Matthew K. Topham

Inflammatory roles of P-selectin.

Polymorphonuclear leukocytes (PMNs) bind rapidly and reversibly to endothelial cells induced to express P-selectin, a glycoprotein that mediates adhesive intercellular interactions. In addition, PMNs adherent to endothelium expressing P-selectin demonstrate an intracellular Ca2+ transient, functionally up-regulate beta-2-integrins (CD11/CD18 glycoproteins), become polarized in shape, and are primed for enhanced degranulation when subsequently stimulated with chemotactic factors. However, P-selectin induces none of these responses directly when used alone, when incorporated into model membranes, or when expressed by transfected cells. The absence of direct activation of the PMNs is not due to competing antiinflammatory effects of P-selectin; instead, purified P-selectin and P-selectin in membranes support agonist-stimulated PMN responses. Furthermore, tethering of PMNs to endothelial surfaces by P-selectin is required for priming to occur efficiently, as shown by experiments with blocking monoclonal antibodies. The priming event is directly mediated by the signaling molecule, platelet-activating factor (PAF), and is inhibited by blocking the PAF receptor on PMNs. Thus, P-selectin and PAF are components of an adhesion and activation cascade, but have distinct roles: P-selectin tethers and captures the PMN, whereas PAF mediates juxtacrine activation. In vivo, selectins may facilitate interaction of target cells with membrane-bound molecules that send intercellular signals, in addition to mediating rolling of leukocytes and other adhesive functions.

Mammalian diacylglycerol kinases, a family of lipid kinases with signaling functions.

Many intracellular signaling pathways are initiated by a simple reaction, the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) 1 (1), which results in a transient rise in the amounts of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The initial signaling has two predictable components: IP3 binds to intracellular receptors to initiate calcium release from intracellular stores (2), and DAG functions as an allosteric activator of protein kinase C (PKC) (3). Both the polar product, IP3, and the lipid messenger, DAG, are converted to inactive products to return the cell to the basal state; the pathways that regulate inositol phosphate levels were reviewed in the fourth minireview in this series (22). In addition to activating PKC, DAG participates in other cellular events. For example, it is a potent activator of the guanine nucleotide exchange factors vav (4) and Ras-GRP (5), indicating a potential role for DAG in regulating Ras and Rho family proteins. In addition to these signaling roles, DAG occupies a central position in the synthesis of major phospholipids (phosphatidylcholine and phosphatidylethanolamine (6)) and triacylglycerols. Thus, to maintain cellular homeostasis, intracellular diacylglycerol levels must be tightly regulated. This is illustrated by evidence that inappropriate accumulation of diacylglycerol contributes to cellular transformation. For example, cell lines that overexpress PLC g have a malignant phenotype (7). Also, cells transformed with one of several oncogenes have elevated DAG levels (8–11), and growth factors that are proto-oncogenes stimulate this pathway. Most of the evidence for this pathological effect centers on excessive and/or prolonged activation of PKC, which is a common feature of the transformed state, both in tumors and in cell cultures (12). PKC function was identified, in part, by virtue of being the target for phorbol esters; these tumor promoters function in the same way as DAG to activate PKC but persist because they are not metabolized (or at least this happens very slowly). Thus, these observations have led to the hypothesis that prolonged elevation of DAG functions as a tumor promoter, the equivalent of an endogenous phorbol ester. This review focuses on a family of enzymes, the diacylglycerol kinases (DGKs) that phosphorylate diacylglycerol to phosphatidic acid (PA) (Fig. 1), which also has signaling functions; it stimulates DNA synthesis (13, 14) and modulates the activity of several enzymes including phosphatidylinositol 5-kinases (PI-5-K) (reviewed by Rameh and Cantley (84), first article in this series), PAK1 (15), PKCz, and Ras-GAP (16). Although the bulk of the signaling “pool” of PA (it, too, is an intermediate in phospholipid synthesis) is thought to derive from the action of phospholipase D (16), DGKs likely contribute to it as well. Thus, DGKs catalyze a reaction that removes DAG and would terminate the PKC-mediated signal but yield a product, PA, that has other functions both in signaling and phospholipid synthesis. The net result on cellular events is, therefore, difficult to predict, but the potential outcomes all support the conclusion that DGKs occupy an interesting niche.

Protein kinase C regulates the nuclear localization of diacylglycerol kinase-ζ

Diacylglycerol kinases (DGKs) terminate signalling from diacylglycerol by converting it to phosphatidic acid. Diacylglycerol regulates cell growth and differentiation, and its transient accumulation in the nucleus may be particularly important in this regulation,. Here we show that a fraction of DGK-ζ is found inthe nucleus, where it regulates the amount of nuclear diacylglycerol. Reducing nuclear diacylglycerol levels by conditional expression of DGK-ζ attenuates cell growth. The nuclear-localization signal of DGK-ζ is located in a region that is homologous to the phosphorylation-site domain of the MARCKS protein. This is, to our knowledge, the first evidence that this domain, which is amajor target for protein kinase C, can localize a protein to thenucleus. Two isoforms of protein kinase C, but not others, regulate the localization of DGK-ζ. Our results define a cycle in which diacylglycerol activates protein kinase C, which then regulates the metabolism of diacylglycerol by alternating the intracellular location of DGK-ζ. This may be a general mechanism to control mitogenic signals that depend on nuclear diacylglycerol.

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-α resulted in a defect in T cell receptor signaling that is characteristic of anergy. We generated DGK-α-deficient mice and found that DGK-α-deficient T cells had more diacylglycerol-dependent T cell receptor signaling. In vivo anergy induction was impaired in DGK-α-deficient mice. When stimulated in anergy-producing conditions, T cells lacking DGK-α or DGK-ζ proliferated and produced interleukin 2. Pharmacological inhibition of DGK-α activity in DGK-ζ-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.

Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.

Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.

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. J. Cell. Biochem.

Regulation and Functions of Diacylglycerol Kinases

4.1. Regulation of Immune Function 6194 4.2. Cell Proliferation and Cancer 6195 4.3. Brain Function 6196 4.4. Cardiac Function 6197 4.5. Glucose Homeostasis 6197 4.6. Vision 6198 5. Inter-relationships 6198 5.1. Role of PA Derived from DGK Activity 6198 5.2. Deacylation of PA To Form LPA Signals 6199 5.3. Acylation and Deacylation of DAG 6200 5.4. Other Sources of DAG: PA-Phosphohydrolases, PLC, Sphingomyelin Synthetase 6201

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.

Mammalian diacylglycerol kinases: Molecular interactions and biological functions of selected isoforms

The mammalian diacylglycerol kinases (DGK) are a group of enzymes having important roles in regulating many biological processes. Both the product and the substrate of these enzymes, i.e. diacylglycerol and phosphatidic acid, are important lipid signalling molecules. Each DGK isoform appears to have a distinct biological function as a consequence of its location in the cell and/or the proteins with which it associates. This review discusses three of the more extensively studied forms of this enzyme, DGKalpha, DGKvarepsilon, and DGKzeta. DGKalpha has an important role in immune function and its activity is modulated by several mechanisms. DGKvarepsilon has several unique features among which is its specificity for arachionoyl-containing substrates, suggesting its importance in phosphatidylinositol cycling. DGKzeta is expressed in many tissues and also has several mechanisms to regulate its functions. It is localized in several subcellular organelles, including the nucleus. The current state of our understanding of the properties and functions of these proteins is reviewed.

Diacylglycerol kinase δ regulates protein kinase C and epidermal growth factor receptor signaling

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG) to terminate its signaling. To study DGKδ, we disrupted its gene in mice and found that DGKδ deficiency reduced EGF receptor (EGFR) protein expression and activity. Similar to EGFR knockout mice, DGKδ-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 DGKδ knockout cells, indicating that DGKδ regulates EGFR by modulating PKC signaling.