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  • All DGKs have at least two cysteine rich regions


    All DGKs have at least two cysteine-rich regions homologous to the C1A and C1B motifs of PKCs [26]. In AM1241 mg theory, these domains may bind DAG, perhaps localizing DGKs to where DAG accumulates. However, no DGK C1 domain has so far been conclusively shown to bind DAG. In fact, structural predictions suggest that most DGK C1 domains may not bind DAG. For example, Hurley et al. [26] noted that the amino AM1241 mg sequences of most DGK C1 domains differed enough from those in PKCs that many DGK C1 domains may not bind DAG. Most DGKs tested were unable to bind long-lived DAG-like analogues such as PDBu [27]. However, the inability of DGK C1 domains to bind DAG-like molecules may simply reflect exquisite selectivity of DGK C1 domains for authentic DAG, unlike other C1 domains such as those in PKCs which appear to be more promiscuous. Houssa and van Blitterswijk [28] noted that in DGKs, the C1 domain closest to the catalytic domain is highly conserved, including an extended motif of 15 amino acids not present in C1 domains of other proteins or in the DGK C1 motifs further from the catalytic domain. They noted that conserved residues in this extended motif may be critical for DAG kinase activity. Taken together, these data suggest that DGK C1 domains are structurally different from C1 domains in other proteins and that the different C1 domains of a single DGK isoform may have unique functions. However, distinct functions of individual C1 domains remain to be definitively demonstrated. In addition to the C1 and catalytic domains, DGKs have other regulatory domains that form the basis for dividing them into five subtypes. Type I DGKs [29], [30], [31] have calcium-binding EF hand motifs and are more active in the presence of calcium [32]. Type II DGKs have pleckstrin homology (PH) domains at their amino termini [23], [24]. This domain in DGKδ has been shown to bind weakly and nonselectively to phosphatidylinositols [33]. Type II DGKs also have sterile alpha motifs (SAM domain) at their carboxy termini. Nagaya et al. [21] demonstrated that the SAM domain of DGKδ helped localize it to the endoplasmic reticulum, and this domain also appeared necessary for homo- and hetero-oligomerization of DGKs δ and η [34], [35]. The physiological significance of oligomerization is not clear at this point, although in some cases, it may suppress DAG kinase activity [34]. DGKε, the only type III enzyme, has an unusual specificity toward acyl chains of DAG, strongly preferring a specific fatty acid—arachidonate—at the sn-2 position [36]. Its preference for arachidonate-DAG suggests that DGKε may be a component of the biochemical pathway that accounts for the enrichment of phosphatidylinositols with arachidonate [37]. Type IV DGKs [38], [39] have domains similar to the phosphorylation site domain of the MARCKS protein. This domain in both DGKs ζ and ι functions as a nuclear localization signal and it is phosphorylated by conventional PKC isoforms[22], [39]. Its phosphorylation in DGKζ not only reduces nuclear localization [22], but also suppresses DAG kinase activity [40] and causes it to dissociate from the PKC [41]. Type IV DGKs also have four ankyrin repeats and carboxy terminal PDZ-binding domains [42]. The type V enzyme, DGKθ, has three C1 domains, a putative PH domain, and a Ras association (RA) domain [43]. The function of its RA domain is not clear, and although most RA domains bind RasGTP, van Blitterswijk found that DGKθ did not bind Ras [44].
    Phosphatidic acid produced by DGKs may have signalling properties Several reports indicate that phosphatidic acid, the product of the DGK reaction, has signalling properties. For example, studies have indicated that PA can stimulate DNA synthesis and is potentially mitogenic [45], [46]. However, these properties may have also been caused by contaminating lysophosphatidic acid. Other work indicates that PA is involved in vesicle trafficking [47] and can bind and regulate the activity of numerous enzymes, including the phosphatidylinositol 5-kinases [48], Ras-GAP [49], PKCζ [50], PAK1 [51], and protein phosphatase 1 [52]. Phosphatidic acid also helps recruit Raf to the Ras signalling complex [53]. While the majority of signalling PA is likely derived from the phospholipase D reaction [54], it is possible that PA produced by DGKs also has a signalling role. This suggests that in some cases after terminating a DAG signal, DGKs subsequently activate a PA signalling event. Flores et al. [55] presented evidence in T lymphocytes suggesting that PA from the DGK reaction had a potential role in progression of cells to S phase of the cell cycle. We recently demonstrated that DGKζ associated with type Iα phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a protein activated by PA [56]. We found that co-expression of DGKζ with the PIP5K increased its phosphatidylinositol kinase activity. In addition to these examples, it is possible that DGKs modulate the activity of other phosphatidic acid protein targets.