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

Abstract

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...

AtDGK2, a Novel Diacylglycerol Kinase from Arabidopsis thaliana, Phosphorylates 1-Stearoyl-2-arachidonoyl-sn-glycerol and 1,2-Dioleoyl-sn-glycerol and Exhibits Cold-inducible Gene Expression

“…It was suggested that the extCRD, in conjunction with the conserved catalytic domain, is essential for the unique enzymatic function of DGK (30,31). Recently, it was demonstrated that C1A domains in animal DGK␤ and DGK␥ isozymes exhibited significant binding to phorbol 12,13-dibutyrate (29). It remains to be tested whether also the plant DGK isozymes are competent for phorbol ester binding.…”

“…The typical core structure of C1 domains in PKC contains two histidines and six cysteines in the order HX 12 CX 2 CX 13-14 CX 2 CX 4 HX 2 CX 7 C (where X represents any amino acid), coordinating two zinc atoms in a tetrahedral geometry. The core of animal DGK C1 domains is slightly different, HX 10 -12 CX 2-6 CX 9 -19 CX 2 CX 4 HX 2-4 CX 5-10 C, but the zinc-coordinating histidine and cysteine residues are present (29). We screened the GenBank TM data base using the AtDGK2 sequence as a bait and retrieved cDNA sequences from Oryza sativa (GenBank TM accession number AK100331) and Zea mays (partial cDNA, resulting from an EST assembly project; AY110054).…”

“…PE can directly stimulate PKC. PE and DAG bind in a zinc-dependent manner to the so-called C1 region of the protein (29). The C1 region contains one or two copies (depending on the PKC isozyme) of a cysteinerich domain (CRD), which is essential for DAG/PE-binding.…”

“…Cluster I DGKs, such as AtDGK1 and AtDGK2, could be potential phorbol ester effectors in plants. Phorbol ester binding has recently been demonstrated for DGK␥ and DGK␤ from animals (29). Whether plant DGKs also bind phorbol esters awaits further biochemical analysis.…”

Translocation of Diacylglycerol Kinase θ from Cytosol to Plasma Membrane in Response to Activation of G Protein-coupled Receptors and Protein Kinase C

“…First, similar to PKC, DAG, generated by PLC in the plasma membrane or the nucleus, may potentially recruit DGK through binding to its CRDs (32). Indeed, the CRDs of DGK␥ and possibly DGK␤ interact with the DAG substitute, phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) (33,34). Furthermore, an undefined DGK activity translocated to artificially enhanced DAG in membranes of some cell systems (reviewed in Ref.…”