Biomedical subjects

Matthew K Topham

Publications and source records attributed to Matthew K Topham.

17 recordsLinked to original sources

PubMed2006

Disruption of diacylglycerol metabolism impairs the induction of T cell anergy.

Anergic T cells have altered diacylglycerol metabolism, but whether that altered metabolism has a causative function in the induction of T cell anergy is not apparent. To test the importance of diacylglycerol metabolism in T cell anergy, we manipulated diacylglycerol kinases (DGKs), which are enzymes that terminate diacylglycerol-dependent signaling. Overexpression of DGK-alpha resulted in a defect in T cell receptor signaling that is characteristic of anergy. We generated DGK-alpha-deficient mice and found that DGK-alpha-deficient T cells had more diacylglycerol-dependent T cell receptor signaling. In vivo anergy induction was impaired in DGK-alpha-deficient mice. When stimulated in anergy-producing conditions, T cells lacking DGK-alpha or DGK-zeta proliferated and produced interleukin 2. Pharmacological inhibition of DGK-alpha activity in DGK-zeta-deficient T cells that received an anergizing stimulus proliferated similarly to wild-type T cells that received CD28 costimulation and prevented anergy induction. Our findings suggest that regulation of diacylglycerol metabolism is critical in determining whether activation or anergy ensues after T cell receptor stimulation.

Animals↗

PubMed2006

Diacylglycerol kinase delta regulates protein kinase C and epidermal growth factor receptor signaling.

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG) to terminate its signaling. To study DGKdelta, we disrupted its gene in mice and found that DGKdelta deficiency reduced EGF receptor (EGFR) protein expression and activity. Similar to EGFR knockout mice, DGKdelta-deficient pups were born with open eyelids and died shortly after birth. PKCs are activated by DAG and phosphorylate EGFR to reduce its expression and activity. We found DAG accumulation, increased threonine phosphorylation of EGFR, enhanced phosphorylation of other PKC substrates, and increased PKC autophosphorylation in DGKdelta knockout cells, indicating that DGKdelta regulates EGFR by modulating PKC signaling.

Animals↗

PubMed2006

Signaling roles of diacylglycerol kinases.

Diacylglycerol kinases (DGKs) attenuate diacylglycerol signaling by converting this lipid to phosphatidic acid (PA). The nine mammalian DGKs that have been identified are widely expressed, but each isoform has a unique tissue and subcellular distribution. Their kinase activity is regulated by mechanisms that modify their access to diacylglycerol, directly affect their kinase activity, or alter their ability to bind to other proteins. In many cases, these enzymes regulate the activity of proteins that are modulated by either diacylglycerol or PA. Experiments using cultured cells and model organisms have demonstrated that DGKs have prominent roles in neuronal transmission, lymphocyte signaling, and carcinogenesis.

Animals↗

PubMed2005

The retinoblastoma family proteins bind to and activate diacylglycerol kinase zeta.

The retinoblastoma protein (pRB) is a tumor suppressor and key regulator of the cell cycle. We have previously shown that pRB interacts with phosphatidylinositol-4-phosphate 5-kinases, lipid kinases that can regulate phosphatidylinositol 4,5-bisphosphate levels in the nucleus. Here, we investigated pRB binding to another lipid kinase in the phosphoinositide cycle, diacylglycerol kinase (DGK) that phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. We found that DGKzeta, but not DGKalpha or DGK, interacts with pRB in vitro and in vivo. Binding of DGKzeta to pRB is dependent on the phosphorylation status of pRB, since only hypophosphorylated pRB interacts with DGKzeta. DGKzeta also binds to the pRB-related pocket proteins p107 and p130 in vitro and in cells. Although DGKzeta did not affect the ability of pRB to regulate E2F-mediated transcription, we found that pRB, p107, and p130 potently stimulate DGKzeta activity in vitro. Finally, overexpression of DGKzeta in pRB-null fibroblasts reconstitutes a cell cycle arrest induced by gamma-irradiation. These results suggest that DGKzeta may act in vivo as a downstream effector of pRB to regulate nuclear levels of diacylglycerol and phosphatidic acid.

Animals↗

PubMed2005

Diacylglycerol kinase iota regulates Ras guanyl-releasing protein 3 and inhibits Rap1 signaling.

To study the physiological function of diacylglycerol (DAG) kinase iota (DGKiota), which converts DAG to phosphatidic acid, we deleted this gene in mice. In contrast to previous studies showing that DGK isoforms decrease Ras activity, signaling downstream of Ras in embryonic fibroblasts was significantly reduced in cells lacking DGKiota. DGKs regulate Ras signaling by attenuating the function of the DAG-dependent Ras guanyl nucleotide-releasing proteins (RasGRPs). We tested whether DGKiota inhibited the four known RasGRPs and found that it inhibited only RasGRP3. In addition to activating Ras, RasGRP3 also activates Rap1, which in some cases can antagonize the function of Ras. We demonstrate that DGKiota bound to RasGRP3 and inhibited its activation of Rap1 by metabolizing DAG. This inhibition consequently affected Ras signaling. We tested the physiological consequence of deleting DGKiota by crossing wild-type or DGKiota-deficient mice with mice carrying a v-Ha-Ras transgene, and then we assessed tumor formation. We observed significantly fewer tumors in DGKiota-deficient mice. Because Rap1 can antagonize the function of Ras, our data are consistent with a model in which DGKiota regulates RasGRP3 with a predominant effect on Rap1 activity. Additionally, we found that DGKzeta, which is structurally similar to DGKiota, inhibited RasGRPs 1, 3, and 4 and predominantly affected Ras signaling. Thus, type IV DGKs regulate RasGRPs, but the downstream effects differ depending on the DGK.

Animals↗

PubMed2005

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.

Adenoviridae↗

PubMed2004

The alpha isoform of diacylglycerol kinase exhibits arachidonoyl specificity with alkylacylglycerol.

We compared the diacylglycerol kinase (DGK) catalyzed phosphorylation of 1-O-hexanoyl-2-oleoylglycerol (HOG) with 1-O-hexanoyl-2-arachidonoylglycerol (HAG). We assayed the activity of DGKalpha and DGKzeta using a liposomal-based assay system. Liposomal assays show that the DGKalpha and, to a lesser extent, DGKzeta preferentially act on substrates containing an arachidonoyl group when this group is incorporated into alkylacylglycerols. The activity of DGKalpha was 82 times greater with HAG compared to HOG. DGKzeta is 10 times more active in catalyzing the phosphorylation of HAG compared to HOG. Although diacylglycerols were better substrates for both DGKalpha and DGKzeta than the alkylacylglycerols, no specificity was exhibited for arachidonoyl-containing diacylglycerols. However, this specificity for HAG over HOG is modulated by the phospholipid composition of the liposome. Addition of cholesterol and/or phosphatidylethanolamine partially reduces the substrate selectivity. We also analyzed the kinetic constants for the phosphorylation of both diacylglycerol and 1-alkyl-2-acylglycerol catalyzed by the alpha, epsilon, or zeta isoforms using a soluble Triton mixed micelle system. We found that all three isoforms of DGK can phosphorylate 1-alkyl-2-acylglycerols but generally at a lower rate than for the corresponding diacylglycerol. The specificity of DGKepsilon for diacylglycerols containing an arachidonoyl group was retained when the ester group in the C-1 position is replaced with an ether linkage. In contrast, DGKalpha and, to a lesser extent, DGKzeta had greater specificity for arachidonoyl-containing 1-alkyl-2-acylglycerols than for arachidonoyl-containing diacylglycerols. This demonstrates that the acyl chain specificity is affected by the structure of the lipid headgroup.

Arachidonic Acid↗

PubMed2004

Lipid modulation of the activity of diacylglycerol kinase alpha- and zeta-isoforms: activation by phosphatidylethanolamine and cholesterol.

Diacylglycerol kinase (DGK) isoforms alpha and zeta were extracted from transfected cells that overexpressed these enzymes. We determined the lipid dependence of the binding of these isoforms to liposomes. The modulation by lipid of the rate of phosphorylation of diacylglycerol by these enzymes was also measured. Incorporation of phosphatidylethanolamine into the liposomes resulted in an increased partitioning of both isoforms of DGK to the membrane as well as an increased catalytic rate. We demonstrate that the increased catalytic rate is a consequence of both increased portioning of the enzyme to the membrane and increased catalytic activity of the membrane-bound form. DGKalpha, a calcium-dependent isoform, can be activated in a calcium-independent fashion in the presence of phosphatidylethanolamine. Similar effects are observed with cholesterol. In contrast, sphingomyelin inhibits the activity of both isoforms of DGK. Our results demonstrate that the translocation to membranes and activity of DGKalpha and DGKzeta are modulated by the composition and properties of the membrane. The enzymes are activated by the presence of lipids that promote the formation of inverted phases. However, the promotion of negative curvature is not the sole factor contributing to the lipid effects on enzyme binding and activity. A truncated form of DGKalphalacking both the E-F hand and the recoverin homology domain is constitutively active and is not further activated by any of the lipids tested or by calcium. However, a truncated form lacking only the recoverin homology domain is partially activated by either calcium or certain lipids.

Animals↗

PubMed2004

Diacylglycerol kinase zeta regulates phosphatidylinositol 4-phosphate 5-kinase Ialpha by a novel mechanism.

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays an important role during actin polymerization and is produced by the type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KI), which are activated by phosphatidic acid (PA). As diacylglycerol kinases (DGKs) generate PA by phosphorylating diacylglycerol (DAG), we investigated whether DGKs were involved in controlling PIP2 levels by regulating PIP5KI activity. Here we show that expression of DGKzeta significantly enhances PIP5KIalpha activity in thrombin-stimulated HEK293 cells, and DGK activity is required for this stimulation. We also observed that DGKzeta co-immunoprecipitated and co-localized with PIP5KIalpha, suggesting that they reside in a regulated signaling complex. To explore the role of DGKzeta in actin polymerization, we examined the subcellular distribution of DGKzeta, PIP5KIalpha and actin, and found that these proteins co-localized with actin in lamellipodial protrusions. Supporting that PIP5KIalpha regulation occurs at the sites of actin polymerization, we found that PIP2 also accumulated in the actin-rich regions of lamellipodia. Significantly, in wounding assays, DGKzeta, PIP5KIalpha and PIP2 accumulated at the leading edge of migrating A172 cells, where massive actin polymerization is known to occur. Combined, these data support a novel function for DGKzeta: by generating PA, it stimulates PIP5KIalpha activity to increase local PIP2, which regulates actin polymerization.

Actins↗

PubMed2004

Diacylglycerol kinases.

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol to form phosphatidic acid. In most cases, members of this large family of enzymes appear to bind and regulate proteins activated by either diacylglycerol or phosphatidic acid. Proteins that appear to be regulated, in part, by DGKs include protein kinase Cs, RasGRPs, and phosphatidylinositol kinases. By modulating the activity of these proteins, DGKs potentially affect a number of biological events including-but likely not limited to-cell growth, neuronal transmission, and cytoskeleton remodeling.

Animals↗

PubMed2003

Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.

Actins↗

PubMed2003

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.

Binding Sites↗

PubMed2003

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.

3T3 Cells↗

PubMed2003

Ceramide-induced alterations in dopamine transporter function.

The purpose of this study was to determine the effects of ceramide on dopamine and serotonin (5-HT, 5-hydroxytryptamine) transporters. Exposure of rat striatal synaptosomes to C2-ceramide caused a reversible, concentration-dependent decrease in plasmalemmal dopamine uptake. In contrast, ceramide exposure increased striatal 5-HT synaptosomal uptake. This increase did not appear to be due to an increased uptake by the 5-HT transporter. Rather, the increase appeared to result from an increase in 5-HT transport through the dopamine transporter, an assertion evidenced by findings that this increase: (1) does not occur in hippocampal synaptosomes (i.e., a preparation largely devoid of dopamine transporters), (2) occurs in striatal synaptosomes prepared from para-chloroamphetamine-treated rats (i.e., a preparation lacking 5-HT transporters), (3) is attenuated by pretreatment with methylphenidate (i.e., a relatively selective dopamine reuptake inhibitor) and (4) is inhibited by exposure to exogenous dopamine (i.e., which presumably competes for uptake with 5-HT). Taken together, these results reveal that ceramide is a novel modulator of monoamine transporter function, and may alter the affinity of dopamine transporters for its primary substrate.

Animals↗

PubMed2002

Differential trafficking of the vesicular monoamine transporter-2 by methamphetamine and cocaine.

High-dose administration of cocaine or methamphetamine to rats acutely (< or = 24 h) alters vesicular dopamine transport. This study elucidates the nature of these changes. Results reveal a differential redistribution of the vesicular monoamine transporter-2 (VMAT-2) within striatal synaptic terminals after drug treatment. In particular, cocaine shifts VMAT-2 protein from a synaptosomal membrane fraction to a vesicle-enriched fraction, as assessed ex vivo in fractions prepared from treated rats. In contrast, methamphetamine treatment redistributes VMAT-2 from a vesicle-enriched fraction to a location that is not retained in a synaptosomal preparation. These data suggest that psychostimulants acutely and differentially affect VMAT-2 subcellular localization.

Animals↗

PubMed2002

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.

Antigens, CD↗

PubMed2002

Diacylglycerol kinases: regulation and signaling roles.

Diacylglycerol kinases (DGKs) are thought to attenuate diacylglycerol signals by converting diacylglycerol to phosphatidic acid. The nine mammalian diacylglycerol kinases that have been identified are widely expressed, but each isoform has a unique tissue and subcellular distribution. The activity of DGKs is regulated by mechanisms that can modify their access to diacylglycerol, affect their activity, or alter their ability to bind to other proteins. Although little is known of the specific function of DGKs in platelets, they likely influence actin reorganization and other signaling events requiring diacylglycerol.

Animals↗