PIASy mediates NEMO sumoylation and NF-kappaB activation in response to genotoxic stress

Protein modification by SUMO (small ubiquitin-like modifier) is an important regulatory mechanism for multiple cellular processes. SUMO-1 modification of NEMO (NF-kappaB essential modulator), the IkappaB kinase (IKK) regulatory subunit, is critical for activation of NF-kappaB by genotoxic agents. However, the SUMO ligase, and the mechanisms involved in NEMO sumoylation, remain unknown. Here, we demonstrate that although small interfering RNAs (siRNAs) against PIASy (protein inhibitor of activated STATy) inhibit NEMO sumoylation and NF-kappaB activation in response to genotoxic agents, overexpression of PIASy enhances these events. PIASy preferentially stimulates site-selective modification of NEMO by SUMO-1, but not SUMO-2 and SUMO-3, in vitro. PIASy-NEMO interaction is increased by genotoxic stress and occurs in the nucleus in a manner mutually exclusive with IKK interaction. In addition, hydrogen peroxide (H2O2) also increases PIASy-NEMO interaction and NEMO sumoylation, whereas antioxidants prevent these events induced by DNA-damaging agents. Our findings demonstrate that PIASy is the first SUMO ligase for NEMO whose substrate specificity seems to be controlled by IKK interaction, subcellular targeting and oxidative stress conditions.     

Calcium-dependent regulation of exocytosis

A rapid increase in intracellular calcium directly triggers regulated exocytosis. In addition, changes in intracellular calcium concentration can adjust the extent of exocytosis (quantal content) or the magnitude of individual release events (quantal size) in both the short- and long-term. It is generally agreed that calcium achieves this regulation via an interaction with a number of different molecular targets located at or near to the site of membrane fusion. We review here the synaptic proteins with defined calcium-binding domains and protein kinases activated by calcium, summarize what is known about their function in membrane fusion and the experimental evidence in support of their involvement in synaptic plasticity.     

Calcium-dependent protein kinase gene expression in response to physical and chemical stimuli in mungbean (Vigna radiata)

Protein kinases are important in eukaryotic signal transduction pathways. In this study we designed degenerate oligonucleotides corresponding to two conserved regions of protein kinases and using the polymerase chain reaction (PCR) have amplified a 141 bp fragment of DNA from mungbeans (Vigna radiata Rwilcz cv. Berken). Sequence analysis of the PCR products indicates that they encode several putative protein kinases with respect to their identity with other known plant protein kinases. Using one of the six fragments (CPK3-8), we isolated a 2022 bp cDNA (VrCDPK-1) from a Vigna radiata gt11 library. VrCDPK-1 has a 96 bp 5-untranslated region and a 465 bp 3-untranslated region and an open reading frame of 1461 bp. VrCDPK-1 contains all of the conserved regions commonly found in calcium dependent protein kinases (CDPK). VrCDPK-1 shares 24 to 89% sequence identity with previously reported sequences for plant CDPKs at the protein level. southern analysis revealed the presence of several copies of the CDPK gene. VrCDPK-1 expression was stimulated when mungbean cuttings were treated with CaCl₂, while treatment with MgCl₂ had no effect. We are reporting for the first time a CDPK gene in mungbean which is inducible by mechanical strain. Cuttings treated with indole-3-acetic acid (IAA) or subjected to salt stress showed an increase in VrCDPK-1 expression. There was a dramatic stimulation in VrCDPK-1 expression 6 h after cuttings were treated with cycloheximide.     

p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.     

Calcium-dependent regulation of NAD kinase.

An activator protein of NAD kinase from the pea, $\text{Pisum}\text{satavum}$ L., has been shown to be Ca²⁺-dependent. This plant activator protein also stimulates the activity of modulator protein dependent-cyclic nucleotide phosphodiesterase from porcine brain. This stimulation is similar to that observed with modulator protein isolated from animal sources. Furthermore, Ca²⁺-dependent modulator proteins isolated from porcine brain, bovine brain, and the coelenterate, $\text{Renilla}$, will regulate the NAD kinase activity of peas. Other common properties of the plant activator protein and animal modulator proteins are their acidic nature, heat stabilities, similar Stokes' radii, and their interactions with troponin I.     

Physiological function as regulation of large transcriptional programs: the cellular response to genotoxic stress

The responses to ionizing radiation and other genotoxic environmental stresses are complex and are regulated by a number of overlapping molecular pathways. One such stress signaling pathway involves p53, which regulates the expression of over 100 genes already identified. It is also becoming increasingly apparent that the pattern of stress gene expression has some cell type specificity. It may be possible to exploit these differences in stress gene responsiveness as molecular markers through the use of a combined informatics and functional genomics approach. The techniques of microarray analysis potentially offer the opportunity to monitor changes in gene expression across the entire set of expressed genes in a cell or organism. As an initial step in the development of a functional genomics approach to stress gene analysis, we have recently demonstrated the utility of cDNA microarray hybridization to measure radiation-stress gene responses and identified a number of previously unknown radiation-regulated genes. The responses of some of these genes to DNA-damaging agents vary widely in cell lines from different tissues of origin and different genetic backgrounds. While this again highlights the importance of a cellular context to genotoxic stress responses, it also raises the prospect of expression-profiling of cell lines, tissues, and tumors. Such profiles may have a predictive value if they can define regions of ‘expression space’ that correlate with important endpoints, such as response to cancer therapy regimens, or identification of exposures to environmental toxins.     

Calcium-dependent regulation of glucose homeostasis in the liver

A major role of the liver is to integrate multiple signals to maintain normal blood glucose levels. The balance between glucose storage and mobilization is primarily regulated by the counteracting effects of insulin and glucagon. However, numerous signals converge in the liver to ensure energy demand matches the physiological status of the organism. Many circulating hormones regulate glycogenolysis, gluconeogenesis and mitochondrial metabolism by calcium-dependent signaling mechanisms that manifest as cytosolic Ca²⁺ oscillations. Stimulus-strength is encoded in the Ca²⁺ oscillation frequency, and also by the range of intercellular Ca²⁺ wave propagation in the intact liver. In this article, we describe how Ca²⁺ oscillations and waves can regulate glucose output and oxidative metabolism in the intact liver; how multiple stimuli are decoded though Ca²⁺ signaling at the organ level, and the implications of Ca²⁺ signal dysregulation in diseases such as metabolic syndrome and non-alcoholic fatty liver disease.     

Regulation of base excision repair: Ntg1 nuclear and mitochondrial dynamic localization in response to genotoxic stress

Numerous human pathologies result from unrepaired oxidative DNA damage. Base excision repair (BER) is responsible for the repair of oxidative DNA damage that occurs in both nuclei and mitochondria. Despite the importance of BER in maintaining genomic stability, knowledge concerning the regulation of this evolutionarily conserved repair pathway is almost nonexistent. The Saccharomyces cerevisiae BER protein, Ntg1, relocalizes to organelles containing elevated oxidative DNA damage, indicating a novel mechanism of regulation for BER. We propose that dynamic localization of BER proteins is modulated by constituents of stress response pathways. In an effort to mechanistically define these regulatory components, the elements necessary for nuclear and mitochondrial localization of Ntg1 were identified, including a bipartite classical nuclear localization signal, a mitochondrial matrix targeting sequence and the classical nuclear protein import machinery. Our results define a major regulatory system for BER which when compromised, confers a mutator phenotype and sensitizes cells to the cytotoxic effects of DNA damage.     

The regulation of glycogen synthase kinase-3 nuclear export by Frat/GBP

Previous studies have shown that nuclear levels of glycogen synthase kinase-3 (GSK-3) are dynamically regulated and may affect access of GSK-3 to its substrates. In this study we show that the GSK-3-binding protein Frat/GBP regulates the nuclear export of GSK-3. We show that Frat/GBP contains a nuclear export sequence that promotes its own nuclear export and that of associated GSK-3. Treating cells with leptomycin B increased nuclear levels of endogenous GSK-3 suggesting that an endogenous process targets GSK-3 for nuclear export. To investigate this further, we used two approaches to disrupt the interaction between GSK-3 and endogenous Frat. First we isolated mutants of GSK-3 that selectively interfered with Frat binding and found that these mutants were poorly exported. Second we expressed a peptide that competes with Frat for GSK-3 binding and found that it caused endogenous GSK-3 to accumulate in the nucleus. Together these data suggest that Frat may be the endogenous factor that targets GSK-3 for nuclear export. The dynamic expression patterns of Frat mRNAs together with the role of Frat in mediating GSK-3 nuclear export have important implications for the control of the substrate access of GSK-3 in several signaling pathways.     

Reconstitution of nuclear protein export in isolated nuclear envelopes

Signal-dependent nuclear protein export was studied in perforated nuclei and isolated nuclear envelopes of Xenopus oocytes by optical single transporter recording. Manually isolated and purified oocyte nuclei were attached to isoporous filters and made permeable for macromolecules by perforation. Export of a recombinant protein (GG-NES) containing the nuclear export signal (NES) of the protein kinase A inhibitor through nuclear envelope patches spanning filter pores could be induced by the addition of GTP alone. Export continued against a concentration gradient, and was NES dependent and inhibited by leptomycin B and GTPgammaS, a nonhydrolyzable GTP analogue. Addition of recombinant RanBP3, a potential cofactor of CRM1-dependent export, did not promote GG-NES export at stoichiometric concentration but gradually inhibited export at higher concentrations. In isolated filter-attached nuclear envelopes, export of GG-NES was virtually abolished in the presence of GTP alone. However, a preformed export complex consisting of GG-NES, recombinant human CRM1, and RanGTP was rapidly exported. Unexpectedly, export was strongly reduced when the export complex contained RanGTPgammaS or RanG19V/Q69L-GTP, a GTPase-deficient Ran mutant. This paper shows that nuclear transport, previously studied in intact and permeabilized cells only, can be quantitatively analyzed in perforated nuclei and isolated nuclear envelopes.     

Analysis of the complex relationship between nuclear export and aryl hydrocarbon receptor-mediated gene regulation

The aryl hydrocarbon receptor (AHR) contains signals for both nuclear import and nuclear export (NES). The purpose of the studies in this report was to determine the relationship between the nuclear export of the AHR and AHR-mediated gene regulation. Blockage of nuclear export in HepG2 cells with leptomycin B (LMB) resulted in increased levels of AHR-AHR nuclear translocator (ARNT) complex in the nucleus and correlative reductions in agonist-stimulated AHR degradation. However, LMB exposure inhibited agonist-mediated induction of numerous AHR-responsive reporter genes by 75 to 89% and also inhibited induction of endogenous CYP1A1. LMB did not transform the AHR to a ligand binding species or affect activation by TCDD (2, 3,7,8-tetrachlorodibenzo-p-dioxin). Mutagenesis of leucines 66 and 71 of the putative AHR NES resulted in a protein with reduced function in dimerization to ARNT and binding to DNA, while alanine substitution at leucine 69 (AHR(A69)) resulted in an AHR that bound with ARNT and associated with DNA. AHR(A69) protein injected directly into the nuclei of E36 cells remained nuclear following 6 h of agonist stimulation. In transient-transfection assays, AHR(A69) accumulated within the nucleus was not degraded efficiently following agonist exposure. Finally, AHR(A69) supported induction of AHR-responsive reporter genes in an agonist-dependent manner. These findings show that it is possible to generate an AHR protein defective in nuclear export that is functional in agonist-mediated gene induction. This implies that the negative effect of LMB on agonist-mediated gene induction is independent of the nuclear export of the AHR.     

Mechanisms of receptor-mediated nuclear import and nuclear export

Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.