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  • These reactions are used to account for variations

    2021-04-06

    These reactions are used to account for variations in protein solubility or vesicle recovery. For example, with DGKθ we have found it necessary to maintain the purified enzyme in a solution of 0.01% DDM (DGK-D dilution buffer) in the control reaction to prevent enzyme loss due to aggregation or adhesion to the ultracentrifuge Eppendorf tubes which occurs over the time required for the high-speed centrifugation step. The following equation can be used to determine the amount of soluble enzyme that pellets with the vesicles. This calculation assumes that all NBD signal comes from a vesicle that has the potential to bind enzyme.where:
    Tips for Nonspecialists
    Introduction Numerous intracellular signalling pathways are initiated when phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) [1]. The products of this reaction, diacylglycerol (DAG) and inositol-1,4,5-triphosphate, transiently rise and then fall back to basal levels. These second messengers initiate two predictable events: inositol-1,4,5-triphosphate binds to intracellular receptors causing calcium release from intracellular stores, while diacylglycerol recruits and often activates signalling proteins that contain cysteine-rich, C1 domains. Several proteins contain C1 domains, the best known are protein kinase C (PKC) isoforms, which regulate a broad array of cell functions [2]. Until recently, most of the physiological effects of DAG were attributed to activation of the PKCs. However, other DAG targets exist that likely also contribute to the downstream effects of diacylglycerol [3]. For example, DAG binds and activates the family of four RasGRP nucleotide exchange factors [4], [5], [6], [7], it recruits the chimaerins—Rac GTPase-activating proteins—to membrane compartments [8], and it can activate some ion retinoic acid receptor [9]. Because DAG can modulate so many signalling proteins—and consequently affects numerous signalling events—it is crucial that intracellular DAG levels be tightly regulated. The adverse effects of excessive and/or prolonged DAG signalling are best illustrated by the tumor-promoting effects of the phorbol esters. These compounds are DAG analogues that can bind C1 domains, but are very slowly metabolized. Their tumorigenic effects are likely due to persistent activation of proteins that bind phorbol esters such as PKC and RasGRP isoforms. The effects of the phorbol esters have led many to hypothesize that prolonged elevation of DAG—which is seen in tumors and in transformed cell lines [10], [11]—is the equivalent of an endogenous phorbol ester.
    The diacylglycerol kinase gene family DGK isoforms have been identified in organisms such as Caenorhabditis elegans[12], [13], Drosophila melanogaster[14], [15], [16], and Arabidopsis thaliana[17], [18], while no DGK gene has been identified in yeast. Bacteria express only one DGK enzyme, and it is an integral membrane protein capable of phosphorylating in vitro other lipids such as ceramide [19]. Bacterial DGK does not appear to have structural elements allowing regulation of its activity, suggesting that it is constitutively active and is limited by access to its substrate(s). Based on shared structural motifs, DGK isoforms in multicellular organisms are classified into five subtypes. Because most of these organisms express at least one DGK ortholog of each subtype, the five DGK subtypes appear to have distinct functions. Nine mammalian DGK isoforms have been identified (Fig. 1). The heterogeneity of this gene family is similar to the PKC and PLC families, suggesting that the DGKs are not simply lipid biosynthetic enzymes, but also have signalling roles, since enzymes involved in biosynthetic pathways usually do not have extended families. All DGK isoforms have a catalytic domain that is necessary for kinase activity. Each catalytic domain has an ATP-binding site similar to protein kinase catalytic domains with the sequence Gly-X-Gly-X-X-Gly. Mutation of the second glycine in this motif to an aspartate or alanine renders the DGK catalytically inactive [20], [21], [22]. Mutation of that glycine to an aspartate was first noted in the Drosophila DGK, dDGK2 [15]. The mutant dDGK2 protein is expressed in the Drosophila strain rdgA, and causes rapid retinal degeneration after birth. Although protein kinase and DAG kinase catalytic domains share some similarities, there are important structural differences between them that may allow DGK catalytic domains to access DAG in lipid bilayers, a property not required for most protein kinases. In most cases, the DGK catalytic domains are composed of a single motif, but DGKs δ and η have bipartite catalytic domains [23], [24], indicating that these and perhaps other DGK catalytic domains may function as two independent units in a coordinated fashion. The DGK catalytic domains may also require other motifs for maximal activity because several DGK catalytic domains have very little DAG kinase activity when expressed as isolated subunits (M.K.T., unpublished observations). However, Sakane et al. [25] demonstrated that when expressed as an isolated subunit, the catalytic domain of DGKα retained about 60% of the activity of the wild-type enzyme. However, to compare their DAG kinase activities, this group used 100,000 g supernatants, which may have eliminated most of the more active, membrane-bound, wild-type enzyme in the 100,000 g pellets. Moreover, DGKα has an inhibitory motif that, when deleted, yields a fully active enzyme (see below). Sakane et al. [25] found that the isolated DGKα catalytic domain had only 1/3 the activity of this fully active mutant. Thus, it appears that mammalian DGK catalytic domains, unlike bacterial DGK, require other motifs such as C1 domains for maximal activity. This suggests that these other motifs function in coordination with the catalytic domain.