Drug-induced and postnatal hypothyroidism impairs the accumulation of diacylglycerol in liver and liver cell plasma membranes

Background  

Thyroid hormones are well known modulators of signal transduction. The effect of hyper- and hypo-thyroidism on diacylglycerol/protein kinase C (DAG/PKC) signaling in cardiomiocytes has been determined. Triiodothyronine (T₃) has been shown to prevent the α1-adrenoreceptor-mediated activation of PKC but does not alter the stimulation of enzyme and hepatic metabolism by phorbol ethers. It has been suggested that the elevation of endogenous DAG in senescent or hypothyroid cells changes the PKC-dependent response of cells to phorbol esters and hormones. In the present study, was examined the formation of DAG and activation of PKC in liver cells from rats of different thyroid status.     

Insights into the behavior of unsaturated diacylglycerols in mixed lipid bilayers in relation to protein kinase C activation—A molecular dynamics simulation study

The lipid second messenger diacylglycerol (DAG) is known for its involvement in many types of cellular signaling, especially as an endogenous agonist for protein kinase C (PKC). Evidence has emerged that the degree of saturation of the DAG molecules can affect PKC activation. DAG molecules with different acyl chain saturation have not only been observed to induce varying extents of PKC activation, but also to express selectivity towards different PKC isozymes. Both qualities are important for precise therapeutic activation of PKC; understanding DAG behavior at the molecular level in different environments has much potential in the development of drugs to target PKC. We used molecular dynamics simulations to study the behavior of two different unsaturated DAG species in lipid environments with varying degrees of unsaturation. We focus on phosphatidylethanolamine (PE) instead of phosphatidylcholine (PC) to more accurately model the relevant biomembranes. The effect of cholesterol (CHOL) on these systems was also explored. We found that both high level of unsaturation in the acyl chains of the DAG species and presence of CHOL in the surrounding membrane increase DAG molecule availability at the lipid–water interface. This can partially explain the previously observed differences in PKC activation strength and specificity, the complete mechanism is, however, likely to be more complex. Our simulations coupled with the current understanding of lipids highlight the need for more simulations of biologically accurate lipid environments in order to determine the correct correlations between molecular mechanisms and biological behavior when studying PKC activation.     

Negative feedback regulation of Gq signaling by protein kinase C is disrupted by diacylglycerol kinase ζ in COS-7 cells

Cellular response to G(q)-linked agonists is shaped by regulatory inputs which determine signal strength and duration. Stimulation of phospholipase C-β (PLC-β) lipase activity results in an increase in the levels of diacylglycerol (DAG) and activation of protein kinase C (PKC) activity. PKC has been implicated in the feedback regulation of G(q) signaling through actions on PLC-β and phospholipase D (PLD) lipase activity. As PKC activity is modulated by multiple layers of regulation, the physiological impact of PKC on G(q) signaling is unclear. PKC signaling can be terminated by diacylglycerol kinases (DGKs) which are regulated in a cell-specific manner. The present studies investigated the contribution of the ubiquitously expressed DGKζ isoform in the regulation of PKC signaling and G(q) response in transfected COS-7 cells. Genetic depletion of DGKζ protein with antisense oligonucleotides dramatically reduced DAG metabolism. The sustained increase in PKC signaling was associated with a pronounced inhibition of carbachol-stimulated lipase activity in cells co-transfected with m1 muscarinic receptor, Gα(q) and either with or without PLC-β(1). The data also reveal that sustained activation of PKC alone does not increase cellular PLD1 activity. Therefore, G(12)-activated RhoA is physiologically important for adequate stimulation of PLD1 activity. These data show that the impact of PKC on G(q) signal transduction is determined by the background of cell-specific processes.     

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.     

Physiologic activation of protein kinase C limits IL-2 secretion

Interaction of Ag, antibodies against the T cell receptor complex, or mitogenic lectins with T lymphocytes induces hydrolysis of membrane phospholipids leading to the production of diacylglycerol (DAG). DAG then activates the Ca2+- and phospholipid-dependent phosphotransferase, protein kinase C (PKC). Increases in DAG concentrations are transient as is the increase in PKC activity. Phorbol esters, which induce potent, prolonged activation of PKC, augment many T lymphocyte responses, including cell proliferation and secretion of the T cell growth factor IL-2. Therefore, it has been suggested that activation of PKC is a positive regulatory signal in T lymphocytes. We have determined the consequences of transient stimulation of PKC, and of depletion of PKC, on early cell activation signals and on production of IL-2 by the murine lymphoma line LBRM 331A5. When this cell line is depleted of PKC overnight incubation in high concentrations of phorbol esters, lectin-induced IL-2 secretion is augmented. Similarly, mitogen-induced changes in [Ca2+]i and phosphoinositide metabolism were augmented in these cells. In contrast, a short preactivation of PKC abrogated these early transmembrane signaling events. This suggested that normal physiologic activation of PKC may limit cell activation and decrease IL-2 production. We compared the effects of phorbol esters and mezerein, which produce prolonged activation of PKC, with those of diacylglycerol analogs, which induce transient activation of PKC. At concentrations that give similar levels of PKC activation, phorbol esters and mezerein, but not DAG analogs, increased IL-2 secretion. This suggests that prolonged, nonphysiologic activation of PKC is required to augment IL-2 secretion. Therefore, physiologic activation of PKC may not augment T cell activation but instead may function to decrease cell activation and limit IL-2 secretion.     

Mitotic lamin disassembly is triggered by lipid-mediated signaling

Disassembly of the nuclear lamina is a key step during open mitosis in higher eukaryotes. The activity of several kinases, including CDK1 (cyclin-dependent kinase 1) and protein kinase C (PKC), has been shown to trigger mitotic lamin disassembly, yet their precise contributions are unclear. In this study, we develop a quantitative imaging assay to study mitotic lamin B1 disassembly in living cells. We find that CDK1 and PKC act in concert to mediate phosphorylation-dependent lamin B1 disassembly during mitosis. Using ribonucleic acid interference (RNAi), we showed that diacylglycerol (DAG)-dependent PKCs triggered rate-limiting steps of lamin disassembly. RNAi-mediated depletion or chemical inhibition of lipins, enzymes that produce DAG, delayed lamin disassembly to a similar extent as does PKC inhibition/depletion. Furthermore, the delay of lamin B1 disassembly after lipin depletion could be rescued by the addition of DAG. These findings suggest that lipins activate a PKC-dependent pathway during mitotic lamin disassembly and provide evidence for a lipid-mediated mitotic signaling event.     

Single cell analysis and temporal profiling of agonist-mediated inositol 1,4,5-trisphosphate, Ca2+, diacylglycerol, and protein kinase C signaling using fluorescent biosensors

The magnitude and temporal nature of intracellular signaling cascades can now be visualized directly in single cells by the use of protein domains tagged with enhanced green fluorescent protein (eGFP). In this study, signaling downstream of G protein-coupled receptor-mediated phospholipase C (PLC) activation has been investigated in a cell line coexpressing recombinant M(3) muscarinic acetylcholine and alpha(1B) -adrenergic receptors. Confocal measurements of changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)), using the pleckstrin homology domain of PLCdelta1 tagged to eGFP (eGFP-PH(PLCdelta)), and 1,2-diacylglycerol (DAG), using the C1 domain of protein kinase Cgamma (PKCgamma) (eGFP-C1(2)-PKCgamma), demonstrated clear translocation responses to methacholine and noradrenaline. Single cell EC(50) values calculated for each agonist indicated that responses to downstream signaling targets (Ca(2+) mobilization and PKC activation) were approximately 10-fold lower compared with respective Ins(1,4,5)P(3) and DAG EC(50) values. Examining the temporal profile of second messenger responses to sub-EC(50) concentrations of noradrenaline revealed oscillatory Ins(1,4,5)P(3), DAG, and Ca(2+) responses. Oscillatory recruitments of conventional (PKCbetaII) and novel (PKCepsilon) PKC isoenzymes were also observed which were synchronous with the Ca(2+) response measured simultaneously in the same cell. However, oscillatory PKC activity (as determined by translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate protein) required oscillatory DAG production. We suggest a model that uses regenerative Ca(2+) release via Ins(1,4,5)P(3) receptors to initiate oscillatory second messenger production through a positive feedback effect on PLC. By acting on various components of the PLC signaling pathway the frequency-encoded Ca(2+) response is able to maintain signal specificity at a level downstream of PKC activation.     

Involvement of PKCα in PMA-induced facilitation of exocytosis and vesicle fusion in PC12 cells

Phorbol-12-myristate-13-acetate, a stable analog of the important signaling membrane lipid diacylglycerol (DAG), is known to potentiate exocytosis and modulate vesicle fusion kinetics in neurons and endocrine cells. The exact mechanisms underlying the actions of PMA, however, is often not clear, largely because of the diversity of the DAG/PMA receptors involved in the exocytotic process, which include, most notably, various isoforms of protein kinase C (PKC). In this study, the roles of PKCα in PMA-mediated regulation of exocytosis were investigated by over-expressing wild-type PKCα (wt-PKCα) or dominant negative PKCα (dn-PKCα). Amperometric measurements based on carbon fiber microelectrodes demonstrated that PKCα has a key role in the PMA-mediated facilitation of exocytosis and vesicle fusion in neuroendocrine PC12 cells.     

Diacylglycerol kinase-epsilon restores cardiac dysfunction under chronic pressure overload: a new specific regulator of Galpha(q) signaling cascade

Galpha(q) protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in cardiac hypertrophy. DAG kinase (DGK) catalyzes DAG phosphorylation and controls cellular DAG levels, thus acting as a regulator of GPCR signaling. It has been reported that DGKepsilon acts specifically on DAG produced by inositol cycling. In this study, we examined whether DGKepsilon prevents cardiac hypertrophy and progression to heart failure under chronic pressure overload. We generated transgenic mice with cardiac-specific overexpression of DGKepsilon (DGKepsilon-TG) using an alpha-myosin heavy chain promoter. There were no differences in cardiac morphology and function between wild-type (WT) and DGKepsilon-TG mice at the basal condition. Either continuous phenylephrine infusion or thoracic transverse aortic constriction (TAC) was performed in WT and DGKepsilon-TG mice. Increases in heart weight after phenylephrine infusion and TAC were abolished in DGKepsilon-TG mice compared with WT mice. Cardiac dysfunction after TAC was prevented in DGKepsilon-TG mice, and the survival rate after TAC was higher in DGKepsilon-TG mice than in WT mice. Phenylephrine- and TAC-induced DAG accumulation, the translocation of PKC isoforms, and the induction of fetal genes were blocked in DGKepsilon-TG mouse hearts. The upregulation of transient receptor potential channel (TRPC)-6 expression after TAC was attenuated in DGKepsilon-TG mice. In conclusion, these results demonstrate the first evidence that DGKepsilon restores cardiac dysfunction and improves survival under chronic pressure overload by controlling cellular DAG levels and TRPC-6 expression. DGKepsilon may be a novel therapeutic target to prevent cardiac hypertrophy and progression to heart failure.     

Defining the role of DAG,mitochondrial function,and lipid deposition in palmitate-induced proinflammatory signaling and its counter-modulation by palmitoleate

Chronic exposure of skeletal muscle to saturated fatty acids, such as palmitate (C16:0), enhances proinflammatory IKK-NFκB signaling by a mechanism involving the MAP kinase (Raf-MEK-ERK) pathway. Raf activation can be induced by its dissociation from the Raf-kinase inhibitor protein (RKIP) by diacylglycerol (DAG)-sensitive protein kinase C (PKC). However, whether these molecules mediate the proinflammatory action of palmitate, an important precursor for DAG synthesis, is currently unknown. Here, involvement of DAG-sensitive PKCs, RKIP, and the structurally related monounsaturated fatty acid palmitoleate (C16:1) on proinflammatory signaling are investigated. Palmitate, but not palmitoleate, induced phosphorylation/activation of the MEK-ERK-IKK axis and proinflammatory cytokine (IL-6, CINC-1) expression. Palmitate increased intramyocellular DAG and invoked PKC-dependent RKIP^Ser153 phosphorylation, resulting in RKIP-Raf1 dissociation and MEK-ERK signaling. These responses were mimicked by PMA, a DAG mimetic and PKC activator. However, while pharmacological inhibition of PKC suppressed PMA-induced activation of MEK-ERK-IKK signaling, activation by palmitate was upheld, suggesting that DAG-sensitive PKC and RKIP were dispensable for palmitate''s proinflammatory action. Strikingly, the proinflammatory effect of palmitate was potently repressed by palmitoleate. This repression was not due to reduced palmitate uptake but linked to increased neutral lipid storage and enhanced cellular oxidative capacity brought about by palmitoleate''s ability to restrain palmitate-induced mitochondrial dysfunction.     

Exercise-induced cardiac performance in autoimmune (Type 1) diabetes is associated with a decrease in myocardial diacylglycerol

One of the fundamental biochemical defects underlying the complications of diabetic cardiovascular system is elevation of diacylglycerol (DAG) and its effects on protein kinase C (PKC) signaling. It has been noted that exercise training attenuates poor cardiac performance in Type 1 diabetes. However, the role of PKC signaling in exercise-induced alleviation of cardiac abnormalities in diabetes is not clear. We investigated the possibility that exercise training modulates PKC-βII signaling to elicit its beneficial effects on the diabetic heart. bio-breeding diabetic resistant rats, a model reminiscent of Type 1 diabetes in humans, were randomly assigned to four groups: 1) nonexercised nondiabetic (NN); 2) nonexercised diabetic (ND); 3) exercised nondiabetic; and 4) exercised diabetic. Treadmill training was initiated upon the onset of diabetes. At the end of 8 wk, left ventricular (LV) hemodynamic assessment revealed compromised function in ND compared with the NN group. LV myocardial histology revealed increased collagen deposition in ND compared with the NN group, while electron microscopy showed a reduction in the viable mitochondrial fraction. Although the PKC-βII levels and activity were unchanged in the diabetic heart, the DAG levels were increased. With exercise training, the deterioration of LV structure and function in diabetes was attenuated. Notably, improved cardiac performance in training was associated with a decrease in myocardial DAG levels in diabetes. Exercise-induced benefits on cardiac performance in diabetes may be mediated by prevention of an increase in myocardial DAG levels.     

Hepatic protein kinase C is not activated despite high intracellular 1,2-sn-diacylglycerol in obese Zucker rats

High intracellular 1,2,-sn-diacylglycerol (DAG) usually activates protein kinase C (PKC). In choline-deficient Fischer 344 rats, we previously showed that fatty liver was associated with elevated hepatic DAG and sustained activation of PKC. Steatosis is a sequelae of many liver toxins, and we wanted to determine whether fatty liver is always associated with accumulation of DAG with activation of PKC. Obese Zucker rats had 11-fold more triacylglycerol in their livers and 2-fold more DAG in their hepatic plasma membrane than did lean control Zucker rats. However, this increased diacylglycerol was not associated with translocation or activation of PKC in hepatic plasma membrane (activity in obese rats was 897 pmol/mg protein×min⁻¹ vs. 780 pmol/mg protein×min⁻¹ in lean rats). No differences in PKC isoform expression were detected between obese and lean rats. In additional studies, we found that choline deficiency in the Zucker rat did not result in activation of PKC in liver, unlike our earlier observations in the choline deficient Fischer rat. This dissociation between fatty liver, DAG accumulation and PKC activation in Zucker rats supports previous reports of abnormalities in PKC signaling in this strain of rats.     

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.     

干预DAG-PKC信号转导通路对糖尿病大鼠心肌细胞JNK1和IRS1基因表达的影响

目的干预二酰甘油-蛋白激酶C(DAG-PKC)信号转导通路后观察JNK1、IRS1等在糖尿病大鼠心肌中的表达情况。方法采用HE染色,masson染色、电镜观察大鼠心肌的病理变化,应用免疫组化、Real-Time PCR检测PKCβ2、JNK1及IRS1在大鼠心肌的表达情况。结果糖尿病模型组PKCβ2、JNK1、p-JNK、IRS1表达明显高于对照组,干预DAG-PKC信号转导通路后明显下调其表达水平。结论 DAG-PKC通路可能是通过G蛋白受体和胰岛素受体途径的共同信号点JNK1影响下游的信号传导而导致糖尿病心肌病的发生发展,DAG-PKC-JNK1-IRS1-Akt/PKB-mTOR-p70S6K1等一系列信号位点可能是DAG-PKC信号转导通路引起糖尿病心肌病可能的潜在途径。     

Intramolecular occlusion of the diacylglycerol-binding site in the C1 domain of munc13-1

Protein kinase C (PKC) isozymes and other receptors of diacylglycerol (DAG) bind to this widespread second messenger through their C(1) domains. These alternative DAG receptors include munc13-1, a large neuronal protein that is crucial for DAG-dependent augmentation of neurotransmitter release. Whereas the structures of several PKC C(1) domains have been determined and have been shown to require little conformational changes for ligand binding, it is unclear whether the C(1) domains from other DAG receptors contain specific structural features with key functional significance. To gain insight into this question, we have determined the three-dimensional structure in solution of the munc13-1 C(1) domain using NMR spectroscopy. The overall structure includes two beta-sheets, a short C-terminal alpha-helix, and two Zn(2+)-binding sites, resembling the structures of PKC C(1) domains. However, the munc13-1 C(1) domain exhibits striking structural differences with the PKC C(1) domains in the ligand-binding site. These differences result in occlusion of the binding site of the munc13-1 C(1) domain by a conserved tryptophan side chain that in PKCs adopts a completely different orientation. As a consequence, the munc13-1 C(1) domain requires a considerable conformational change for ligand binding. This structural distinction is expected to decrease the DAG affinity of munc13-1 compared to that of PKCs, and is likely to be critical for munc13-1 function. On the basis of these results, we propose that augmentation of neurotransmitter release may be activated at higher DAG levels than PKCs as a potential mechanism for uncoupling augmentation of release from the multitude of other signaling processes mediated by DAG.     

Activation and translocation of PKCdelta is necessary for VEGF-induced ERK activation through KDR in HEK293T cells

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-gamma-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCdelta knock-down using siRNA method showed that PKCdelta played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCdelta specific inhibitor, or human PKCdelta siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCdelta-knockdown HEK293T cells, but co-expression of rat PKCdelta-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCdelta-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCdelta-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCdelta-knockdown HEK293T cell. These results indicate that PKCdelta is involved in VEGF-KDR-induced ERK activation via C1B domain.     

PDGF initiates two distinct phases of protein kinase C activity that make unequal contributions to the G0 to S transition

BACKGROUND: Platelet-derived growth factor (PDGF) promotes cell-cycle progression by engaging signaling enzymes such as phospholipase Cgamma (PLCgamma). When activated, PLCgamma cleaves phosphatidylinositol-4,5-bisphosphate to produce inositol-1,4, 5-trisphosphate (IP(3)) and diacylglycerol (DAG). IP(3) stimulates the release of calcium from intracellular stores, which together with DAG activate some protein kinase C (PKC) family members. In this study we focused on putative downstream effectors of PLCgamma - PKC family members. We investigated whether, and when, DAG-responsive PKCs contribute to PDGF-dependent DNA synthesis. RESULTS: In HepG2 cells expressing wild-type PDGF beta receptors (betaPDGFRs), PDGF activated at least one PKC family member (PKCepsilon) at two distinct times - within 10 minutes after PDGF stimulation, and then for a longer duration between 5 and 9 hours. Blocking the early burst of PKC activity had no effect on PDGF-dependent DNA synthesis. In contrast, the DNA-synthesis response was reduced by 60-80% when the second phase of PKC activity was blocked. Similarly, DAG rescued PDGF-dependent DNA synthesis in the cells expressing a mitogenically incompetent mutant betaPDGFR, but only when DAG was added at times corresponding to the late phase of PKC activity. Our studies also indicate that the late phase of PKCepsilon activity can be induced by either phosphoinositide 3-kinase-dependent or DAG-dependent pathways in PDGF-stimulated HepG2 cells. CONCLUSIONS: We conclude that PDGF activates PKCs at two distinct times and that these two intervals of PKC activity make unequal contributions to the mitogenic response. The late phase of PKC activity is required for PDGF-dependent DNA synthesis, whereas the early phase of activity is dispensable.     

C1 domains exposed: from diacylglycerol binding to protein-protein interactions

C1 domains, cysteine-rich modules originally identified in protein kinase C (PKC) isozymes, are present in multiple signaling families, including PKDs, chimaerins, RasGRPs, diacylglycerol kinases (DGKs) and others. Typical C1 domains bind the lipid second messenger diacylglycerol (DAG) and DAG-mimetics such as phorbol esters, and are critical for governing association to membranes. On the contrary, atypical C1 domains possess structural determinants that impede phorbol ester/DAG binding. C1 domains are generally expressed as twin modules (C1A and C1B) or single domains. Biochemical and cellular studies in PKC and PKD isozymes revealed that C1A and C1B domains are non-equivalent as lipid-binding motifs or translocation modules. It has been recently determined that individual C1 domains have unique patterns of ligand recognition, driven in some cases by subtle structural differences. Insights from recent 3-D studies on beta2-chimaerin and Munc13-1 revealed that their single C1 domains are sterically blocked by intramolecular interactions, suggesting that major conformational changes would be required for exposing the site of DAG interaction. Thus, it is clear that the protein context plays a major role in determining whether binding of DAG to the C1 domain would lead to enzyme activation or merely serves as an anchoring mechanism.     

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.     

Sealing of Transected Neurites of Rat B104 Cells Requires a Diacylglycerol PKC-Dependent Pathway and a PKA-Dependent Pathway

To survive, neurons and other eukaryotic cells must rapidly repair (seal) plasmalemmal damage. Such repair occurs by an accumulation of intracellular vesicles at or near the plasmalemmal disruption. Diacylglycerol (DAG)-dependent and cAMP-dependent proteins are involved in many vesicle trafficking pathways. Although recent studies have implicated the signaling molecule cAMP in sealing, no study has investigated how DAG and DAG-dependent proteins affect sealing. By means of dye exclusion to assess Ca²⁺-dependent vesicle-mediated sealing of transected neurites of individually identifiable rat hippocampal B104 cells, we now report that, compared to non-treated controls, sealing probabilities and rates are increased by DAG and cAMP analogs that activate PKC and Munc13-1 and PKA. Sealing is decreased by inhibiting DAG-activated novel protein kinase C isozymes ?? (nPKC??) and ?? (nPKC??) and Munc13-1, the PKC effector myristoylated alanine rich PKC substrate (MARCKS) or phospholipase C (PLC). DAG-increased sealing is prevented by inhibiting MARCKS or protein kinase A (PKA). Sealing probability is further decreased by simultaneously inhibiting nPKC??, nPKC??, and PKA. Extracellular Ca²⁺, DAG, or cAMP analogs do not affect this decrease in sealing. These and other data suggest that DAG increases sealing through MARCKS and that nPKC??, nPKC??, and PKA are all required to seal plasmalemmal damage in B104 and likely all eukaryotic cells.