Identification and characterization of two splice variants of human diacylglycerol kinase eta

Diacylglycerol kinase (DGK) participates in regulating the intracellular concentrations of two bioactive lipids, diacylglycerol and phosphatidic acid. DGK eta (eta 1, 128 kDa) is a type II isozyme containing a pleckstrin homology domain at the amino terminus. Here we identified another DGK eta isoform (eta 2, 135 kDa) that shared the same sequence with DGK eta 1 except for a sterile alpha motif (SAM) domain added at the carboxyl terminus. The DGK eta 1 mRNA was ubiquitously distributed in various tissues, whereas the DGK eta 2 mRNA was detected only in testis, kidney, and colon. The expression of DGK eta 2 was suppressed by glucocorticoid in contrast to the marked induction of DGK eta 1. DGK eta 2 was shown to form through its SAM domain homo-oligomers as well as hetero-oligomers with other SAM-containing DGKs (delta 1 and delta 2). Interestingly, DGK eta 1 and DGK eta 2 were rapidly translocated from the cytoplasm to endosomes in response to stress stimuli. In this case, DGK eta 1 was rapidly relocated back to the cytoplasm upon removal of stress stimuli, whereas DGK eta 2 exhibited sustained endosomal association. The experiments using DGK eta mutants suggested that the oligomerization of DGK eta 2 mediated by its SAM domain was largely responsible for its sustained endosomal localization. Similarly, the oligomerization of DGK eta 2 was suggested to result in negative regulation of its catalytic activity. Taken together, alternative splicing of the human DGK eta gene generates at least two isoforms with distinct biochemical and cell biological properties responding to different cellular metabolic requirements.     

Dynamics of diacylglycerol kinase zeta translocation in living T-cells. Study of the structural domain requirements for translocation and activity

The diacylglycerol kinases (DGK) regulate diacylglycerol-based signals by phosphorylating this key lipid intermediate to phosphatidic acid. Here, we have investigated the spatial and temporal regulation of diacylglycerol kinase zeta (DGK zeta) in living Jurkat T-cells expressing a muscarinic type I receptor. Using real time confocal videomicroscopy, we show the rapid translocation of a green fluorescent protein-tagged enzyme from the cytosol to the plasma membrane following receptor stimulation. The generation of a panel of truncations, deletions, and point mutations of the enzyme allowed us to examine the requirements of the different structural motifs for both activity and receptor-regulated translocation. The data show that DGK zeta has strict requirements for intact zinc fingers and the conserved catalytic domain for full enzymatic activity. Protein kinase C-driven myristoylated alanine-rich C kinase substrate domain phosphorylation and intact zinc fingers are in turn essential for plasma membrane translocation. DGK zeta does not translocate to the membrane following stimulation of the endogenous T-cell receptor, and our data demonstrate that the specificity in terms of receptor response is provided by the regulatory motifs present at the C-terminal domain of the protein. This is the first report that shows in vivo DGK zeta translocation in response to agonist stimulation and establishes the role of the different domains in enzymatic activity and the selectivity of the response to receptors.     

Multiple polymer architectures of human polyhomeotic homolog 3 sterile alpha motif

The self‐association of sterile alpha motifs (SAMs) into a helical polymer architecture is a critical functional component of many different and diverse array of proteins. For the Drosophila Polycomb group (PcG) protein Polyhomeotic (Ph), its SAM polymerization serves as the structural foundation to cluster multiple PcG complexes, helping to maintain a silenced chromatin state. Ph SAM shares 64% sequence identity with its human ortholog, PHC3 SAM, and both SAMs polymerize. However, in the context of their larger protein regions, PHC3 SAM forms longer polymers compared with Ph SAM. Motivated to establish the precise structural basis for the differences, if any, between Ph and PHC3 SAM, we determined the crystal structure of the PHC3 SAM polymer. PHC3 SAM uses the same SAM–SAM interaction as the Ph SAM sixfold repeat polymer. Yet, PHC3 SAM polymerizes using just five SAMs per turn of the helical polymer rather than the typical six per turn observed for all SAM polymers reported to date. Structural analysis suggested that malleability of the PHC3 SAM would allow formation of not just the fivefold repeat structure but also possibly others. Indeed, a second PHC3 SAM polymer in a different crystal form forms a sixfold repeat polymer. These results suggest that the polymers formed by PHC3 SAM, and likely others, are dynamic. The functional consequence of the variable PHC3 SAM polymers may be to create different chromatin architectures. Proteins 2014; 82:2823–2830. © 2014 Wiley Periodicals, Inc.     

Shh signaling, negatively regulated by BMP signaling, inhibits the osteo/dentinogenic differentiation potentials of mesenchymal stem cells from apical papilla

Type I diacylglycerol kinase (DGK) isozymes (α, β, and γ) contain recoverin homology domains and calcium-binding EF-hand motifs at their N-termini. The γ-isoform of DGK is abundantly expressed in retinal and Purkinje cells; however, its function in neuronal cells remains unknown. Here, we report that the mRNA and protein levels of DGKγ, but not DGKα or β, were markedly increased in N1E-115 neuroblastoma cells upon cellular differentiation by serum starvation. Interestingly, overexpression of wild-type DGKγ, which was partially located at the plasma membrane, considerably induced the formation of slender, filopodia-like cytoplasmic projections from N1E-115 cell bodies. Deletion of the recoverin homology domain and the EF-hand motifs, which potentiated the plasma membrane localization of the isozyme, significantly enhanced the formation of the filopodia-like protrusions. Intriguingly, the catalytic activity of the isozyme is not essential for the protrusion formation. The N-terminal half of the catalytic domain and a short stretch of amino acid residues at the C-terminus are responsible for plasma membrane localization and filopodia-like process formation. Taken together, we have described a potentially novel morphological function of the C-terminal DGKγ catalytic region that is independent of its enzymatic activity.     

Diacylglycerol Kinase δ Suppresses ER-to-Golgi Traffic via Its SAM and PH Domains

We report here that the anterograde transport from the endoplasmic reticulum (ER) to the Golgi was markedly suppressed by diacylglycerol kinase delta (DGKdelta) that uniquely possesses a pleckstrin homology (PH) and a sterile alpha motif (SAM) domain. A low-level expression of DGKdelta in NIH3T3 cells caused redistribution into the ER of the marker proteins of the Golgi membranes and the vesicular-tubular clusters (VTCs). In this case DGKdelta delayed the ER-to-Golgi traffic of vesicular stomatitis virus glycoprotein (VSV G) and also the reassembly of the Golgi apparatus after brefeldin A (BFA) treatment and washout. DGKdelta was demonstrated to associate with the ER through its C-terminal SAM domain acting as an ER-targeting motif. Both of the SAM domain and the N-terminal PH domain of DGKdelta were needed to exert its effects on ER-to-Golgi traffic. Kinase-dead mutants of DGKdelta were also effective as the wild-type enzyme, suggesting that the catalytic activity of DGK was not involved in the present observation. Remarkably, the expression of DGKdelta abrogated formation of COPII-coated structures labeled with Sec13p without affecting COPI structures. These findings indicate that DGKdelta negatively regulates ER-to-Golgi traffic by selectively inhibiting the formation of ER export sites without significantly affecting retrograde transport.     

Phorbol ester-regulated oligomerization of diacylglycerol kinase delta linked to its phosphorylation and translocation

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. In yeast two-hybrid screening, we unexpectedly found a self-association of the C-terminal part of DGKdelta containing a sterile alpha-motif (SAM) domain. We then bacterially expressed the SAM domain fused with maltose-binding protein and confirmed the formation of dimeric and tetrameric structures. Moreover, gel filtration and co-immunoprecipitation analyses demonstrated that DGKdelta formed homo-oligomeric structures in intact cells and that the SAM domain was critically involved in the oligomerization. Interestingly, phorbol ester stimulation induced dissociation of the oligomeric structures with concomitant phosphorylation of DGKdelta. Furthermore, we found that DGKdelta was translocated from cytoplasmic vesicles to the plasma membrane upon phorbol ester stimulation. In this case, DGKdelta mutants lacking the ability of self-association were localized at the plasma membranes even in the absence of phorbol ester. A protein kinase C inhibitor, staurosporine, blocked all of the effects of phorbol ester, i.e. oligomer dissociation, phosphorylation, and translocation. We confirmed that tumor-promoting phorbol esters did not directly bind to DGKdelta. The present studies demonstrated that the formation and dissociation of oligomers serve as the regulatory mechanisms of DGKdelta and that DGKdelta is a novel downstream effector of phorbol ester/protein kinase C signaling pathway.     

Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR

The simultaneous observation of interdependent reactions within different phases as catalyzed by membrane-bound enzymes is still a challenging task. One such enzyme, the Escherichia coli integral membrane protein diacylglycerol kinase (DGK), is a key player in lipid regulation. It catalyzes the generation of phosphatidic acid within the membrane through the transfer of the γ-phosphate from soluble MgATP to membrane-bound diacylglycerol. We demonstrate that time-resolved (31)P magic angle spinning NMR offers a unique opportunity to simultaneously and directly detect both ATP hydrolysis and diacylglycerol phosphorylation. This experiment demonstrates that solid-state NMR provides a general approach for the kinetic analysis of coupled reactions at the membrane interface regardless of their compartmentalization. The enzymatic activity of DGK was probed with different lipid substrates as well as ATP analogs. Our data yield conclusions about intersubunit cooperativity, reaction stoichiometries and phosphoryl transfer mechanism and are discussed in the context of known structural data.     

Subtype-specific translocation of diacylglycerol kinase alpha and gamma and its correlation with protein kinase C

We examined the translocation of diacylglycerol kinase (DGK) alpha and gamma fused with green fluorescent protein in living Chinese hamster ovary K1 cells (CHO-K1) and investigated temporal and spatial correlations between DGK and protein kinase C (PKC) when both kinases are overexpressed. DGKalpha and gamma were present throughout the cytoplasm of CHO-K1 cells. Tetradecanoylphorbol 13-acetate (TPA) induced irreversible translocation of DGKgamma, but not DGKalpha, from the cytoplasm to the plasma membrane. The (TPA)-induced translocation of DGKgamma was inhibited by the mutation of C1A but not C1B domain of DGKgamma and was not inhibited by staurosporine. Arachidonic acid induced reversible translocation of DGKgamma from the cytoplasm to the plasma membrane, whereas DGKalpha showed irreversible translocation to the plasma membrane and the Golgi network. Purinergic stimulation induced reversible translocation of both DGKgamma and alpha to the plasma membrane. The timing of the ATP-induced translocation of DGKgamma roughly coincided with that of PKCgamma re-translocation from the membrane to the cytoplasm. Furthermore, re-translocation of PKCgamma was obviously hastened by co-expression with DGKgamma and was blocked by an inhibitor of DGK (R59022). These results indicate that DGK shows subtype-specific translocation depending on extracellular signals and suggest that PKC and DGK are orchestrated temporally and spatially in the signal transduction.     

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.     

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.     

Tandem SAM domain structure of human Caskin1: a presynaptic, self-assembling scaffold for CASK

The synaptic scaffolding proteins CASK and Caskin1 are part of the fibrous mesh of proteins that organize the active zones of neural synapses. CASK binds to a region of Caskin1 called the CASK interaction domain (CID). Adjacent to the CID, Caskin1 contains two tandem sterile α motif (SAM) domains. Many SAM domains form polymers so they are good candidates for forming the fibrous structures seen in the active zone. We show here that the SAM domains of Caskin1 form a new type of SAM helical polymer. The Caskin1 polymer interface exhibits a remarkable segregation of charged residues, resulting in a high sensitivity to ionic strength in?vitro. The Caskin1 polymers can be decorated with CASK proteins, illustrating how these proteins may work together to organize the cytomatrix in active zones.     

Role of the diacylglycerol kinase alpha-conserved domains in membrane targeting in intact T cells

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKalpha is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKalpha domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr(335). This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.     

Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase

The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca(2+) or Mg(2+) ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 degrees C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent K(m) = 6.5 mol %) and saturation kinetics with respect to CTP (apparent K(m) = 0.3 mm). dCTP was both a substrate (apparent K(m) = 0.4 mm) and competitive inhibitor (apparent K(i) = 0.4 mm) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.     

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.     

The growth-suppressive function of the polycomb group protein polyhomeotic is mediated by polymerization of its sterile alpha motif (SAM) domain

Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.     

T cell activation in vivo targets diacylglycerol kinase alpha to the membrane: a novel mechanism for Ras attenuation

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid, leading to decreased and increased levels, respectively, of these two lipid messengers that play a central role in T cell activation. Nine DGK isoforms, grouped into five subtypes, are found in higher organisms; all contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. In this study, we have researched in vivo the regulation of DGK alpha, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells. We demonstrate that DGK alpha, highly expressed in resting T lymphocytes, is subject to complex control at the mRNA and protein levels during in vivo T cell activation. Subcellular fractionation of T lymphocytes shortly after in vivo engagement of the TCR shows rapid translocation of cytosolic DGK alpha to the membrane fraction. At early time points, DGK alpha translocation to the membrane correlates with rapid translocation of Ras guanyl nucleotide-releasing protein (RasGRP), a nucleotide exchange activator for Ras that associates to the membrane through a diacylglycerol-binding domain. To demonstrate a causal relationship between DGK alpha activity and RasGRP relocation to the membrane, we determined RasGRP translocation kinetics in a T cell line transiently transfected with constitutive active and dominant-negative DGK alpha mutants. We show that membrane localization of DGK alpha is associated with a negative regulatory signal for Ras activation by reversing RasGRP translocation. This study is the first demonstration of in vivo regulation of DGK alpha, and provides new insight into the functional role of a member of this family of lipid kinases in the regulation of the immune response.     

Diacylglycerol kinase gamma is one of the specific receptors of tumor-promoting phorbol esters.

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two different enzyme families that interact with diacylglycerol. Both enzymes contain cysteine-rich C1 domains with a zinc finger-like structure. Most of the C1 domains of PKCs show strong phorbol-12,13-dibutyrate (PDBu) binding with nanomolar dissociation constants (K(d)'s). However, there has been no experimental evidence that phorbol esters bind to the C1 domains of DGKs. We focused on DGK gamma because its C1A domain has a high degree of sequence homology to those of PKCs, and because DGK gamma translocates from the cytoplasm to the plasma membrane following 12-O-tetradecanoylphorbol-13-acetate treatment similar to PKCs. Two C1 domains of DGK gamma (DGK gamma-C1A and DGK gamma-C1B) were synthesized and tested for their PDBu binding along with whole DGK gamma (Flag-DGK gamma) expressed in COS-7 cells. DGK gamma-C1A and Flag-DGK gamma showed strong PDBu binding affinity, while DGK gamma-C1B was completely inactive. Scatchard analysis of DGK gamma-C1A and Flag-DGK gamma gave K(d)'s of 3.1 and 4.4 nM, respectively, indicating that the major PDBu binding site of DGK gamma is C1A. This is the first evidence that DGK gamma is a specific receptor of tumor-promoting phorbol esters.