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.

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).

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 (Aβ42). 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

Diacylglycerol kinase gamma serves as an upstream suppressor of Rac1 and lamellipodium formation.

Nine diacylglycerol kinase (DGK) isozymes have been identified. However, our knowledge of their individual functions is still limited. Here, we demonstrate the role of DGKgamma in regulating Rac1-governed cell morphology.

Identification and characterization of two splice variants of human diacylglycerol kinase eta.

Diacylglycerol kinase (DGK) participates in regulating the intracellular concentrations of two bioactive lipids, diacylglycerol and phosphatidic acid. DGK eta (eta 1, 128 kDa) is a type II isozyme containing a pleckstrin homology domain at the amino terminus. Here we identified another DGK eta isoform (eta 2, 135 kDa) that shared the same sequence with DGK eta 1 except for a sterile alpha motif (SAM) domain added at the carboxyl terminus.

Regulatory role of diacylglycerol kinase gamma in macrophage differentiation of leukemia cells.

Although nine diacylglycerol kinase (DGK) isozymes have been identified, our knowledge of their individual functions is still limited. Here we report that the levels of DGKgamma mRNA/protein in human leukemia HL-60 and U937 cells were rapidly and markedly decreased upon cellular differentiation into macrophages. In contrast, the enzyme expression remained almost unchanged in granulocytic differentiation pathway.

Alternative splicing of the human diacylglycerol kinase delta gene generates two isoforms differing in their expression patterns and in regulatory functions.

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. DGKdelta (type II isozyme) contains a pleckstrin homology domain at the N terminus and a sterile alpha motif domain at the C terminus. We identified another DGKdelta isoform (DGKdelta2, 135 kDa) that shared the same sequence with DGKdelta previously cloned (DGKdelta1, 130 kDa) except for the 52 residues N-terminally extended.

Phorbol ester-regulated oligomerization of diacylglycerol kinase delta linked to its phosphorylation and translocation.

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. In yeast two-hybrid screening, we unexpectedly found a self-association of the C-terminal part of DGKdelta containing a sterile alpha-motif (SAM) domain. We then bacterially expressed the SAM domain fused with maltose-binding protein and confirmed the formation of dimeric and tetrameric structures.