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Recent reports have described the involvement of the diacylglycerol kinase (DGK) family in various pathological conditions. In an animal model of transient ischemia, DGKζ containing a nuclear localization signal (NLS) is shown to translocate quickly from the nucleus to the cytoplasm in hippocampal neurons and to disappear gradually after reperfusion. Those neurons die a delayed neuronal death because of glutamate excitotoxicity. This study investigated the molecular mechanism and functional relation linking DGKζ and neuronal death. In primary cultured neurons, transient exposure to excitotoxic concentration of glutamate led to cytoplasmic accumulation of DGKζ followed by its down-regulation. Results showed that DGKζ down-regulation was caused by proteolytic degradation through the ubiquitin–proteasome system (UPS) rather than transcriptional inhibition. DGKζ polyubiquitination was inhibited in the presence of nuclear export inhibitor leptomycin B. Furthermore, NLS-deleted mutant DGKζΔNLS, which mainly localizes to the cytoplasm, was ubiquitinated more heavily than wild-type DGKζ. From a functional perspective, in vitro gene silencing of DGKζ via specific siRNA enhanced DNA fragmentation in cultured neurons after glutamate exposure. At the organismal level, hippocampal neurons of DGKζ-deficient mice showed vulnerability to kainate-induced seizures. In addition, DGKζ-deficient hippocampus exhibited a significant increase in Ser807/811 phosphorylated retinoblastoma protein levels together with up-regulation of the expression of type D and E cyclins, indicative of cell cycle reentry. Collectively, these results suggest that 1) glutamate excitotoxicity induces nucleocytoplasmic translocation of DGKζ followed by its degradation through the cytoplasmic UPS in hippocampal neurons and that 2) DGKζ-deficient neurons do not succumb directly to apoptosis, although they are more vulnerable to excitotoxicity because of aberrant cell cycle reentry.

Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGKζ-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGKζ or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKζ−/− mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGKζ is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages.

Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGKζ, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGKζ. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGKζ binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGKζ and NAP1Ls prohibits nuclear import of DGKζ because binding of NAP1Ls to DGKζ blocks import carrier proteins, Qip1 and NPI1, to interact with DGKζ, leading to cytoplasmic tethering of DGKζ. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGKζ and provide a clue to examine functional significance of its translocation under pathological conditions.

The effects of geometric properties of membranes on the structure of the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain were investigated using solid state (13)C NMR spectroscopy. Conformations of the PLC-delta1 PH domain at the surfaces of multilamellar vesicles (MLV), small unilamellar vesicles (SUV), and micelles were examined to evaluate the effects of membrane curvature on the PH domain. An increase in curvature of the water-hydrophobic layer interface hinders membrane-penetration of the amphipathic alpha2-helix of the PH domain that assists the membrane-association of the PH domain dominated by the phosphatidylinositol 4,5-bisphosphate (PIP(2)) specific lipid binding site. The solid state (13)C NMR signal of Ala88 located at the alpha2-helix indicates that the conformation of the alpha2-helix at the micelle surface is similar to the solution conformation and significantly different from those at the MLV and SUV surfaces which were characterized by membrane-penetration and re-orientation. The signal of Ala112 which flanks the C-terminus of the beta5/beta6 loop that includes the alpha2-helix, showed downfield displacement with decrease in the interface curvature of the micelles, SUV and MLV. This reveals that the conformation of the C-terminus of the beta5/beta6 loop connecting the beta-sandwich core containing the PIP(2) binding site and the amphipathic alpha2-helix is sensitive to alterations of the curvature of lipid bilayer surface. It is likely that these alterations in the conformation of the PLC-delta1 PH domain contribute to the regulatory mechanisms of the intracellular localization of PLC-delta1 in a manner dependent upon the structure of the molecular complex containing PIP(2).

START-GAP2, also termed as DLC2, is a START domain-containing RhoGAP and a negative regulator of RhoA and Cdc42. Although it was reported as a tumor suppresser gene product, the molecular basis for function of START-GAP2 remains to be clarified. Here, we demonstrate that START-GAP2 is localized in focal adhesions through a "FAT (focal adhesion targeting)" region in the N-terminal half. START-GAP2 competes with START-GAP1/DLC1, another START domain-containing RhoGAP, in focal adhesion targeting. Moreover, the C-terminus of tensin2, one of focal adhesion components and reported to bind START-GAP1, also directly interacts with START-GAP2. These results suggest that START-GAP2 and START-GAP1 share the same molecular mechanism in targeting to focal adhesions.

Dynamin 2 has been reported to be implicated in phagocytosis. However, the mode of action of dynamin is poorly understood. In this study, we examined whether dynamin 2 participates in actin assembly during phagocytosis in Sertoli cells. In the presence of dynasore, a dynamin inhibitor, phagocytosis was reduced by 60-70% in Sertoli cells and macrophages. Scanning electron microscopy revealed that Sertoli cells treated with dynasore were unable to form phagocytic cups. In addition, dysfunction of dynamin 2 reduced both actin polymerization and recruitment of actin and dynamin 2 to phosphatidylinositol (4,5) bisphosphate [PI(4,5)P{sub 2}]-containing liposomes. The formation of dynamin 2-positive ruffles of Sertoli cells was decreased by 60-70% by sequestering PI(4,5)P{sub 2} either by expression of PH domain of PLC{delta} or treatment with neomycin. These results strongly suggest that dynamin 2 is involved in actin dynamics and the formation of dynamin 2-positive ruffles during phagocytosis.

Two types of immunosuppressants, cycloprodigiosin hydrochloride (cPrG) and l -leucyl- l -leucine methyl ester (LeuLeuOMe), both have the ability to selectively inhibit the lysosomal function, and a related compound to cPrG, prodigiosin 25-C, and LeuLeuOMe have been reported to selectively inhibit the T cell function in vitro. We therefore examined the cell-type specificity of cPrG and LeuLeuOMe using murine splenocytes. Concanavalin A (Con A)- and lentil lectin-induced proliferation was suppressed by cPrG more profoundly than lipopolysaccharide-induced proliferation. At the optimal concentration, Con A induced the proliferation of both CD4+ and CD8+ cells, whereas at a supra-optimal concentration Con A induced rather selective proliferation of CD8+ cells. Irrespective of the dose of Con A, CD4+ and CD8+ cells were equally affected by cPrG. In contrast, LeuLeuOMe induced the selective loss of CD8+ cells. cPrG enhanced the apoptosis of murine splenocytes and nylon fiber column-purified T cells cultured in the presence of Con A, as shown by the decrease in cell size and/or DNA fragmentation. Overall, this study revealed that the cell-type specificity of cPrG is different from that of LeuLeuOMe, and that the immunosuppression by cPrG is associated with apoptosis.

We have previously observed, using a green fluorescent protein (GFP) fusion system, that PLC-delta1 is localized mainly at the plasma membrane and in the cytosol, whereas little is present in the nucleus in Madin-Darby canine kidney cells (Fujii, M., Ohtsubo, M., Ogawa, T., Kamata, H., Hirata, H., and Yagisawa, H. (1999) Biochem. Biophys. Res. Commun. 254, 284-291). Herein, we demonstrate that PLC-delta1 has a functional nuclear export signal (NES) sequence in amino acid residues 164-177 of the EF-hand domain. The fluorescence of NES-disrupted GFP/PLC-delta1 expressed in Madin-Darby canine kidney cells was present not only at the plasma membrane and in the cytosol but also in the nucleus. Moreover, treatment with leptomycin B, a specific inhibitor of NES-dependent nuclear export, resulted in the accumulation of GFP/PLC-delta1 in the nucleus. A site-directed mutant containing a pleckstrin homology domain, which does not bind inositol 1,4,5-trisphosphate and cannot hydrolyze phosphatidylinositol 4,5-bisphosphate in vitro, accumulated in the nucleus to a much greater extent than wild-type GFP/PLC-delta1 after treatment with leptomycin B. These results suggest that PLC-delta1 is shuttled between the cytoplasm and the nucleus; its nuclear export is dependent on the leucine-rich NES sequence and its active nuclear import is regulated by an unidentified signal(s).

Green fluorescent protein (GFP)-tagged phospholipase C (PLC)-delta1 and its mutants were expressed in Madin-Darby canine kidney (MDCK) cells. GFP-PLC-delta1 or the GFP-tagged pleckstrin homology (PH) domain of PLC-delta1 itself was found to be predominantly localized at the plasma membrane. The DeltaPH mutant or a site-directed mutant containing a PH domain which does not bind inositol 1,4, 5-trisphosphate and cannot hydrolyze phosphatidylinositol 4, 5-bisphosphate in vitro was seen only in the cytosol. In living MDCK cells hypo-osmotic stress caused a rapid dissociation of GFP-PLC-delta1 from the plasma membrane, which coincided with phosphoinositide breakdown. A PLC inhibitor, U73122, blocked this translocation, but depletion of extracellular Ca2+ had no effect. The translocation was reversed by replacement with an iso-osmotic buffer. Our results demonstrate that the PH domain plays a critical role in the membrane targeting of PLC-delta1 and that the intracellular distribution of the enzyme is regulated by osmotic stress-driven phosphoinositide turnover.

The efficacy of Interferon (IFN) therapy against hepatitis C is greatly affected by viral factors such as the level of viremia and the HCV genotype. The prognosis of those with high serum HCV-RNA levels in genotype 1b is poor. In the present report, we carried out a randomized controlled study of two IFN doses for patients with genotype 1b chronic hepatitis C whose serum HCV-RNA levels were at least 10 6 copies/ml as determined by multicyclic polymerase chain reaction (PCR) assay. These patients were randomly divided into two groups. Group A received 6 million units (MIU) of IFN 6 days per week for 8 weeks, followed by the same dose 3 days per week for 16 weeks (total IFN dose: 576 MIU). Group B received 6 MIU of IFN 6 days per week for 2 weeks, followed by the same dose 3 days per week for 22 weeks (total IFN dose: 480 MIU). The number of complete responders was four of 24 (16.6%) in group A and three of 20 (15.0%) in group B. There was no significant difference between the groups. On the basis of these findings, it appears that there is no advantage in lengthening the initial period of consecutive IFN administration to 8 weeks or in increasing the total dose by 100 MIU for those with genotype 1b hepatitis C and high virus levels.

The cellular redox state is thought to play an important role in a wide variety cellular signaling pathways. Here, we investigated the involvement of redox regulation in the nerve growth factor (NGF) signaling pathway and neuronal differentiation in PC12 cells. N-acetyl-L-cysteine (NAC), which acts as a reductant in cells both by its direct reducing activity and by increasing the synthesis of the cellular antioxidant glutathione, inhibited neuronal differentiation induced by NGF or by the expression of oncogenic ras in PC12 cells. NAC suppressed NGF-induced c-fos gene expression and AP-1 activation. These results suggest that neuronal differentiation and NGF signaling are subject to regulation by the cellular redox state. NAC also suppressed the NGF-induced activation of mitogen-activated protein kinases (MAPKs) and decreased the amount of tyrosine phosphorylation of MAPKs. The suppression of MAPK by NAC was independent of glutathione synthesis. In parallel with the suppression of MAPK, the activation of MAPK kinase kinase activity was also suppressed in the presence of NAC. In contrast, NGF-induced activation of Ras was not inhibited by NAC. The inhibitory effect of NAC on the MAPK cascade was independent of transcription and translation. Thus, NAC suppresses NGF-induced neuronal differentiation by uncoupling the signal transduction from Ras to the MAP kinase cascade in PC12 cells.

Objective: We undertook this study to investigate the appearance of the diaphragmatic echo behind liver tumors and to analyze the possible mechanism of this artifact by using a microcomputer simulation model. Subjects and methods: (1) Sonograms of 76 liver tumors in the right intercostal scanning plane were analyzed. These 76 tumors consisted of 22 hepatocellular carcinomas (HCC), 23 liver metastasis and 31 hemangiomas. The appearances of the post-tumoral diaphragmatic echo were evaluated with respect to the remaining diaphragmatic echo. (2) We evaluated how the sound refraction through the liver tumor affects the displayed diaphragmatic echo, by using microcomputer simulation model. Results: (1) A clockwise rotation of the diaphragmatic echo was frequently seen in the metastasis group in comparison with the HCC or hemangioma groups (P

We demonstrated that a 65-kDa cytosolic protein (pp65) was most heavily phosphorylated in murine macrophages in response to bacterial lipopolysaccharide ( J. Immunol. 146:3617,1991). The pp65 phosphorylation closely correlated with cellular responses in the macrophages. We have thus performed amino acid sequence analysis of the two pp65-derived peptide fragments in order to elucidate its structure and function. The results indicated that pp65 is a novel protein existing almost exclusively in hemopoitetic cells and has the strong homology with human L-plastin, a substrate which has recently been identified as a novel protein transformation-dependently expressed in neoplastic human cells. Our pp65 is apparently the first purified protein whose phosphorylation has been augmented by stimulation with bacterial lipopolysaccharide and whose primary structure has been clarified. Because L-plastin possesses the unique functional domains, detailed investigation of the pp65 phosphorylation should lead to progress in our understanding of the mechanisms of macrophage activation.

It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-immobilized matrix was the delta 1 isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-delta 1 in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P3 binding. The lysate was applied to Ins(1,4,5)P3-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100-kDa protein showed high Ins(1,4,5)P3 binding. The lysate was also purified to near homogeneity using a glutathione-Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme. PLC-delta 1 consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme. Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P3 binding domain in the molecule. Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding. A 76-kDa proteolytic product of the expressed PLC-delta 1 which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P3 binding activity. A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-delta 1, which contains 6 basic amino acids, binds to an Ins(1,4,5)P3-immobilized matrix. Moreover, Ins(1,4,5)P3 binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-delta 1 is important for the binding of both Ins(1,4,5)P3 and phosphatidylinositol 4,5-bisphosphate.

A human astrocytoma cell line, U373, and its subclones showed a high proliferative response to IL1 alpha or IL1 beta with concomitant IL6 production and IL6 mRNA accumulation. IL1 itself raised neither the cAMP level nor the intracellular Ca2+ level nor did it induce phosphatidylinositol (PI) breakdown. Chorela toxin (CT) and pertussis toxin (PT)-pretreatment markedly inhibited IL1-induced proliferation, while CT enhanced IL1-induced IL6 mRNA expression significantly. Drugs raising cellular cAMP level or cAMP analogues augmented IL1-mediated IL6 mRNA expression but much less. Although cAMP is not directly involved in the IL1 action, cAMP thus has a modulatory effect on IL1-mediated IL6 gene activation.

Role of protein kinase C (PKC) in interleukin (IL) 2-induced proliferation was investigated by utilizing two murine IL 2-dependent cell lines, CT6 and CTLL-2 cell lines. CT6 cells showed a marked proliferative response to phorbol 12-myristate 13-acetate (PMA), while CTLL-2 did not. PMA induced PKC translocation from cytosol to membrane only in a PMA-responsive cell line. IL 2 failed to stimulate PKC translocation in both cell lines. H-7, a potent and specific PKC inhibitor, however, inhibited the proliferation of both cell lines induced by IL 2. Taken collectively, IL 2 may induce PKC activation without its translocation.