specifically regulate the cellular lipid microenvironment resulting in PKC activation.     

Regulation of T cell receptor-induced activation of the Ras-ERK pathway by diacylglycerol kinase zeta

T cell development in the thymus and activation of mature T cells in the periphery depend on signals stimulated by engagement of the T cell antigen receptor (TCR). Among the second messenger cascades initiated by TCR ligation include the phosphatidylinositol pathway where the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, is hydrolyzed to inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Inositol 1,4,5-trisphosphate signals a rise in intracellular free calcium, leading to translocation of nuclear factor of activated T cells into the nucleus. DAG activates RasGRP and protein kinase C theta. Because both RasGRP and protein kinase C theta are essential for thymocyte and T cell function, it is critical to understand how DAG is regulated. In this report, we demonstrate expression of DAG kinase zeta (DGKzeta, the enzyme that catalyzes the conversion of DAG to phosphatidic acid) in multiple lymphoid organs, with highest expression observed within the T cell compartment. Overexpression studies in Jurkat T cells indicate that DGKzeta interferes with TCR-induced Ras and ERK activation, AP-1 induction, and expression of the activation marker CD69. In contrast, TCR-stimulated calcium influx is not altered. Mutational analysis indicates that the kinase and DAG binding domains, but not the ankyrin repeats of DGKzeta, are required for its inhibitory effects. Collectively these studies demonstrate a potential role of DGKzeta to function as a selective negative regulator of DAG signaling on T cell activation and provide the first structure/function analysis of this enzyme in T cells.     

Cardiac-specific overexpression of diacylglycerol kinase zeta attenuates left ventricular remodeling and improves survival after myocardial infarction

Left ventricular (LV) remodeling, including cardiomyocyte necrosis, scar formation, LV geometric changes, and cardiomyocyte hypertrophy, contributes to cardiac dysfunction and mortality after myocardial infarction (MI). Although precise cellular signaling mechanisms for LV remodeling are not fully elucidated, G(q) protein-coupled receptor signaling pathway, including diacylglycerol (DAG) and PKC, are involved in this process. DAG kinase (DGK) phosphorylates DAG and controls cellular DAG levels, thus acting as a negative regulator of PKC and subsequent cellular signaling. We previously reported that DGK inhibited angiotensin II and phenylephrine-induced activation of the DAG-PKC signaling and subsequent cardiac hypertrophy. The purpose of this study was to examine whether DGK modifies LV remodeling after MI. Left anterior descending coronary artery was ligated in transgenic mice with cardiac-specific overexpression of DGKzeta (DGKzeta-TG) and wild-type (WT) mice. LV chamber dilatation (4.12 +/- 0.10 vs. 4.53 +/- 0.32 mm, P < 0.01), reduction of LV systolic function (34.8 +/- 8.3% vs. 28.3 +/- 4.8%, P < 0.01), and increases in LV weight (95 +/- 3.6 vs. 111 +/- 4.1 mg, P < 0.05) and lung weight (160 +/- 15 vs. 221 +/- 25 mg, P < 0.05) at 4 wk after MI were attenuated in DGKzeta-TG mice compared with WT mice. In the noninfarct area, fibrosis fraction (0.51 +/- 0.04, P < 0.01) and upregulation of profibrotic genes, such as transforming growth factor-beta1 (P < 0.01), collagen type I (P < 0.05), and collagen type III (P < 0.01), were blocked in DGKzeta-TG mice. The survival rate at 4 wk after MI was higher in DGKzeta-TG mice than in WT mice (61% vs. 37%, P < 0.01). In conclusion, these results demonstrate the first evidence that DGKzeta suppresses LV structural remodeling and fibrosis and improves survival after MI. DGKzeta may be a potential novel therapeutic target to prevent LV remodeling after MI.     

The rickettsia surface cell antigen 4 applies mimicry to bind to and activate vinculin

Pathogenic Rickettsia species cause high morbidity and mortality, especially R. prowazekii, the causative agent of typhus. Like many intracellular pathogens, Rickettsia exploit the cytoskeleton to enter and spread within the host cell. Here we report that the cell surface antigen sca4 of Rickettsia co-localizes with vinculin in cells at sites of focal adhesions in sca4-transfected cells and that sca4 binds to and activates vinculin through two vinculin binding sites (VBSs) that are conserved across all Rickettsia. Remarkably, this occurs through molecular mimicry of the vinculin-talin interaction that is also seen with the IpaA invasin of the intracellular pathogen Shigella, where binding of these VBSs to the vinculin seven-helix bundle head domain (Vh1) displaces intramolecular interactions with the vinculin tail domain that normally clamp vinculin in an inactive state. Finally, the vinculin·sca4-VBS crystal structures reveal that vinculin adopts a new conformation when bound to the C-terminal VBS of sca4. Collectively, our data define the mechanism by which sca4 activates vinculin and interacts with the actin cytoskeleton, and they suggest important roles for vinculin in Rickettsia pathogenesis.     

Erythropoietin and interleukin-2 activate distinct JAK kinase family members.

The erythropoietin (EPO) receptor and the interleukin-2 (IL-2) receptor beta-chain subunit are members of the cytokine receptor superfamily. They have conserved primary amino acid sequences in their cytoplasmic domains and activate phosphorylation of common substrates, suggesting common biochemical signaling mechanisms. We have generated a cell line, CTLL-EPO-R, that contains functional cell surface receptors for both EPO and IL-2. CTLL-EPO-R cells demonstrated similar growth kinetics in EPO and IL-2. Stimulation with EPO resulted in the rapid, dose-dependent tyrosine phosphorylation of JAK2. In contrast, stimulation with IL-2 or the related cytokine IL-4 resulted in the rapid, dose-dependent tyrosine phosphorylation of JAK1 and an additional 116-kDa protein. This 116-kDa protein was itself immunoreactive with a polyclonal antiserum raised against JAK2 and appears to be a novel member of the JAK kinase family. Immune complex kinase assays confirmed that IL-2 and IL-4 activated JAK1 and EPO activated JAK2. These results demonstrate that multiple biochemical pathways are capable of conferring a mitogenic signal in CTLL-EPO-R cells and that the EPO and IL-2 receptors interact with distinct JAK kinase family members within the same cellular background.     

The sustained second phase of hormone-stimulated diacylglycerol accumulation does not activate protein kinase C in GH3 cells

Numerous hormones activate cells through receptor-regulated hydrolysis of phosphoinositides resulting in elevated cellular diacylglycerol (DAG), an activator of protein kinase C (PKC). Our previous studies showed that thyrotropin-releasing hormone (TRH) treatment of GH3 cells stimulated a rapid (less than 10 s) but transient (less than 60 s) association of cytosolic PKC with the membrane. In this study, we investigated the roles of hormone-stimulated Ca2+ and DAG levels in initiating and terminating the membrane association of PKC. The initial effects of TRH were not mimicked by elevating CA2+ levels, however, inhibiting TRH-stimulated Ca2+ increases blocked hormone-stimulated PKC translocation. Hence, the TRH stimulation of both Ca2+ and DAG levels were essential for the initial PKC translocation. The termination of PKC membrane association could not be attributed to proteolysis of PKC nor to limiting Ca2+ levels. Treatment of cells with phorbol diesters potentiated and prolonged the effects of TRH on PKC translocation, suggesting that DAG levels limited the membrane association of PKC. Since TRH stimulated a sustained increase in DAG levels, DAG composition was analyzed. There was a marked shift in DAG from tetraenoic (at 15 s) to more saturated DAGs at longer times. In addition, increases in plasma membrane DAG in response to TRH were transient rather than sustained. We propose that the TRH stimulation of PKC translocation is short-lived due to the metabolism of plasma membrane DAGs which are effective in promoting PKC activation. In contrast, DAGs which accumulate in intracellular membranes during the sustained phase of TRH treatment appear to be ineffective as activators of PKC.     

Designing ligands to bind proteins

The ability to design drugs (so-called 'rational drug design') has been one of the long-term objectives of chemistry for 50 years. It is an exceptionally difficult problem, and many of its parts lie outside the expertise of chemistry. The much more limited problem - how to design tight-binding ligands (rational ligand design) - would seem to be one that chemistry could solve, but has also proved remarkably recalcitrant. The question is 'Why is it so difficult?' and the answer is 'We still don't entirely know'. This perspective discusses some of the technical issues - potential functions, protein plasticity, enthalpy/entropy compensation, and others - that contribute, and suggests areas where fundamental understanding of protein-ligand interactions falls short of what is needed. It surveys recent technological developments (in particular, isothermal titration calorimetry) that will, hopefully, make now the time for serious progress in this area. It concludes with the calorimetric examination of the association of a series of systematically varied ligands with a model protein. The counterintuitive thermodynamic results observed serve to illustrate that, even in relatively simple systems, understanding protein-ligand association is challenging.     

Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA.

Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2-dioleoyl-sn-glycerol is observed in the absence of added Ca2+. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2-Dioleoyl-sn-glycerol eliminates the Ca(2+)-dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2-dioleoyl-sn-glycerol in this respect. This suggests that the 1,2-dioleoyl-sn-glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2-dioleoyl-sn-glycerol on the phosphatidic-acid-stimulated protein kinase C activity is dependent on the molar fraction of 1,2-dioleoyl-sn-glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2-dioleoyl-sn-glycerol concentrations to calcium inhibition at higher concentrations of 1,2-dioleoyl-sn-glycerol. Phosphatidylserine-stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2-dioleoyl-sn-glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2+ to high affinity sites on the enzyme. Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2-dioleoyl-sn-glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2+. There is no isozyme specificity in this binding. These results suggest that a less-tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.     

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.     

The multigenic family of monogalactosyl diacylglycerol synthases

Because the synthesis of monogalactosyldiacylglycerol (MGDG) is unique to plants, identified as an important marker of the plastid envelope, involved in a key step of plastid biogenesis and is the most abundant lipid on earth, MGDG synthase activity was extensively analysed at the biochemical and physiological levels. In the present paper, we present our current knowledge on the MGDG synthase's function, structure and topology in envelope membranes, and discuss possible roles in plant cell glycerolipid metabolism. The recent discovery of a multigenic family of MGDG synthases raised the possibility that multiple isoenzymes might carry out MGDG synthesis in various tissues and developmental stages.     

Regulation of neurite outgrowth in N1E-115 cells through PDZ-mediated recruitment of diacylglycerol kinase zeta

Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase zeta (DGK-zeta), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-zeta and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-zeta in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-zeta kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-zeta mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-zeta promotes neurite outgrowth through association with the GTPase Rac1. DGK-zeta colocalized with Rac1 in neuronal processes and DGK-zeta-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-zeta directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-zeta mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1(V12)) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-zeta with Rac1(V12), suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-zeta binding to active Rac1. Collectively, these findings suggest DGK-zeta, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells.     

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.     

Specific postsynaptic density proteins bind tubulin and calmodulin-dependent protein kinase type II

Cytoskeletal interactions which contribute to the assembly of the postsynaptic density (PSD) were investigated. PSDs bound 125I-tubulin specifically with an apparent Km of 2 X 10(-7) M and a Bmax of about 1 nmol/mg of protein. 125I-Tubulin blots revealed that a group of polypeptides between Mr 135,000 and 147,000 (P-140) was a major tubulin-binding PSD component. The P-140 polypeptides were highly enriched in the PSD fraction of purified synaptosomes and could not be detected in crude brain cytoplasm preparations. These polypeptides were subject to phosphorylation by endogenous calmodulin-dependent protein kinase type II, with a concomitant reduction in 125I-tubulin binding. The tubulin-binding polypeptides could also associate with the radiolabeled alpha- and beta-subunits of the calmodulin-dependent protein kinase. These observations are consistent with a role for the P-140 polypeptides in organizing the molecular structure of the PSD. The data also suggest that this structure may be modified by Ca2+-sensitive phosphorylation, thus permitting neuronal activity to modulate the cytoskeletal interactions of the PSD.     

Carbonyl compounds cross-link cellular proteins and activate protein-tyrosine kinase p60c-Src

Glyoxal, a dicarbonyl compound, is produced under oxidative stress by the autoxidation of glucose and reacts with the protein amino group to form Schiff base. In vitro treatment of murine thymocytes and fibroblasts with glyoxal induced extensive tyrosine phosphorylation of multiple proteins, which was drastically inhibited by the addition of OPB-9195, an inhibitor of the carbonyl reaction with proteins. Glyoxal induced cross-linking of a number of cellular proteins, including glycosylphosphatidylinositol (GPI)-anchored cell surface Thy-1. We then demonstrated that treatment of cells with glyoxal promptly induced activation of non-receptor protein-tyrosine kinase c-Src, which was partially inhibited by OPB-9195. It is suggested from these results that carbonyl amine reaction quickly activates c-Src, possibly through cross-linkage of GPI-anchored proteins or putative specific receptors.