Yukio Okano

Inhibitory action of cyclic GMP on secretion, polyphosphoinositide hydrolysis and calcium mobilization in thrombin-stimulated human platelets

The effect of cyclic GMP (cGMP) on human platelet activation was investigated, using its metabolically stable analogue, 8-bromo cGMP (8-bcGMP). Thrombin-induced serotonin secretion was inhibited by pretreatment with 8bcGMP in a dose-dependent manner. Production of inositol trisphosphate (IP3), a Ca2+ releaser was inhibited by 8bcGMP pretreatment of platelets. Preincubation of platelets with 8bcGMP was without effect on the basal level of cytosolic free Ca2+, measured by fluorescent indicator quin2, but suppressed its thrombin-induced enhancement independently of extracellular Ca2+. These results indicate that cGMP may be implicated in phospholipase C activation and Ca2+ mobilization (both influx through the plasma membrane and efflux from internal stores) in thrombin-activated human platelets.

The RING finger protein RNF8 recruits UBC13 for lysine 63‐based self polyubiquitylation

The heterodimeric ubiquitin conjugating enzyme (E2) UBC13‐UEV mediates polyubiquitylation through lysine 63 of ubiquitin (K63), rather than lysine 48 (K48). This modification does not target proteins for proteasome‐dependent degradation. Searching for potential regulators of this variant polyubiquitylation we have identified four proteins, namely RNF8, KIA00675, KF1, and ZNRF2, that interact with UBC13 through their RING finger domains. These domains can recruit, in addition to UBC13, other E2s that mediate canonical (K48) polyubiquitylation. None of these RING finger proteins were known previously to recruit UBC13. For one of these proteins, RNF8, we show its activity as a ubiquitin ligase that elongates chains through either K48 or K63 of ubiquitin, and its nuclear co‐localization with UBC13. Thus, our screening reveals new potential regulators of non‐canonical polyubiquitylation.

Sphingosine Kinase Isoforms Regulate Oxaliplatin Sensitivity of Human Colon Cancer Cells through Ceramide Accumulation and Akt Activation

The relationship between sphingosine kinase (SPHK), cellular ceramide concentration and chemosensitivity was investigated in human colon cancer cell lines. Among nine colon cancer cell lines, SPHK1 and SPHK2 activity and protein expression was highest in RKO cells and lowest in HCT116 cells. A viability assay revealed that HCT116 cells were sensitive to the effects of oxaliplatin (l-OHP), whereas RKO cells were resistant to those of l-OHP. Treatment with 5μg/ml l-OHP induced a marked time-dependent increase in various ceramides (C16, C24, C24:1) in HCT116 cells but not in RKO cells, as indicated by liquid chromatography/mass spectrometry. The increase in ceramide and caspase activation induced by l-OHP in the sensitive HCT116 cells was abolished by pretreatment with a neutral sphingomyelinase inhibitor, suggesting that the ceramide formation was due to the activation of neutral, rather than acid, sphingomyelinase. In contrast, in l-OHP-resistant RKO cells, treatment with an SPHK inhibitor or SPHK1 and SPHK2 silencing by RNA interference suppressed cell viability and increased caspase activity and cellular ceramide formation after l-OHP treatment. The elevated ceramide formation induced by SPHK inhibition and l-OHP was inhibited by fumonisin B1 but not myriocin, suggesting that ceramide formation was through the salvage pathway. Endogenous phosphorylated Akt levels were much higher in the resistant RKO cells than in the sensitive HCT116 cells. Either SPHK1 or SPHK2 silencing in RKO cells decreased phosphorylated Akt levels and increased p53 and p21 protein levels as well as poly(ADP-ribose) polymerase cleavage in response to l-OHP treatment. These findings indicate that SPHK isoforms and neutral sphingomyelinase contribute to the regulation of chemosensitivity by controlling ceramide formation and the downstream Akt pathway in human colon cancer cells.

Bradykinin-induced generation of inositol 1,4,5-trisphosphate in fibroblasts and neuroblastoma cells: Effect of pertussis toxin, extracellular calcium, and down-regulation of protein kinase C

The net content of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was measured in bradykinin (BK)-stimulated NIH3T3 fibroblasts and neuroblastoma-glioma hybrid cells (NG108-15). BK-mediated production of Ins(1,4,5)P3 was not affected by replacing the medium with Ca2+-free medium, but addition of EGTA (1mM) to Ca2+-free medium markedly prevented production of Ins(1,4,5)P3. Although pertussis toxin (PT) treatment caused ADP-ribosylation in both NIH3T3 cells and NG108-15 cells, the BK-induced Ins(1,4,5)P3 formation was considerably reduced in the former cells but not in the latter cells, suggesting that PT-sensitive and PT-insensitive GTP-binding proteins are involved in phosphoinositide phospholipase C (PI-PLC) activation in fibroblasts and neuroblastoma cells, respectively. In NG108-15 cells down-regulated in protein kinase C (PKC) by long-term exposure to phorbol 12-myristate 13-acetate (PMA), BK-stimulated Ins(1,4,5)P3 accumulation was significantly enhanced compared to control cells.

A wasp venom mastoparan‐induced polyphosphoinositide breakdown in rat peritoneal mast cells

The phospholipid metabolism of rat peritoneal mast cells stimulated with mastoparan, a secretagogue purified from wasp venom, was investigated. Mastoparan at 20 caused a rapid secretion of histamine. Mastoparan induced a transient decrease of phosphatidylinositol 4,5‐bisphosphate on 32P labeling and generation of a water‐soluble degradation product, inositol trisphosphate on [3H]inositol labeling, suggesting the activation of phospholipase C upon stimulation.

Inhibition by cyclic AMP of guanine nucleotide‐induced activation of phosphoinositide‐specific phospholipase C in human platelets

Phosphoinositide‐specific phospholipase C (PLC) activity of human platelet membranes was activated by the nonhydrolyzable guanine nucleotide GTPγS. This activation did not occur in either membranes prepared from dibutyryl cyclic AMP‐pretreated platelets (A‐membranes) or those prepared from untreated cells and subsequently incubated with cyclic AMP (cAMP) (B‐membranes). This cAMP‐mediated inhibition was abolished in the presence of inhibitors of cAMP‐dependent protein kinase (A‐kinase), suggesting that the inhibition was due to phosphorylation of (a) protein component(s). No significant differences were observed in the basal PLC activity and the extent of pertussis toxin‐catalyzed ADP‐ribosylation among control membranes and the two types of phosphorylated membranes (A‐ and B‐membranes). GTP‐binding activities of Gs, Gi and GTP‐binding proteins of lower molecular masses were not altered by the phosphorylation of the membranes. These findings suggest that a GTP‐binding protein is involved in the GTPγS‐mediated activation of PLC and that cAMP (plus A‐kinase) inhibits this activation by phosphorylating a membrane protein (probably a 240‐kDa protein), rather than the GTP‐binding protein or PLC itself. It is likely that this phosphorylation uncouples the GTP‐binding protein from PLC.

Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108‐15 cells

Effects on [Ca2+]i levels of endothelin‐1 (ET) and vasoactive intestinal contractor peptide (VIC), which is a novel member of the endothelin family, were examined in fura 2‐loaded neuroblastoma NG108‐15 cells. VIC was found to be a very effective stimulus for intracellular Ca2+ mobilization and to be more potent than ET. Intracellular calcium response to sequential addition of two stimulants exhibited the homologous desensitization of either ET or VIC, but no heterologous desensitization between ET and VIC. This indicates evidence suggesting that these two peptides act through distinct receptors.

Mitotic catastrophe and cell death induced by depletion of centrosomal proteins

Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, γ-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization.

The ram: A novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library

A novel low Mr GTP‐binding protein cDNA was isolated from a rat megakaryocyte cDNA library with a synthetic oligonucleotide probe corresponding to an 8‐amino acid sequence specific for c25KG, a GTP‐binding protein previously isolated from human platelet cytosol fraction [(1989) J. Biol. Chem. 264, 17000–17005]. The cDNA has an open reading frame encoding a protein of 221 amino acids with a calculated Mr of 25068. The protein is designated as ram (ras‐related gene from megakaryocyte) protein (ram p25). The amino acid sequence deduced from the ram cDNA contains the consensus sequences for GTP‐binding and GTPase domains. ram p25 shares about 23%, 39% and 80% amino acid homology with the H‐ras, smg25A and c25KG proteins, respectively. The 3.5‐kb ram mRNA was detected abundantly in spleen cells.

Different protein kinase C isozymes could modulate bradykinin-induced extracellular calcium-dependent and -independent increases in osteoblast-like MC3T3-E1 cells

Effects of protein kinase C (PKC) on bradykinin (BK)-induced intracellular calcium mobilization, consisting of rapid Ca2+ release from internal stores and a subsequent sustained Ca2+ inflow, were examined in Fura-2-loaded osteoblast-like MC3T3-E1 cells. The sustained Ca2+ inflow as inferred with Mn2+ quench method was blocked by Ni2+ and a receptor-operated Ca2+ channel blocker SK&F 96365, but not by nifedipine. The short-term pretreatment with phorbol 12-myristate 13-acetate (PMA), inhibited BK-stimulated Ca2+ inflow, and the prior treatment with PKC inhibitors, H-7 or staurosporine, enhanced the initial internal release and reversed the PMA effect. Moreover, 6 h pretreatment with PMA caused similar effect on the BK-induced inflow to that obtained with PKC inhibitors, whereas 24 h pretreatment was necessary to affect the internal release. On the other hand, the translocation and down-regulation of PKC isozymes were examined after PMA treatment of MC3T3-E1 cells by immunoblot analyses of PKCs with the isozyme-specific antibodies. 6 h treatment with PMA induced down-regulation of PKC beta, whereas longer treatment was needed for down-regulation of PKC alpha. Taken together, it was suggested that the BK-induced initial Ca2+ peak and the sustained Ca2+ inflow through the activation of a receptor-operated Ca2+ channel, are differentially regulated by PKC isozymes alpha and beta, respectively, in osteoblast-like MC3T3-E1 cells.