A Domain with Homology to Neuronal Calcium Sensors Is Required for Calcium-dependent Activation of Diacylglycerol Kinase α

Abstract

Hydrolysis of phosphatidylinositol 4,5-bisphosphate is a common mechanism of stimulus transduction (1). Diacylglycerol (DAG) 1 released in this reaction activates protein kinase C (PKC) and is then rapidly metabolized back to phosphatidylinositol in a series of reactions initiated by a diacylglycerol kinase (DGK). As such, DGKs attenuate DAG-mediated PKC activation (2). Recent studies indicate that DGKs are also activated by mechanisms independent of phosphoinositide turnover (3, 4). Diacylglycerol kinases catalyze the ATP-dependent phosphorylation of sn-1,2-diacylglycerol to form phosphatidic acid (PA), which is also a lipid mediator (5, 6). Several DGK isoforms have been cloned (7). All these sequences share a homologous catalytic domain and two or three C1 protein kinase C homology domains (7-9). Some DGKs contain EF hands, which are Ca 2ϩ -binding sites (7). These DGKs also have a domain at their N termini with homology to the recoverin family of neuronal calcium sensors (Fig. 1). We term this the recoverin homology (RVH) domain. In S-modulin, the frog orthologue of recoverin, this domain associates with the EF hands to mediate Ca 2ϩ -dependent inhibition of rhodopsin kinase (10).The varied structures of DGK regulatory domains suggest divergent mechanisms of regulation. Several studies have shown variation among DGKs with regard to activation by phospholipids, sphingosine, or Ca 2ϩ (11)(12)(13)(14)(15). Kanoh and coworkers (16 -18) have studied DGK␣, a Ca 2ϩ -activated isoform highly expressed in oligodendrocytes and thymocytes. They have shown that Ca 2ϩ binds the EF hand region of the enzyme and that deletion of the EF hands results in constitutive enzyme activation (19,20). We have now examined a series of DGK␣ mutants in which the RVH and EF hand domains are sequentially deleted. Our results indicate that the N-terminal RVH domain is required for Ca 2ϩ to activate this enzyme. In contrast to the constitutive activation seen with deletion of the EF hands, DGKs with deletions involving only the RVH domain expressed activity similar to that of wild-type enzyme in the absence of Ca 2ϩ . Sites within both the EF hands and RVH domain were protected from trypsin proteolysis by Ca 2ϩ , indicating that both domains participate in a Ca 2ϩ -induced conformational change. A cationic amphiphile, octadecyltrimethylammonium chloride, markedly stimulated DGK␣ activity in vitro. This effect, like Ca 2ϩ -dependent activation, was dependent on the RVH domain. The DGK␣ RVH domain does not itself bind Ca 2ϩ . However, it does appear to function together with the EF hands to couple Ca 2ϩ binding to release of EF handmediated autoinhibition of DGK␣. EXPERIMENTAL PROCEDURES Materials-Restriction

Diacylglycerol Kinase γ Serves as an Upstream Suppressor of Rac1 and Lamellipodium Formation

“…We confirmed that DGK␥-G494D possessed no detectable DGK catalytic activity in vitro (data not shown). The deletion of the N-terminal 259 residues, acting as an autoinhibitory site of type I DGK (22,37), resulted in only a 2-fold increase in DGK activity when assayed in vitro (data not shown). However, this mutant showed constitutive localization at the cell periphery ( Fig.…”

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

“…Regarding the specific activity, values of 3-20 pmol PA min Ϫ1 g of protein Ϫ1 were previously reported in biochemical studies for wheat (37) and tomato (17) DGK isoforms, both being significantly higher than the specific activity observed here for Arabidopsis AtDGK2. A specific activity of 0.4 pmol of PA min Ϫ1 g of protein Ϫ1 has been reported for the human DGK isoform (38), whereas another human DGK isoform, DGK␣, showed values higher than 20 pmol of PA min Ϫ1 g of protein Ϫ1 (39).…”

“…A similar, previously unrecognized, aggregation of basic amino acids is present in the amino-terminal part of AtDGK1 ( 45 YTAFQWRRNINLSWTK-AIARSKKNPKARHKVP 76 ). The two monocot DGKs also contain basic amino acid segments, which are almost identical in sequence, upstream of DAG/PE-binding domain 1 (rice, 39 YT-VNQLNKNISLSLMKAIRARARKYKKLKDKVP 71 ; maize, n Y-TVNQLNKNISLSLIRAIKARAKRYKKSKDKVP nϩ32 , exact position not known, because of a missing 5Ј part in the assembled cDNA). In all four plant enzymes analyzed here, the distances between the upstream basic region and the first copy of the DAG/PE-binding domain (3 amino acids) and between the two copies of the DAG/PE-binding domains (12 amino acid residues), respectively, were strongly conserved.…”

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

“…T cells from DGK␣-deficient mice have enhanced responses (19), further confirming the relevance of this enzyme in regulating immune function. Structural-functional analyses showed that the regulatory N-terminal domain of the enzyme, containing a recoverin-like domain and a tandem of EF hand motifs, acts as a negative regulator of enzyme activation and receptor-induced membrane translocation (8,20). Translocation of wild type (wt) DGK␣ is a very rapid and transient event, whereas a catalytically inactive mutant form remains at the membrane for a longer period of time (18).…”