Cilostazol inhibits interleukin-1-induced ADAM17 expression through cAMP independent signaling in vascular smooth muscle cells.

Increased A disintegrin and metalloprotease 17 (ADAM17) expression in vascular smooth muscle cells (VSMC) is implicated in the development of cardiovascular diseases including atherosclerosis and hypertension. Although cilostazol, type III phosphodiesterase (PDE III) inhibitor, has recently been found to inhibit VSMC proliferation, the mechanisms remain largely unclear. Here, we hypothesized that cilostazol regulates the ADAM17 expression in VSMC. In cultured VSMC, interleukin (IL)-1α and IL-1ß significantly increased ADAM17 expression. MEK inhibitor U0126, NF-κB inhibitor BAY-11-7085, and siRNA targeting p65/RelA significantly inhibited IL-1α or IL-ß-induced ADAM17 expression. Cilostazol significantly inhibited IL-1α or IL-1ß-induced extracellular signal-regulated kinase (ERK) phosphorylation and ADAM17 expression. Unexpectedly, cilostamide, dibutryl cAMP, and forskolin did not affect IL-1-induced ADAM17 expression. Our results clearly demonstrated that IL-1 induces ADAM17 expression through ERK/NF-κB activation in VSMCs. Moreover, the inhibitory effects of cilostazol on IL-1-induced ADAM17 expression may be independent of the cAMP signaling pathway in VSMC. These novel findings may provide important clues to understanding the expression mechanisms of ADAM17 and the inhibitory mechanisms of cilostazol in VSMC proliferation.

Significant increase of plasma tetranectin in ovx mice as defined by proteomics analysis.

BACKGROUND: Proteomics is recognized as a useful tool in the dynamic screening of plasma protein expression. This study aimed to identify increased expressions of novel plasma proteins in ovariectomized mice (ovx) using selective reaction monitoring (SRM) validation in combination with electrospray ionized-quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) screening. MATERIALS AND METHODS: Twenty-week-old female C57BL/6 mice were ovariectomized or subjected to surgical exposure of the ovaries alone (sham). Blood plasma protein at 4 weeks after these operations was pooled for the ovx and sham group each and separated on SDS-PAGE, and then digested by peptides, which were first differentially displayed by ESI-Q-TOF-MS analysis. Mass spectra of peptides upregulated more than twofold in ovx compared to sham mice were selected for protein identification by ESI-Q-TOF-MS. The selected peptides were further validated in independent samples by SRM using electrospray ionized-triple quadrupole-linear ion trap mass spectrometry (ESI-QqLIT-MS). Optimum transitions for SRM were manually chosen for their high specificity in identifying peptides derived from the candidate proteins. RESULTS: Differential analysis of peptides revealed 1,108 upregulated peptides in ovx compared with sham control mice. Among the upregulated peptides, 231 nonredundant proteins were identified. Validation analysis for the potential use of these proteins as markers of bone turnover was performed using ESI-QqLIT-MS. The four proteins from the plasma samples, namely mannose-binding lectin-C, major urinary protein 2, type I collagen alpha 2 chain, and tetranectin, were evaluated in a blinded manner. A statistically significant elevation of all four proteins in the plasma of ovx mice was confirmed by SRM. Of the four upregulated plasma proteins, tetranectin increased by almost 50 times in the ovx mice compared with the sham mice. CONCLUSIONS: On the basis of proteomics analysis, this study demonstrated that four plasma proteins were significantly elevated in the ovx mice; of these, tetranectin was markedly upregulated by almost 50 times compared with the sham mice.

Evaluation of annexin A5 as a biomarker for Alzheimer's disease and dementia with lewy bodies.

BACKGROUND: Alzheimer's disease (AD) differs from other forms of dementia in its relation to amyloid beta peptide (Aß42). Using a cell culture model we previously identified annexin A5, a Ca(2+), and phospholipid binding protein, as an AD biomarker. Plasma level of annexin A5 was significantly higher in AD patients compared to that in a control group. On the other hand, AD has been identified to share a number of clinical and pathological features with Dementia with Lewy bodies (DLB). The present study was done to examine whether or not plasma annexin A5 is a specific marker for AD, when being compared with the levels of DLB patients. As Apolipoprotein E (ApoE) gene subtype ε4 (ApoE-ε4) has been noticed as the probable genetic factor for AD, we also examined and compared ApoE genotype in both AD and DLB. METHODS: Blood samples were obtained from 150 patients with AD (aged 77.6 ± 6.5 years), 50 patients of DLB (79.4 ± 5.0) and 279 community-dwelling healthy elderly individuals of comparable age and sex (75.6 ± 8.1). All AD patients met NINCDS-ADRDA criteria and all DLB patients were diagnosed as probable DLB according to the latest consensus diagnostic criteria. Quantification was done using the Chemiluminescent Enzyme Immunoassay (CLEIA) Technique (SphereLight assay) using the monoclonal antibodies against annexin A5. DNA genotyping of ApoE was performed by distinguishing unique combinations of Hha1 fragments of PCR-amplified genomic DNA products. RESULTS: The plasma level of annexin A5 was significantly higher in AD patients than in the healthy individuals (control) (P < 0.0001). The plasma annexin A5 level was also significantly higher in DLB patients than in the control group (P < 0.0001). From the ROC curves with plasma annexin A5 concentrations, the mean areas under the curve were 0.863 and 0.838 for the AD/control and DLB/control, respectively. The rate of ApoE4 carrier status and the frequency of the ε4 allele were significantly higher in AD or DLB than in control and there was no significant difference between AD and DLB. CONCLUSIONS: These results suggest that both annexin A5 and ApoE4 are common markers for AD and DLB.

Metallothionein deficiency in the injured peripheral nerves of complex regional pain syndrome as revealed by proteomics.

Complex regional pain syndrome (CRPS) is characterized by persistent and severe pain after trauma or surgery; however, its molecular mechanisms in the peripheral nervous system are poorly understood. Using proteomics, we investigated whether injured peripheral nerves of CRPS patients have altered protein profiles compared with control nerves. We obtained nerve samples from 3 patients with CRPS-2 who underwent resection of part of an injured peripheral nerve. Sural nerves from fresh cadavers with no history of trauma or neuropathic pain served as controls. Proteomic analysis showed that the number and functional distribution of proteins expressed in CRPS and control nerves was similar. Interestingly, metallothionein was absent in the injured nerves of CRPS-2, although it was readily detected in control nerves. Western blotting further confirmed the absence of metallothionein in CRPS-2 nerves, and immunohistochemistry corroborated the deficiency of metallothionein expression in injured nerves from 5 of 5 CRPS patients and 2 of 2 patients with painful neuromas. In contrast, all control nerves, including 5 sural nerves from fresh cadavers and 41 nerves obtained from surgically resected tumors, expressed MT. Furthermore, expression of S100 as a marker for Schwann cells, and neurofilament M as a marker of axons was comparable in both CRPS-2 and controls. Metallothioneins are zinc-binding proteins that are probably involved in protection against injury and subsequent regeneration after CNS damage. Their absence from the injured peripheral nerves of patients with CRPS-2 suggests a potential pathogenic role in generating pain in the damaged peripheral nerves.

Early expression of plasma CCL8 closely correlates with survival rate of acute graft-vs.-host disease in mice.

OBJECTIVE: To elucidate the significance of early expression of CC-chemokine ligand motif 8 (CCL8) in mice with graft-vs.-host disease (GVHD), we investigated its induction mechanisms and correlation with overall survival rate in GVHD mice. Plasma CCL8 increases on day 5 of allogeneic transplantation, when signs of GVHD are barely detectable. Increase of allogeneic splenocytes in grafts exacerbates GVHD and leads to upregulation of plasma CCL8 on day 5. Overall survival is the gold standard in determining the severity of acute GVHD in mice, but the absence of clinical and/or pathological manifestations in the early phase make it difficult to estimate vital outcomes at this stage of allogeneic marrow transplantation. MATERIALS AND METHODS: After lethal irradiation, BALB/c mice received bone marrow transplantation from C57BL/6 mice. Survival rate was monitored and clinical and pathological scores of GVHD were examined. Coculture of BALB/c-derived dendritic cells and C57BL/6-derived splenocytes was performed. CCL8 was measured by immunoassay. RESULTS: The plasma CCL8 level at day 5 of transplantation was closely correlated with survival rate and clinical/pathological scores on day 14. In vitro study revealed that the BALB/c-derived dendritic cells expressed CCL8 upon stimulation of C57BL/6 CD4(+) T cells by cell interactions through major histocompatibility complex class II molecules. CONCLUSIONS: These investigations indicate that early and preclinical expression of CCL8 in plasma predicts overall survival of GVHD mice. Together with an involvement of allo-recognition in CCL8 expression, it suggests that CCL8 plays an important role in GVHD pathology.

Investigation of annexin A5 as a biomarker for Alzheimer's disease using neuronal cell culture and mouse model.

Alzheimer's disease (AD) differs from other forms of dementia in its relation to amyloid beta peptide (Abeta). Abeta, a proteolytic product of amyloid precursor proteins (APP), has a toxic effect on neuronal cells, which involves perturbation of their Ca(2+) homeostasis. This effect implies that changes of protein expression in neuronal cells with calcium stress should provide a molecular marker for this disease. In the present study, we used the supernatant from a neuronal cell culture after incubation with or without Abeta and isolated a Ca(2+)-dependent acidic phospholipid binding fraction to perform a proteomic study. Several unique proteins were identified after incubation with Abeta. We focused on annexin A5, among these proteins, because it binds both Ca(2+) and lipids likely to be involved in calcium homeostasis. Tg2576 transgenic mice (AD model) overexpressing mutant human APP showed a significant increase of annexin A5 in the brain cortex but not in other organs, including liver, kidney, lung, and intestine. In human plasma samples, the level of annexin A5 was significantly increased in a proportion of AD patients compared with a control group (P < 0.0001 in the logistic regression analysis). From the receiver operating characteristic (ROC) curve with plasma annexin A5 concentrations, the mean area under the curve (AUC 0.898) suggests that annexin A5 is a favorable marker for AD.

Diacylglycerol kinase alpha enhances protein kinase Czeta-dependent phosphorylation at Ser311 of p65/RelA subunit of nuclear factor-kappaB.

We recently reported that diacylglycerol kinase (DGK) alpha enhanced tumor necrosis factor-alpha (TNF-alpha)-induced activation of nuclear factor-kappaB (NF-kappaB). However, the signaling pathway between DGKalpha and NF-kappaB remains unclear. Here, we found that small interfering RNA-mediated knockdown of DGKalpha strongly attenuated protein kinase C (PKC) zeta-dependent phosphorylation of a large subunit of NF-kappaB, p65/RelA, at Ser311 but not PKCzeta-independent phosphorylation at Ser468 or Ser536. Moreover, knockdown and overexpression of PKCzeta suppressed and synergistically enhanced DGKalpha-mediated NF-kappaB activation, respectively. These results strongly suggest that DGKalpha positively regulates TNF-alpha-dependent NF-kappaB activation via the PKCzeta-mediated Ser311 phosphorylation of p65/RelA.

Diacylglycerol kinase eta augments C-Raf activity and B-Raf/C-Raf heterodimerization.

The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase eta (DGKeta) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKeta1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKeta serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKeta-silenced HeLa cells, this study revealed that DGKeta activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKeta enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKeta physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKeta-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKeta acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.

Diacylglycerol kinase delta associates with receptor for activated C kinase 1, RACK1.

The delta-isozyme (type II) of diacylglycerol kinase (DGK) is known to positively regulate growth factor receptor signaling. DGKdelta, which is distributed to clathrin-coated vesicles, interacts with DGKdelta itself, protein kinase C and AP2alpha. To search for additional DGKdelta-interacting proteins, we screened a yeast two-hybrid cDNA library from HepG2 cells using aa 896-1097 of DGKdelta as a bait. We identified aa 184-317 (WD40 repeats 5-7) of receptor for activated C kinase 1 (RACK1), which interacts with various important signaling molecules, as a novel binding partner of DGKdelta. Co-immunoprecipitation analysis, using COS-7 cells co-expressing RACK1 and DGKdelta, revealed that RACK1 selectively interacted with DGKdelta, but not with type I DGKs, in mammalian cells. The interaction was dynamically regulated by phorbol ester. Intriguingly, DGKdelta appeared to recruit RACK1 to clathrin-coated vesicles and co-localized with RACK1. These results suggest that DGKdelta serves as an adaptor protein to regulate the localization of the versatile scaffold protein, RACK1.

Diacylglycerol kinases as emerging potential drug targets for a variety of diseases.

Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating (consuming) DAG to yield PA. Ten mammalian DGK isozymes have been identified to date. In addition to two or three cysteine-rich C1 domains (protein kinase C-like zinc finger structures) commonly conserved in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile alpha motif domain, a MARCKS (myristoylated alanine-rich C kinase substrate) phosphorylation site domain and ankyrin repeats. Recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of mammalian signal transduction pathways conducting growth factor/cytokine-dependent cell proliferation and motility, seizure activity, immune responses, cardiovascular responses and insulin receptor-mediated glucose metabolism. It is suggested that several DGK isozymes can serve as potential drug targets for cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension and type II diabetes. Unfortunately, there are no DGK isozyme-specific inhibitors/activators at present. Development of these compounds is eagerly awaited for the development of novel drugs targeting DGKs.

Regulation of enzyme localization by polymerization: polymer formation by the SAM domain of diacylglycerol kinase delta1.

The diacylglycerol kinase (DGK) enzymes function as regulators of intracellular signaling by altering the levels of the second messengers, diacylglycerol and phosphatidic acid. The DGK delta and eta isozymes possess a common protein-protein interaction module known as a sterile alpha-motif (SAM) domain. In DGK delta, SAM domain self-association inhibits the translocation of DGK delta to the plasma membrane. Here we show that DGK delta SAM forms a polymer and map the polymeric interface by a genetic selection for soluble mutants. A crystal structure reveals that DGKSAM forms helical polymers through a head-to-tail interaction similar to other SAM domain polymers. Disrupting polymerization by polymer interface mutations constitutively localizes DGK delta to the plasma membrane. Thus, polymerization of DGK delta regulates the activity of the enzyme by sequestering DGK delta in an inactive cellular location. Regulation by dynamic polymerization is an emerging theme in signal transduction.

Rab7 regulates maturation of melanosomal matrix protein gp100/Pmel17/Silv.

Melanosome biogenesis consists of multistep processes that involve synthesis of melanosomal protein, which is followed by vesicle transport/fusion and post-translational modifications such as glycosylation, proteolysis, and oligomerization. Because of its complexity, the details of the molecular mechanism of melanosome biogenesis are not yet fully understood. Here, we report that, in MMAc melanoma cells, wild-type (WT) Rab7 and its dominant-active mutant (Rab7-Q67L), but not its dominant-negative mutant (Rab7-T22N), were colocalized in the perinuclear region with granules containing Stage I melanosomes, where the full-length, immature gp100/Pmel17/Silv was present. It was also found that overexpression of Rab7-Q67L and, to a lesser extent, Rab7-WT increased the amount of proteolytically processed, mature gp100. However, Rab7-T22N did not show such an effect. Moreover, siRNA-mediated Rab7 knockdown considerably inhibited gp100 maturation. These results collectively suggest that the GTP-bound form of Rab7 promotes melanogenesis through the regulation of gp100 maturation in melanoma cells.

Phorbol ester and hydrogen peroxide synergistically induce the interaction of diacylglycerol kinase gamma with the Src homology 2 and C1 domains of beta2-chimaerin.

DGKgamma (diacylglycerol kinase gamma) was reported to interact with beta2-chimaerin, a GAP (GTPase-activating protein) for Rac, in response to epidermal growth factor. Here we found that PMA and H2O2 also induced the interaction of DGKgamma with beta2-chimaerin. It is noteworthy that simultaneous addition of PMA and H2O2 synergistically enhanced the interaction. In this case, PMA was replaceable by DAG (diacylglycerol). The beta2-chimaerin translocation from the cytoplasm to the plasma membrane caused by PMA plus H2O2 was further enhanced by the expression of DGKgamma. Moreover, DGKgamma apparently enhanced the beta2-chimaerin GAP activity upon cell stimulation with PMA. PMA was found to be mainly required for a conversion of beta2-chimaerin into an active form. On the other hand, H2O2 was suggested to induce a release of Zn2+ from the C1 domain of beta2-chimaerin. By stepwise deletion analysis, we demonstrated that the SH2 (Src homology 2) and C1 domains of beta2-chimaerin interacted with the N-terminal half of catalytic region of DGKgamma. Unexpectedly, the SH2 domain of beta2-chimaerin contributes to the interaction independently of phosphotyrosine. Taken together, these results suggest that the functional link between DGKgamma and beta2-chimaerin has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein-protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in co-operation with the C1 domain.

Tyrosine phosphorylation of beta2-chimaerin by Src-family kinase negatively regulates its Rac-specific GAP activity.

beta2-Chimaerin, an intracellular receptor for the second messenger diacylglycerol and phorbol esters, is a GTPase-activating protein (GAP) specific for Rac. beta2-Chimaerin negatively controls many Rac-dependent pathophysiological events including tumor development. However, the regulatory mechanism of beta2-chimaerin remains largely unknown. Here we report that beta2-chimaerin is tyrosine-phosphorylated by Src-family kinases (SFKs) upon cell stimulation with epidermal growth factor (EGF). Mutational analysis identified Tyr-21 in the N-terminal regulatory region as a major phosphorylation site. Intriguingly, the addition of SFK inhibitor and the replacement of Tyr-21 with Phe (Y21F) markedly enhanced Rac-GAP activity of beta2-chimaerin in EGF-treated cells. Moreover, the Y21F mutant inhibited integrin-dependent cell spreading, in which Rac1 plays a critical role, more strongly than wild-type beta2-chimaerin. These results suggest Tyr-21 phosphorylation as a novel, SFK-dependent mechanism that negatively regulates beta2-chimaerin Rac-GAP activity.

Diacylglycerol kinases: why so many of them?

Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating DAG to yield PA. To date, ten mammalian DGK isozymes have been identified. In addition to the C1 domains (protein kinase C-like zinc finger structures) conserved commonly in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile alpha motif domain and ankyrin repeats. Beyond our expectations, recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of signal transduction pathways conducting development, neural and immune responses, cytoskeleton reorganization and carcinogenesis. Moreover, there has been rapidly growing evidence indicating that individual DGK isoforms exert their specific roles through interactions with unique partner proteins such as protein kinase Cs, Ras guanyl nucleotide-releasing protein, chimaerins and phosphatidylinositol-4-phosphate 5-kinase. Therefore, an emerging paradigm for DGK is that the individual DGK isoforms assembled in their own signaling complexes should carry out spatio-temporally segregated tasks for a wide range of biological processes via regulating local, but not global, concentrations of DAG and/or PA.

Diacylglycerol kinase alpha suppresses tumor necrosis factor-alpha-induced apoptosis of human melanoma cells through NF-kappaB activation.

We investigated the implication of diacylglycerol kinase (DGK) alpha (type I isoform) in melanoma cells because we found that this DGK isoform was expressed in several human melanoma cell lines but not in noncancerous melanocytes. Intriguingly, the overexpression of wild-type (WT) DGKalpha, but not of its kinase-dead (KD) mutant, markedly suppressed tumor necrosis factor (TNF)-alpha-induced apoptosis of AKI human melanoma cells. In the reverse experiment, siRNA-mediated knockdown of DGKalpha significantly enhanced the apoptosis. The overexpression of other type I isoforms (DGKbeta and DGKgamma) had, on the other hand, no detectable effects on the apoptosis. These results indicate that DGKalpha specifically suppresses the TNF-alpha-induced apoptosis through its catalytic action. We found that the overexpression of DGKalpha-WT, but not of DGKalpha-KD, further enhanced the TNF-alpha-stimulated transcriptional activity of an anti-apoptotic factor, NF-kappaB. Conversely, DGKalpha-knockdown considerably inhibited the NF-kappaB activity. Moreover, an NF-kappaB inhibitor blunted the anti-apoptotic effect of DGKalpha overexpression. Together, these results strongly suggest that DGKalpha is a novel positive regulator of NF-kappaB, which suppresses TNF-alpha-induced melanoma cell apoptosis.

Diacylglycerol kinase gamma interacts with and activates beta2-chimaerin, a Rac-specific GAP, in response to epidermal growth factor.

Diacylglycerol kinase (DGK)gamma was shown to act as an upstream suppressor of Rac1. Here we report that, in COS7 cells stimulated with epidermal growth factor (EGF), DGKgamma specifically interacts and co-localizes at the plasma membrane with beta2-chimaerin, a GTPase-activating protein (GAP) for Rac. Moreover, DGKgamma enhanced EGF-dependent translocation of beta2-chimaerin to the plasma membrane. Interestingly, DGKgamma markedly augmented EGF-dependent GAP activity of beta2-chimaerin through its catalytic action. These results indicate that DGKgamma is a novel regulator of beta2-chimaerin, and thus suggest that beta2-chimaerin is an effector molecule, linking DGKgamma functionally with Rac1.

Lipid phosphate phosphatases 1 and 3 are localized in distinct lipid rafts.

Lipid phosphate phosphatases (LPPs), integral membrane proteins with six transmembrane domains, dephosphorylate a variety of extracellular lipid phosphates. Although LPP3 is already known to bind to Triton X-100-insoluble rafts, we here report that LPP1 is also associated with lipid rafts distinct from those harboring LPP3. We found that LPP1 was Triton X-100-soluble, but CHAPS-insoluble in LNCaP cells endogenously expressing LPP1 and several LPP1 cDNA-transfected cells including NIH3T3 fibroblasts. In addition to the non-ionic detergent insolubility, LPP1 further possessed several properties formulated for raft-localizing proteins as follows: first, the CHAPS-insolubility was resistant to the actin-disrupting drug cytochalasin D; second, the CHAPS-insoluble LPP1 floated in an Optiprep density gradient; third, the CHAPS insolubility of LPP1 was lost by cholesterol depletion; and finally, the subcellular distribution pattern of LPP1 exclusively overlapped with that of a raft marker, cholera toxin B subunit. Interestingly, confocal microscopic analysis showed that LPP1 was distributed to membrane compartments distinct from those of LPP3. Analysis using various LPP1/LPP3 chimeras revealed that their first extracellular regions determine the different Triton X-100 solubilities. These results indicate that LPP1 and LPP3 are distributed in distinct lipid rafts that may provide unique microenvironments defining their non-redundant physiological functions.

Identification and characterization of a novel human type II diacylglycerol kinase, DGK kappa.

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. Here we identified a tenth member of the DGK family designated DGK kappa. The kappa-isozyme (1271 amino acids, calculated molecular mass, 142 kDa) contains a pleckstrin homology domain, two cysteine-rich zinc finger-like structures, and a separated catalytic region as have been found commonly for the type II isozymes previously cloned (DGKdelta and DGKeta). The new DGK isozyme has additionally 33 tandem repeats of Glu-Pro-Ala-Pro at the N terminus. Reverse transcriptase-PCR showed that the DGK kappa mRNA is most abundant in the testis, and to a lesser extent in the placenta. DGK kappa, when expressed in HEK293 cells, was persistently localized at the plasma membrane even in the absence of cell stimuli. Deletion analysis revealed that the short C-terminal sequence (amino acid residues 1199-1268) is necessary and sufficient for the plasma membrane localization. Interestingly, DGK kappa, but not other type II DGKs, was specifically tyrosine-phosphorylated at Tyr78 through the Src family kinase pathway in H2O2-treated cells. Moreover, H2O2 selectively inhibited DGK kappa activity in a Src family kinase-independent manner, suggesting that the isozyme changes the balance of signaling lipids in the plasma membrane in response to oxidative stress. The expression patterns, subcellular distribution, and regulatory mechanisms of DGK kappa are distinct from those of DGKdelta and DGKeta despite high structural similarity, suggesting unique functions of the individual type II isozymes.

The plasma membrane translocation of diacylglycerol kinase delta1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the pleckstrin homology domain.

DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKdelta1 or its PH (pleckstrin homology) domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKdelta1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKdelta1 and its PH domain are still persistently translocated by PMA treatment, suggesting that the PH domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKdelta1 and the PH domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKdelta1 PH domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKdelta1.