Subtype-specific Translocation of Diacylglycerol Kinase α and γ and Its Correlation with Protein Kinase C

Nine diacylglycerol kinase (DGK) isozymes have been identified. However, our knowledge of their individual functions is still limited. Here, we demonstrate the role of DGK␥ in regulating Rac1-governed cell morphology. We found that the expression of kinase-dead DGK␥, which acts as a dominant-negative mutant, and inhibition of endogenous DGK␥ activity with R59949 induced lamellipodium and membrane ruffle formation in NIH3T3 fibroblasts in the absence of growth factor stimulation. Reciprocally, lamellipodium formation induced by plateletderived growth factor was significantly inhibited upon expression of constitutively active DGK␥. Moreover, the constitutively active DGK␥ mutant suppressed integrinmediated cell spreading. These effects are isoform-specific because, in the same experiments, none of the corresponding mutants of DGK␣ and DGK␤, closely related isoforms, affected cell morphology. These results suggest that DGK␥ specifically participates in the Rac1-mediated signaling pathway leading to cytoskeletal reorganization. In support of this, DGK␥ co-localized with dominant-active Rac1 especially in lamellipodia. Moreover, we found that endogenous DGK␥ was physically associated with cellular Rac1. Dominant-negative Rac1 expression blocked the lamellipodium formation induced by kinase-dead DGK␥, indicating that DGK␥ acts upstream of Rac1. This model is supported by studies demonstrating that kinase-dead DGK␥ selectively activated Rac1, but not Cdc42. Taken together, these results strongly suggest that DGK␥ functions through its catalytic action as an upstream suppressor of Rac1 and, consequently, lamellipodium/ruffle formation.It is well recognized that a variety of lipid second messengers in low abundance carry out specific tasks for a wide range of biological processes in eukaryotic cells. The cellular concentrations of such signaling lipids must be strictly regulated by the action of metabolic enzymes. Diacylglycerol kinase (DGK) 1 phosphorylates diacylglycerol (DAG) to yield phosphatidic acid (PA) (1). DAG is an established activator of conventional and novel protein kinases C (PKCs), Unc-13, and Ras guanyl nucleotide-releasing protein (2-6). PA has also been reported to regulate a number of signaling proteins such as phosphatidylinositol-4-phosphate 5-kinase, Ras GTPase-activating protein, Raf-1 kinase, atypical PKC, and chimaerins (6 -8). Moreover, specific PA-binding sites have recently been identified in several important proteins such as Raf-1 (9), mTOR (mammalian target of rapamycin) (10), and p47 phox (11). Thus, DGK can potentially participate in a diverse range of cellular events through modulating the balance between two bioactive lipids, DAG and PA.Mammalian DGK is known to exist as a large protein family consisting of nine isozymes classified into five subtypes according to their structural features (12-15). These subfamilies can be characterized by the presence of a variety of regulatory domains of known and/or predicted functions, clearly indicating their distinct functions and regulatory mecha...

Section: Dgk␥ Operates As An Upstreamsupporting

confidence: 76%

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

Tsushima

Kai

Yamada

et al. 2004

“…Most relevant here, however, are reports on UFAs. In CHO cells, Ն200 M AA and oleic acid cause rapid, transient shifts in EFFP-PKC␥ from cytosol to plasma membrane, whereas 50 M AA causes a slow, sustained shift in cytosol EGFP-PKC⑀ to the perinuclear area (66,67). We are unaware of reports on the effect of UFAs on other EGFP-PKC isoforms.…”

Section: Pdb Binding-[mentioning

confidence: 82%

Protein Kinases C Translocation Responses to Low Concentrations of Arachidonic Acid

O’Flaherty¹,

Chadwell²,

Kearns

et al. 2001

Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a >20 M concentration of the fatty acid. We find that human polymorphonuclear neutrophils ( or PKC␦ fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKC␤ translocation from cytosol to plasma membrane at >0.5 M, and EGFP-PKC␦ translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nM. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca 2؉ or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.

“…However, there was circumstantial evidence that DGK␥ binds phorbol esters; TPA induced the translocation of DGK␥ from the cytoplasm to the plasma membrane of CHO-K1 cells (27). We have recently synthesized the cysteine-rich sequences of all conventional and novel PKC isozyme C1 domains by the solid-phase Fmoc strategy in high purity (23).…”

Section: Discussionmentioning

confidence: 99%

“…The plasmid (BS465) encoding rat-DGK␥ (27) with an XhoI site in the 5Ј terminus and a SmaI site in the 3Ј terminus was used as a template. The sense and antisense primers for producing a C1A-deleted mutant (⌬C1A-DGK␥) were 5Ј-GTGAAAACATACTCCAAAGCCAAAAGG-3Ј and 5Ј-GCGTCCATCCCCCTTGGAG-3Ј, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Sakane et al (26) also detected no specific PDBu binding in either rat-DGK␤ or human-DGK␥ expressed in COS-7 cells. On the other hand, a recent investigation by Shirai et al (27) clearly showed that DGK␥ expressed in Chinese hamster ovary-K1 (CHO-K1) cells was translocated from the cytoplasm to the plasma membrane irreversibly following 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment. They also suggested that the C1A domain of DGK␥ is responsible for this translocation based on the point mutation of each C1 domain.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Shindo¹,

Irie

Masuda

et al. 2003

Self Cite

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...