Molecular Cloning and Characterization of the Human Diacylglycerol Kinase β (DGKβ) Gene

106

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. DGK␦ (type II isozyme) contains a pleckstrin homology domain at the N terminus and a sterile ␣ motif domain at the C terminus. We identified another DGK␦ isoform (DGK␦2, 135 kDa) that shared the same sequence with DGK␦ previously cloned (DGK␦1, 130 kDa) except for the 52 residues N-terminally extended. Analysis of panels of human normal and tumor tissue cDNAs revealed that DGK␦2 was ubiquitously expressed in all normal and tumor tissues examined, whereas the transcript of DGK␦1 was detected only in ovary and spleen, and in a limited set of tumor-derived cells. The expression of DGK␦2 was induced by treating cells with epidermal growth factor and tumor-promoting phorbol ester. In contrast, the levels of mRNA and protein of DGK␦1 were suppressed by phorbol ester treatment. It thus becomes clear that the two DGK␦ isoforms are expressed under distinct regulatory mechanisms. DGK␦1 was translocated through its pleckstrin homology domain from the cytoplasm to the plasma membrane in response to phorbol ester stimulation, whereas DGK␦2 remained in the cytoplasm even after stimulation. Further experiments showed that the ␦2-specific N-terminal sequence blocks the phorbol ester-dependent translocation of this isoform. Co-immunoprecipitation analysis of differently tagged DGK␦1 and DGK␦2 proteins showed that they were able to form homo-as well as heterooligomers. Taken together, alternative splicing of the human DGK␦ gene generates at least two isoforms, differing in their expressions and regulatory functions.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Alternative Splicing of the Human Diacylglycerol Kinase δ Gene Generates Two Isoforms Differing in Their Expression Patterns and in Regulatory Functions

Sakane¹,

Imai²,

Yamada

et al. 2002

106

“…In fact, Caricasole et al (15) have reported recently that human-DGK␤ was redistributed from the cytoplasm to the plasma membrane of HEK-293 cells upon TPA treatment. However, we could not confirm this translocation in our assay system using CHO-K1 cells because almost all GFP-DGK␤ exists in the plasma membrane and not in the cytoplasm (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…To date, nine subtypes of mammalian DGKs have been cloned (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). All DGK isozymes consist of a conserved catalytic domain and two or three cysteine-rich C1 domains designated as C1A, C1B, and C1C (16).…”

mentioning

confidence: 99%

Synthesis and Phorbol Ester Binding of the Cysteine-rich Domains of Diacylglycerol Kinase (DGK) Isozymes

Shindo¹,

Irie

Masuda

et al. 2003

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGK␥-C1A and DGK␤-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGK␥-C1A, human-DGK␥-C1A, and human-DGK␤-C1A gave K d values of 3.6, 2.8, and 14.6 nM, respectively, suggesting that DGK␥ and DGK␤ are new targets of phorbol esters. An A12T mutation of human-DGK␤-C1A enhanced the affinity to bind PDBu, indicating that the ␤-hydroxyl group of Thr-12 significantly contributes to the binding. The K d value for PDBu of FLAG-tagged whole rat-DGK␥ (4.4 nM) was nearly equal to that of rat-DGK␥-C1A (3.6 nM). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGK␥ and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGK␥ to cause its translocation.Diacylglycerol kinase (DGK) 1 and protein kinase C (PKC) both interact with the second messenger diacylglycerol (DG) (1, 2). DGK phosphorylates DG to produce phosphatidic acid, whereas PKC is allosterically activated by DG in the presence of phosphatidylserine. Therefore, DGK may inhibit the activation of PKC by attenuating DG levels, contributing to the regulation of intracellular signal transduction.To date, nine subtypes of mammalian DGKs have been cloned (3-15). All DGK isozymes consist of a conserved catalytic domain and two or three cysteine-rich C1 domains designated as C1A, C1B, and C1C (16). These isozymes are classified into five classes according to the other functional domains (Fig. 1). The class I isozymes (DGK␣, -␤, and -␥) have calcium binding domains (EF-hands). The class II isozymes (DGK␦ and -) have a pleckstrin homology domain at the N terminus, and their catalytic region is split into two domains unlike the other DGK isozymes. DGK⑀ has a simple structure and is classified as a class III isozyme. The class IV isozymes DGK and have a myristoylated alanine-rich C kinase substrate homology domain and four ankyrin repeats. DGK , which has three C1 domains unlike other DGK and PKC isozymes, is the only isozyme in class V.The similarity between DGK and PKC isozymes in structure is in the cysteine-rich C1 domains. Recent investigations using NMR spectroscopy and x-ray crystallography have revealed the three-dimensional structure of C1B domains of PKC␣, PKC␥,. Each PKC C1 domain has six conserved cysteines and two histidines in the typical core structure HX 12 CX 2 CX 13-14 CX 2 CX 4 HX 2 CX 7 C (where X is any amino acid) that coordinates two atoms of zinc in...

“…Deletion of this C-terminal region greatly alters plasma membrane localization of this isoform in vivo (27). Sequence analysis of the C-terminal sequence of type I enzymes indicated conservation of a Pro-rich sequence followed by an amino acid stretch with no obvious conservation among distinct subtypes (Fig.…”

Section: Deletion Of the Dgk␣ C-terminal Region Prevents Constitutivementioning

confidence: 99%

Role of the Diacylglycerol Kinase α-Conserved Domains in Membrane Targeting in Intact T Cells

Merino¹,

Sanjuán²,

Moraga

et al. 2007

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. DGK␣ 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 DGK␣ 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.The transient generation of lipids at the cell membrane by the concerted action of lipases, kinases, and phosphatases is a very effective mechanism for the localization and activation of signaling molecules. The majority of lipid-modifying enzymes are cytosolic proteins that must in turn translocate to the membrane to modify their substrates. Characterization of the mechanism that governs membrane translocation of these proteins is, thus, essential to assess precisely their role in the regulation of cell responses.The diacylglycerol kinases (DGK) 5 are a family of enzymes that phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA); this modulates the levels of these two lipids, both of which have recognized second messenger functions. Early experiments showed that DGK activity occupies a central position in phospholipid synthesis; this enzyme attracted additional interest when it was shown to participate in intracellular signaling (1). DGK activity is found in organisms from bacteria to mammals, although the protein identified as a DGK in bacteria is an integral membrane protein (2) and has little structural homology with the DGK characterized in multicellular organisms (3). Ten DGK isoforms have been identified in mammals and grouped into five subtypes based on the presence of various domains in their primary sequences, which define distinct regulatory motifs.Translocation from one subcellular compartment to another appears to be a general theme in the regulation of DGK proteins. Many of the early data regarding DGK activity regulation refer to changes in DGK activity or localization based on enzymatic studies without specifying the isoform concerned (4, 5). Characterization of the DGK isoforms has broadened the concept that these proteins reg...