Activated Notch1 interacts with p53 to inhibit its phosphorylation and transactivation.

We propose a biochemical mechanism for the negative role of Notch signaling on p53 transactivation function. Expression of the intracellular domain of human Notch1 (Notch1-IC) inhibits the expression of p53-responsive genes p21, mdm2, and bax in HCT116 p53(-/-) cells. Furthermore, Notch1-IC expression inhibits the phosphorylation of ectopically expressed p53 in HCT116 p53(-/-) cells as well as the phosphorylation of endogenous p53 in UV-treated HCT116 p53(+/+) cells. Transcriptional downregulation of p53-responsive genes by Notch1-IC was confirmed both by chromatin immunoprecipitation assay and Northern blot analysis. We found the intracellular interaction between Notch1-IC and p53 in HCT116 p53(+/+) cells and suggest that activated Notch1 interaction with p53 is an important cellular event for the inhibition of p53-dependent transactivation. The N-terminal fragment of Notch1-IC, which can interacts with p53, inhibits p53 phosphorylation and represses p53 transactivation. In addition, Notch signaling downregulated p53-dependent apoptosis induced by UV irradiation.

Tob-mediated cross-talk between MARCKS phosphorylation and ErbB-2 activation.

The biochemical path for the activation of ErbB-2 by PKC activator was investigated in MDA-MB-231 human breast cancer cells. We found that PMA-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) increased its binding with Tob that exerts an anti-proliferative effect through the binding with ErbB-2. The phosphorylation site domain (PSD) of MARCKS was relevant to its interaction with Tob. Decreased binding of Tob with ErbB-2 and subsequent activation of ErbB-2 were observed in MDA-MB-231 cells in response to PMA treatment. The present study proposes that MARCKS phosphorylation by PKC removes Tob from ErbB-2 by increasing its binding affinity with Tob, and thereby activates the ErbB-2 mediated signal transduction.

Negative regulation of the SAPK/JNK signaling pathway by presenilin 1.

Presenilin 1 (PS1) plays a pivotal role in Notch signaling and the intracellular metabolism of the amyloid beta-protein. To understand intracellular signaling events downstream of PS1, we investigated in this study the action of PS1 on mitogen-activated protein kinase pathways. Overexpressed PS1 suppressed the stress-induced stimulation of stress-activated protein kinase (SAPK)/c-Jun NH(2)-terminal kinase (JNK) in human embryonic kidney 293 cells. Interestingly, two functionally inactive PS1 mutants, PS1(D257A) and PS1(D385A), failed to inhibit UV-stimulated SAPK/JNK. Furthermore, H(2)O(2-) or UV-stimulated SAPK activity was higher in mouse embryonic fibroblast (MEF) cells from PS1-null mice than in MEF cells from PS(+/+) mice. MEF(PS1(-/-)) cells were more sensitive to the H(2)O(2)-induced apoptosis than MEF(PS1(+/+)) cells. Ectopic expression of PS1 in MEF(PS1(-/-)) cells suppressed H(2)O(2)-stimulated SAPK/JNK activity and apoptotic cell death. Together, our data suggest that PS1 inhibits the stress-activated signaling by suppressing the SAPK/JNK pathway.

Role of receptor-interacting protein in tumor necrosis factor-alpha -dependent MEKK1 activation.

Receptor-interacting protein (RIP), a death domain serine/threonine kinase, has been shown to play a critical role in tumor necrosis factor-alpha (TNF-alpha)-induced activation of the nuclear factor-kappaB signaling pathway. We demonstrate here that ectopically expressed RIP induces I-kappaB kinase-beta (IKKbeta) activation in intact cells and that RIP-induced IKKbeta activation can be blocked by a kinase-inactive form of MEKK1, MEKK1(K1253M). Interestingly, RIP physically associated with MEKK1 both in vitro and in vivo. RIP phosphorylated MEKK1 at Ser-957 and Ser-994. Our data also indicate that RIP induced the stimulation of MEKK1 but not MEKK1(S957A/S994A) in transfected cells. Furthermore, overexpressed MEKK1(S957A/S994A) inhibited the RIP-induced activation of both IKKbeta and nuclear factor-kappaB. We also demonstrated that the TNF-alpha-induced MEKK1 activation was defective in RIP-deficient Jurkat cells. Taken together, our results suggest that RIP phosphorylates and activates MEKK1 and that RIP is involved in TNF-alpha-induced MEKK1 activation.

Nitric oxide is involved in the immunomodulating activities of acidic polysaccharide from Panax ginseng.

The effects of an acidic polysaccharide isolated from the ethanol-insoluble and water-soluble fraction of Panax ginseng C. A. Meyer on immunomodulating activities were investigated. A high output nitric oxide synthase (iNOS) was shown in female BALB/c mice administered intraperitoneally with the acidic polysaccharide from ginseng. Newly synthesized iNOS protein was also observed in peritoneal macrophages cultured with interferon-gamma and the acidic polysaccharide. Spleen cells from acidic polysaccharide-treated mice did not proliferate in response to concanavalin A, but restored the responsiveness by the cotreatment of NG-monomethyl-L-arginine (NMMA) with concanavalin A. The treatment of mice with aminoguanidine, a specific iNOS inhibitor, alleviated the acidic polysaccharide-induced suppression of antibody response to sheep red blood cells. Present results suggest that the immunomodulating activities of the acidic polysaccharide were mediated by the production of nitric oxide.

Activation of death-inducing signaling complex (DISC) by pro-apoptotic C-terminal fragment of RIP.

The two opposite signaling pathways that stimulate NF-kappaB activation and apoptosis are both mediated by tumor necrosis factor receptor 1 (TNFR1) and its cytosolic associated proteins. In this study, we demonstrate that the proteolytic cleavage of receptor interacting protein (RIP) by caspase-8 during TNF-induced apoptosis abrogates the stimulatory role of RIP on TNF-induced NF-kappaB activation. The uncleavable RIPD324A mutant was less apoptotic, but its ability to activate NF-kappaB activation was greater than the wild type counterpart. Ectopic expression of the pro-apoptotic C-terminal fragment of RIP inhibited TNF-induced NF-kappaB activation by suppressing the activity of I-kappaB kinasebeta (IKKbeta) which phosphorylates I-kB, an inhibitor of NF-kappaB, and triggers its ubiquitin-mediated degradation. The C-terminal fragment of RIP also enhanced the association between TNFR1 and death domain proteins including TNFR1 associated death domain (TRADD) and Fas associated death domain (FADD), resulting in the activation of caspase-8 and stimulation of apoptosis. The present study suggest that the C-terminal fragment of RIP produced by caspase-8 activates death-inducing signaling complex (DISC), attenuates NF-kappaB activation, and thereby amplifies the activation of caspase-8 which initiates the downstream apoptotic events. Oncogene (2000) 19, 4491 - 4499.

Rb protein down-regulates the stress-activated signals through inhibiting c-Jun N-terminal kinase/stress-activated protein kinase.

The Rb protein is the product of the retinoblastoma susceptibility gene and loss of Rb function is detected in many types of human cancers. Rb plays important roles in the regulation of cell proliferation, differentiation, senescence, and apoptotic cell death. Here we show that Rb can physically interact with c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK), thereby inhibiting intracellular signals mediated by JNK/SAPK. Both in vitro binding and in vitro kinase studies suggest that a carboxyl-terminal domain of Rb containing amino acids 768-928 might be crucial for inhibiting JNK/SAPK. In comparison, Rb did not affect enzymatic activity of either extracellular signal-regulated kinase 1 or p38. Ectopically expressed Rb also abrogated the apoptotic cell death induced by ultraviolet radiation or the activation of MEKK1, an upstream kinase that can stimulate the JNK/SAPK cascade. JNK/SAPK inhibition highlights a novel function of Rb, which may provide a new mechanism by which Rb regulates cell death. JNK/SAPK is a major protein kinase that can be stimulated in response to a variety of cellular stresses. Our results, therefore, suggest that Rb, by inhibiting JNK/SAPK, may act as a negative regulator in stress-activated intracellular signaling cascades.

Inhibition of homodimerization of poly(ADP-ribose) polymerase by its C-terminal cleavage products produced during apoptosis.

The biochemical role of the C-terminal fragment of poly(ADP-ribose) polymerase (PARP) was investigated in HeLa cells undergoing UV-mediated apoptosis. During the course of apoptosis, the C-terminal cleavage product of PARP interacted with intact PARP and down-regulated PARP activity by blocking the homodimerization of PARP. The basic leucine zipper motif in the auto-modification domain of the C-terminal fragment of PARP represented the site of association, and Leu(405) was critical to the ability of the basic leucine zipper motif to associate with intact PARP. The expression of the C-terminal fragment of PARP stimulated UV-mediated apoptosis. These results suggest that the C-terminal cleavage product of PARP produced during apoptosis blocks the homodimerization of PARP and inhibits the cellular PARP activity. The inhibition of the cellular PARP activity might prevent cellular NAD(+) depletion and stimulate apoptosis by maintaining the basal cellular energy level required for the completion of apoptosis.

DNA-binding activity of the N-terminal cleavage product of poly(ADP-ribose) polymerase is required for UV mediated apoptosis.

The role of the N-terminal cleavage product of poly(ADP-ribose) polymerase (PARP) on UV mediated apoptosis was investigated in cultured HeLa cells. Ultrastructural analysis of cells expressing caspase-resistant PARP (PARP(D214A)) revealed the typical features of necrosis following UV treatment. However, cells co-expressing PARP(D214A) with the N-terminal fragment of PARP containing the DNA-binding domain underwent apoptosis instead of necrosis. In this study, we have demonstrated that the DNA-binding activity of the N-terminal fragment of PARP is important for the execution of apoptosis. Point mutations were introduced in the DNA-binding sites of the N-terminal fragment. Cells co-expressing PARP(D214A) with the mutated N-terminal fragments neither stimulated apoptosis nor prevented necrosis in response to UV irradiation. The present study proposes that the DNA-binding activity of the N-terminal fragment of PARP in UV treated cells prevents cellular ATP depletion, a mechanism by which necrotic cell death is triggered.

Photomovement of the gliding cyanobacterium Synechocystis sp. PCC 6803.

Using a computerized videomicroscope motion analysis system, we investigated the photomovements of two Synechocystis sp. (PCC 6803 and ATCC 27184). Synechocystis sp. PCC 6803 displays a relatively slow gliding motion. The phototactic and photokinetic speeds of this cyanobacterium in liquid media were 5 microns/min and 15.8 microns/min, respectively, at 3 mumol/m2/s of stimulant white light. Synechocystis sp. PCC 6803 senses light direction rather than intensity for phototaxis. Synechocystis sp. ATCC 27184 showed a weak photokinesis but no phototaxis. Analysis of Synechocystis sp. ATCC 27184 suggests that the loss of phototaxis results from spontaneous mutation during several years of subculture. When directional irradiation was applied, the cell population of Synechocystis sp. PCC 6803 began to deviate from random movement and reached maximum orientation at 5 min after the onset of stimulant white light. Synechocystis sp. PCC 6803 showed high sensitivity to the stimulant white light of fluence rates as low as 0.002 mumol/m2/s. Neither 1,3-dichlorophenyldimethyl urea nor cyanide affected phototactic orientation, whereas cyanide inhibited gliding speed. This result suggests that the phototaxis of Synechocystis sp. PCC 6803 is independent of photosynthetic phosphorylation and that its gliding movement is primarily powered by oxidative phosphorylation. In the visible wavelength region, 560 nm, 660 nm and even 760 nm caused positive phototaxis. However, 360 nm light induced strikingly negative phototaxis. Therefore, at least two independent photoreceptors may exist to control phototaxis. The photoreceptor for positive phototaxis appears likely to be a phytochrome-like tetrapyrrole rather than chlorophyll a.

Lysophosphatidic acid increases intracellular H2O2 by phospholipase D and RhoA in rat-2 fibroblasts.

We have investigated the possible roles of phospholipase D (PLD) and RhoA in the production of intracellular H2O2 and actin polymerization in response to lysophosphatidic acid (LPA) in Rat-2 fibroblasts. LPA increased intracellular H2O2, with a maximal increase at 30 min, which was blocked by the catalase from Aspergillus niger. The LPA-stimulated production of H2O2 was inhibited by 1-butanol or PKC-downregulation, but not by 2-butanol. Purified phosphatidic acid (PA) also increased intracellular H2O2 and the increase was inhibited by the catalase. The role of RhoA was studied by the scrape-loading of C3 transferase into the cells. The C3 toxin, which inhibited stress fiber formation stimulated by LPA, blocked the H2O2 production in response to LPA or PA, but had no inhibitory effect on the activation of PLD by LPA. Exogenous H2O2 increased F-actin content by stress fiber formation. In addition, catalase inhibited actin polymerization activated by LPA, PA, or H2O2, indicated the role of H2O2 in actin polymerization. These results suggest that LPA increased intracellular H2O2 by the activation of PLD and RhoA, and that intracellular H2O2 was required for the LPA-stimulated stress fiber formation.

Inhibition of poly(ADP-ribose)polymerase binding to DNA by thymidine dimer.

The ability of poly(ADP-ribose)polymerase to bind damaged DNA was assessed by electrophoretic mobility shift assay. DNA binding domain of poly(ADP-ribose)polymerase (PARPDBD) binds to synthetic deoxyribonucleotide duplex 10-mer. However, the synthetic deoxyribonucleotide duplex containing cys-syn thymidine dimer which produces the unwinding of DNA helix structure lost its affinity to PARPDBD. It was shown that the binding of PARPDBD to the synthetic deoxyribonucleotide duplex was not affected by O6-Me-dG which causes only minor distortion of DNA helix structure. This study suggests that the stabilized DNA helix structure is important for poly(ADP-ribose)polymerase binding to DNA breaks, which are known to stimulate catalytic activity of poly(ADP-ribose)polymerase.

Improved cycle sequencing of GC-rich DNA template.

Even when DNA sequencing of purified DNA template failed under the optimal condition, it can be generally contributed to high GC content. GC-rich region of template causes a secondary structure to produce shorter readable sequence. To solve this problem, the sequencing reaction was modified by using dimethyl sulfoxide (DMSO). It was found that 5% (v/v) of DMSO in the reaction mixture recovers sequencing signal intensity with reduced frequency of ambiguous bases. When DMSO was added to sequencing reaction of DNA template with normal GC content, it did not show any adverse effect. Sequencing accuracy and unambiguous base frequency were significantly improved at concentration of 2% to 5% (v/v) DMSO in GC-rich DNA template. DMSO has been empirically introduced to enhance the efficiency of PCR in GC-rich templates. However, the underlying mechanism of improved cycle sequencing by DMSO is unknown. Thus, cycle sequencing reaction was remodified with other additives such as N-methyl imidazole, N-methyl2-pyrrolidone, N-methyl-2-pyridone and glycerol, possessing the similar chemical properties as DMSO. Most of methyl nitrogen ring-containing chemicals did not improve sequencing accuracy, whereas only glycerol mimicked the positive effect of DMSO by the same extent. In the present study, we suggest that the treatment of DMSO improve cycle sequencing by the alteration of structural conformation of GC-rich DNA template.

Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation.

The regulation of gap junctional permeability by phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.

Transfer of second messengers through gap junction connexin 43 channels reconstituted in liposomes.

Gap junction channels reconstituted in liposomes provide a pathway for the transfer of second messengers. Gap junction channels were formed in the artificial unilamellar liposomes using immunoaffinity-purified connexin 43 gap junction protein from rat brain. Sucrose-permeable and -impermeable liposomes were separated on the basis of sucrose permeability in the iso-osmolar sucrose density gradient. The liposomes permeable to sucrose were also permeable to a communicating dye molecule, Lucifer yellow. In the present study, we examined the transfer of second messengers through the connexin 43 channels reconstituted in liposomes and first report the direct evidence that the gap junction channels are permeable to second messengers including adenosine 3',5'-cyclic phosphate and inositol 1,4,5-trisphosphate.

Modulation of protein kinase C activity in NIH 3T3 cells by plant glycosides from Panax ginseng.

The involvement of ginsenosides in the signal cascade that stimulates cellular growth was investigated. It was found that ginsenosides Rh1 and Rh2 extracted from the root of Panax ginseng inhibited cellular proliferation in NIH 3T3 fibroblasts. Both ginsenosides Rh1 and Rh2 effectively reduced phospholipase C activity resulting in a decrease in the intracellular level of diacylglycerol, an endogenous activator of protein kinase C. The treatment of cells with Rh1 or Rh2 was thus found to reduce intracellular protein kinase C activity. We also observed that the phosphorylation of myristoylated alanine-rich C kinase substrate, one of the major substrates of protein kinase C in cells, was inhibited by the ginsenosides. Data suggest that the ginsenoside Rh1 or Rh2 exerts antiproliferative effects by inhibiting phospholipase C, which produces second messengers necessary for the activation of protein kinase C.

PKC phosphorylation disrupts gap junctional communication at G0/S phase in clone 9 cells.

Gap junctional communication during the progression of cell cycle from quiescent G0 to S phase was examined in cultured clone 9 rat liver cells. The transfer of scrape-loaded fluorescent dye was suppressed immediately after the stimulation of cell cycle progression in a synchronized cell population. Northern blot analysis showed that the temporal disturbance of gap junctional communication in cells passing from G0 to S phase did not result from transcriptional down-regulation of connexin 43. It was also found that the PKC inhibitor, calphostin C, was able to restore intercellular communication in serum stimulated cells. Data suggest a control mechanism by PKC mediated phosphorylation in the regulation of gap junction function which is vulnerable to cell cycling. The loss of gap junctional communication correlated with the increased phosphorylation of connexin 43 on serine residues in clone 9 cells.

Identification and expression of uvi31+, a UV-inducible gene from Schizosaccharomyces pombe.

The Schizosaccharomyces pombe uvi31+ gene has been previously isolated as a UV-inducible gene [Lee JK et al. (1994) Biochem Biophys Res Commun 202:1113-1119]. This gene encodes a protein of about 12 kDa with 57% amino acid sequence similarity to Escherichia coli BolA protein which is known to be involved in switching between the cell elongation and septation systems during the cell division cycle. The putative Mlul cell cycle box (MCB), SWI4/6-dependent cell cycle box (SCB), and gear-box elements are found in the upstream region of uvi31+ gene, suggesting that this gene shows the cell cycle-regulated and growth phase-dependent expression. Interestingly, the level of uvi31+ transcript varies throughout the cell cycle, peaking in G1 phase before septation, and also shows the growth phase-dependent pattern during cellular growth, increasing maximally at the diauxic shift phase just before stationary phase. Furthermore, the transcript level of this gene is raised after S phase arrest, and is also increased maximally at 4 hr after UV irradiation of 240 J/m2. These results suggest that the delayed induction of uvi31+ gene after UV irradiation may be caused by cell cycle control of this gene after DNA replication checkpoint arrest. Thus, the uvi31+ gene may play a role in controlling the progress of the cell cycle after DNA damage (UV irradiation).

Regulation of the activity of M-phase promoting factor through protein kinase A-mediated pathway in LP1-1 cells.

Treatment of cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, was found to inhibit the proliferation of mouse fibroblast LP1-1 cells and the p34cdc2 kinase activity of M-phase promoting factor (MPF). However, it showed relatively little effect on expression of the cyclin B1 and cdc2 genes. On the other hand, when the nuclear extracts obtained from the cells at early G2 phase were treated with cAMP analogs, the kinase activity was significantly decreased as compared to the untreated control. Furthermore, the inhibitory effect of cAMP analogs could be reversed upon treating with okadaic acid even in the presence of the cAMP analogs, implying that cdc25 remains in an active form. In addition, the treatment of okadaic acid stimulated the cell progression. These results suggest that down-regulation of MPF activity through protein kinase A-mediated pathway is under post-translational control and cdc25 activation pathway involving okadaic acid-sensitive phosphatase play a role in the regulation of MPF activity.

Poly(ADP-ribosyl)ation of histone H1 correlates with internucleosomal DNA fragmentation during apoptosis.

The biochemical role of poly(ADP-ribosyl)ation on internucleosomal DNA fragmentation associated with apoptosis was investigated in HL 60 human premyelocytic leukemia cells. It was found that UV light and chemotherapeutic drugs including adriamycin, mitomycin C, and cisplatin increased poly(ADP-ribosyl)ation of nuclear proteins, particularly histone H1. A poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, prevented both internucleosomal DNA fragmentation and histone H1 poly(ADP-ribosyl)ation in cells treated with the apoptosis inducers. When nuclear chromatin was made accessible to the exogenous nuclease in a permeabilized cell system, chromatin of UV-treated cells was more susceptible to micrococcal nuclease than the chromatin of control cells. Suppression of histone H1 poly(ADP-ribosyl)ation by 3-aminobenzamide reduced the micrococcal nuclease digestibility of internucleosomal chromatin in UV-treated cells. These results suggest that the poly(ADP-ribosyl)ation of histone H1 correlates with the internucleosomal DNA fragmentation during apoptosis mediated by DNA damaging agents. This suggestion is supported by the finding that xeroderma pigmentosum cells which are defective in introducing incision at the site of DNA damage, failed to induce DNA fragmentation as well as histone H1 poly(ADP-ribosyl)ation after UV irradiation. We propose that poly(ADP-ribosyl)ation of histone H1 protein in the early stage of apoptosis facilitates internucleosomal DNA fragmentation by increasing the susceptibility of chromatin to cellular endonuclease.