Masahiro Fujimuro

S -nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) influences cytotoxicity, translocating to the nucleus during apoptosis. Here we report a signalling pathway in which nitric oxide (NO) generation that follows apoptotic stimulation elicits S-nitrosylation of GAPDH, which triggers binding to Siah1 (an E3 ubiquitin ligase), nuclear translocation and apoptosis. S-nitrosylation of GAPDH augments its binding to Siah1, whose nuclear localization signal mediates translocation of GAPDH. GAPDH stabilizes Siah1, facilitating its degradation of nuclear proteins. Activation of macrophages by endotoxin and of neurons by glutamate elicits GAPDH–Siah1 binding, nuclear translocation and apoptosis, which are prevented by NO deletion. The NO–S-nitrosylation–GAPDH–Siah1 cascade may represent an important molecular mechanism of cytotoxicity.

p53 Mediates Cellular Dysfunction and Behavioral Abnormalities in Huntington’s Disease

We present evidence for a specific role of p53 in the mitochondria-associated cellular dysfunction and behavioral abnormalities of Huntingtons disease (HD). Mutant huntingtin (mHtt) with expanded polyglutamine (polyQ) binds to p53 and upregulates levels of nuclear p53 as well as p53 transcriptional activity in neuronal cultures. The augmentation is specific, as it occurs with mHtt but not mutant ataxin-1 with expanded polyQ. p53 levels are also increased in the brains of mHtt transgenic (mHtt-Tg) mice and HD patients. Perturbation of p53 by pifithrin-alpha, RNA interference, or genetic deletion prevents mitochondrial membrane depolarization and cytotoxicity in HD cells, as well as the decreased respiratory complex IV activity of mHtt-Tg mice. Genetic deletion of p53 suppresses neurodegeneration in mHtt-Tg flies and neurobehavioral abnormalities of mHtt-Tg mice. Our findings suggest that p53 links nuclear and mitochondrial pathologies characteristic of HD.

A novel viral mechanism for dysregulation of β-catenin in Kaposi's sarcoma–associated herpesvirus latency

The Kaposis sarcoma–associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is expressed in all KSHV-associated tumors, including Kaposis sarcoma (KS) and primary effusion lymphoma (PEL). We found that β-catenin is overexpressed in both PEL cells and KS tissue. Introduction of anti-LANA small interfering RNA (siRNA) into PEL cells eliminated β-catenin accumulation; LANA itself upregulated expression of β-catenin in transfected cells. LANA stabilizes β-catenin by binding to the negative regulator GSK-3β, causing a cell cycle–dependent nuclear accumulation of GSK-3β. The LANA C terminus contains sequences similar to the GSK-3β-binding domain of Axin. Disruption of this region resulted in a mutant LANA that failed to re-localize GSK-3β or stabilize β-catenin. The importance of this pathway to KSHV-driven cell proliferation was highlighted by the observation that LANA, but not mutant LANA, stimulates entry into S phase. Redistribution of GSK-3β can therefore be a source of β-catenin dysregulation in human cancers.

Production and characterization of monoclonal antibodies specific to multi-ubiquitin chains of polyubiquitinated proteins

Polyubiquitinated proteins tagged with multi‐ubiquitin chains are substrates preferred by the 26 S proteasome (a ubiquitin/ATP‐dependent proteolytic complex). Here, we developed a simple method for the efficient preparation of polyubiquitinated proteins which are degraded by the 26 S proteasome in an ATP‐dependent manner. Our efficient method enabled us to produce ten monoclonal antibodies that recognized the multi‐ubiquitin chains of the polyubiquitinated proteins, but not free ubiquitin or the protein moieties. Eight of the antibodies recognized only the multi‐ubiquitin chains of the polyubiquitinated proteins, while the other two antibodies cross‐reacted with mono‐ubiquitin and methyl‐ubiquitin, both of which are linked to proteins via an isopeptide bond, as well as with the multi‐ubiquitin chains. Thus these antibodies are novel and useful tools for the identification and quantification of polyubiquitinated proteins in various cells and tissues under physiological and pathological conditions.

SKIP, a CBF1-Associated Protein, Interacts with the Ankyrin Repeat Domain of NotchIC To Facilitate NotchIC Function

ABSTRACT Notch proteins are transmembrane receptors that mediate intercell communication and direct individual cell fate decisions. The activated intracellular form of Notch, NotchIC, translocates to the nucleus, where it targets the DNA binding protein CBF1. CBF1 mediates transcriptional repression through the recruitment of an SMRT-histone deacetylase-containing corepressor complex. We have examined the mechanism whereby NotchIC overcomes CBF1-mediated transcriptional repression. We identified SKIP (Ski-interacting protein) as a CBF1 binding protein in a yeast two-hybrid screen. Both CBF1 and SKIP are highly conserved evolutionarily, and the SKIP-CBF1 interaction is also conserved in assays using the Caenorhabditis elegans andDrosophila melanogaster SKIP homologs. Protein-protein interaction assays demonstrated interaction between SKIP and the corepressor SMRT. More surprisingly, SKIP also interacted with NotchIC. The SMRT and NotchIC interactions were mutually exclusive. In competition binding experiments SMRT displaced NotchIC from CBF1 and from SKIP. Contact with SKIP is required for biological activity of NotchIC. A mutation in the fourth ankyrin repeat that abolished Notch signal transduction did not affect interaction with CBF1 but abolished interaction with SKIP. Further, NotchIC was unable to block muscle cell differentiation in myoblasts expressing antisense SKIP. The results suggest a model in which NotchIC activates responsive promoters by competing with the SMRT-corepressor complex for contacts on both CBF1 and SKIP.

Protein Interactions Targeting the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus to Cell Chromosomes

ABSTRACT Maintenance of Kaposis sarcoma-associated herpesvirus (KSHV) latent infection depends on the viral episomes in the nucleus being distributed to daughter cells following cell division. The latency-associated nuclear antigen (LANA) is constitutively expressed in all KSHV-infected cells. LANA binds sequences in the terminal repeat regions of the KSHV genome and tethers the viral episomes to chromosomes. To better understand the mechanism of chromosomal tethering, we performed glutathione S-transferase (GST) affinity and yeast two-hybrid assays to identify LANA-interacting proteins with known chromosomal association. Two of the interactors were the methyl CpG binding protein MeCP2 and the 43-kDa protein DEK. The interactions of MeCP2 and DEK with LANA were confirmed by coimmunoprecipitation. The MeCP2-interacting domain was mapped to the previously described chromatin binding site in the N terminus of LANA, while the DEK-interacting domain mapped to LANA amino acids 986 to 1043 in the C terminus. LANA was unable to associate with mouse chromosomes in chromosome spreads of transfected NIH 3T3 cells. However, LANA was capable of targeting to mouse chromosomes in the presence of human MeCP2 or DEK. The data indicate that LANA is tethered to chromosomes through two independent chromatin binding domains that interact with different protein partners.

The Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Manipulates the Activity of Glycogen Synthase Kinase-3β

ABSTRACT The latency-associated nuclear antigen (LANA) of Kaposis sarcoma-associated herpesvirus (KSHV) is expressed in all KSHV-associated malignancies. LANA is essential for replication and maintenance of the viral episomes during latent infection. However, LANA also has a transcriptional regulatory role and can affect gene expression both positively and negatively. A previously performed yeast two-hybrid screen identified glycogen synthase kinase 3 (GSK-3) as a LANA-interacting protein. Interaction with both GSK-3α and GSK-3β was confirmed in transfected cells with coprecipitation assays. GSK-3β also interacted with the herpesvirus saimiri homolog ORF73. GSK-3β is an intermediate in the Wnt signaling pathway and a negative regulator of β-catenin. In transfected cells, LANA was shown to overcome GSK-3β-mediated degradation of β-catenin. Examination of primary effusion lymphoma (PEL) cells found increased levels of β-catenin relative to KSHV-negative B cells, and this translated into increased activity of a β-catenin-responsive reporter containing Tcf/Lef binding sites. In tetradecanoyl phorbol acetate-treated PEL cells, loss of LANA expression correlated temporally with loss of detectable β-catenin. LANA was found to alter the intracellular distribution of GSK-3β so that nuclear GSK-3β was more readily detectable in the presence of LANA. Mapping experiments with coimmunoprecipitation assays revealed that both N-terminal and C-terminal LANA sequences were required for efficient GSK-3β interaction. LANA mutants that were defective for GSK-3β interaction were unable to mediate GSK-3β relocalization or activate a β-catenin-responsive Tcf-luciferase reporter. This study identified manipulation of GSK-3β activity as a mechanism by which LANA may modify transcriptional activity and contribute to the phenotype of primary effusion lymphoma.

A Role for SKIP in EBNA2 Activation of CBF1-Repressed Promoters

ABSTRACT EBNA2 is essential for Epstein-Barr virus (EBV) immortalization of B lymphocytes. EBNA2 functions as a transcriptional activator and targets responsive promoters through interaction with the cellular DNA binding protein CBF1. We have examined the mechanism whereby EBNA2 overcomes CBF1-mediated transcriptional repression. A yeast two-hybrid screen performed using CBF1 as the bait identified a protein, SKIP, which had not previously been recognized as a CBF1-associated protein. Protein-protein interaction assays demonstrated contacts between SKIP and the SMRT, CIR, Sin3A, and HDAC2 proteins of the CBF1 corepressor complex. Interestingly, EBNA2 also interacted with SKIP in glutathioneS-transferase affinity and mammalian two-hybrid assays and colocalized with SKIP in immunofluorescence assays. Interaction with SKIP was not affected by mutation of EBNA2 conserved region 6, the CBF1 interaction region, but was abolished by mutation of conserved region 5. Mutation of conserved region 5 also severely impaired EBNA2 activation of a reporter containing CBF1 binding sites. Thus, interaction with both CBF1 and SKIP is necessary for efficient promoter activation by EBNA2. A model is presented in which EBNA2 competes with the SMRT-corepressor complex for contacts on SKIP and CBF1.

Role of CCAAT/Enhancer-Binding Protein Alpha (C/EBPα) in Activation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic-Cycle Replication-Associated Protein (RAP) Promoter in Cooperation with the KSHV Replication and Transcription Activator (RTA) and RAP

ABSTRACT The Kaposis sarcoma-associated herpesvirus (KSHV)-encoded replication-associated protein (RAP, or K8) has been shown to induce both CCAAT/enhancer binding protein alpha (C/EBPα) and p21CIP-1 expression, resulting in G0/G1 cell cycle arrest during the lytic cycle. RAP and C/EBPα are also known to interact strongly both in vitro and in lytically infected cells. We recognized two potential consensus C/EBP binding sites in the RAP promoter and performed electrophoretic mobility shift assay (EMSA) analysis with in vitro-translated C/EBPα; this analysis showed that one of these sites has a very high affinity for C/EBPα. Luciferase (LUC) assays performed with a target RAP promoter-LUC reporter gene confirmed that C/EBPα can transcriptionally activate the RAP promoter up to 50-fold. Although RAP had no effect on its own promoter by itself, the addition of RAP and C/EBPα together resulted in a threefold increase in activity over that obtained with C/EBPα alone. Importantly, the introduction of exogenous Flag-tagged C/EBPα triggered RAP expression in BCBL-1 cells latently infected with KSHV, as detected by both reverse transcription-PCR and double-label immunofluorescence assay analyses, suggesting the presence of a self-reinforcing loop with C/EBPα and RAP activating each other. The RAP promoter can also be activated 50- to 120-fold by the KSHV lytic-cycle-triggering protein known as replication and transcription activator (RTA). C/EBPα and RTA together cooperated to elevate RAP promoter activity four- to sixfold more than either alone. Furthermore, the addition of RAP, C/EBPα, and RTA in LUC reporter cotransfection assays resulted in 7- to 15-fold more activation than that seen with either C/EBPα or RTA alone. Site-specific mutational analysis of the RAP promoter showed that the strong C/EBP binding site is crucial for C/EBPα-mediated transactivation of the RAP promoter. However, the C/EBP binding site also overlaps the previously reported 16-bp RTA-responsive element (RRE), and the same mutation also both reduced RTA-mediated transactivation and abolished the cooperativity between C/EBPα and RTA. Furthermore, in vitro-translated RTA, although capable of binding directly to the polyadenylated nuclear RNA (PAN) RRE motif, failed to bind to the RAP RRE and interfered with RRE-bound C/EBPα in EMSA experiments. Partial RTA responsiveness but no cooperativity could be transferred to a heterologous promoter containing added consensus C/EBP binding sites. A chromatin immunoprecipitation assay showed that all three proteins associated specifically with RAP promoter DNA in vivo and that, when C/EBPα was removed from a tetradecanoyl phorbol acetate-treated JSC-1 primary effusion lymphoma cell lysate, the levels of association of RTA and RAP with the RAP promoter were reduced 3- and 13-fold, respectively. Finally, RTA also proved to physically interact with both C/EBPα and RAP, as assayed both in vitro and by immunoprecipitation. Binding to C/EBPα occurred within the N-terminal DNA binding domain of RTA, and deletion of a 17-amino-acid basic motif of RTA abolished both the C/EBPα and DNA binding activities as well as all RTA transactivation and the cooperativity with C/EBPα. Therefore, we suggest that RTA transactivation of the RAP RRE is mediated by an interaction with DNA-bound C/EBPα but that full activity requires more than just the core C/EBP binding site.

Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae

The nin1‐1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1‐1 strains fail to activate histone H1 kinase after release from alpha factor‐imposed G1 block and after release from hydroxyurea‐imposed S block. The nin1‐1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28‐IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP‐Mg2+, and (ii) nin1‐1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p.