Phosphorylation Of Nuclear Proteins Research Articles

Radiation-Induced Bystander Effect Mediated by Exosomes Involves the Replication Stress in Recipient Cells.

Exosomes released by irradiated cells mediate the radiation-induced bystander effect, which is manifested by DNA breaks detected in recipient cells; yet, the specific mechanism responsible for the generation of chromosome lesions remains unclear. In this study, naive FaDu head and neck cancer cells were stimulated with exosomes released by irradiated (a single 2 Gy dose) or mock-irradiated cells. Maximum accumulation of gamma H2A.X foci, a marker of DNA breaks, was detected after one hour of stimulation with exosomes from irradiated donors, the level of which was comparable to the one observed in directly irradiated cells (a weaker wave of the gamma H2A.X foci accumulation was also noted after 23 h of stimulation). Exosomes from irradiated cells, but not from control ones, activated two stress-induced protein kinases: ATM and ATR. Noteworthy is that while direct irradiation activated only ATM, both ATM and ATR were activated by two factors known to induce the replication stress: hydroxyurea and camptothecin (with subsequent phosphorylation of gamma H2A.X). One hour of stimulation with exosomes from irradiated cells suppressed DNA synthesis in recipient cells and resulted in the subsequent nuclear accumulation of RNA:DNA hybrids, which is an indicator of impaired replication. Interestingly, the abovementioned effects were observed before a substantial internalization of exosomes, which may suggest a receptor-mediated mechanism. It was observed that after one hour of stimulation with exosomes from irradiated donors, phosphorylation of several nuclear proteins, including replication factors and regulators of heterochromatin remodeling as well as components of multiple intracellular signaling pathways increased. Hence, we concluded that the bystander effect mediated by exosomes released from irradiated cells involves the replication stress in recipient cells.

A splicing factor phosphorylated by protein kinase A is increased in HL60 cells treated with retinoic acid

Retinoic acid (RA) induces the differentiation of human promyelocytic leukemia HL60 cells into granulocytic cells and inhibits proliferation. Certain of actions of RA are mediated by RA nuclear receptors that regulate gene expression. However, it is also known that direct protein modification by RA (retinoylation) can occur. One such retinoylated protein in HL60 cells is a regulatory subunit of protein kinase A (PKA), which is increased in the nucleus following RA treatment and which then increases phosphorylation of other nuclear proteins. However, a complete understanding of which nuclear proteins are phosphorylated is lacking. In the current study, we employed mass spectrometry to identify one of the PKA-phosphorylated proteins as a serine/arginine-rich splicing factor 1 (SF2, SRSF1). We found that RA treatment increased the level of PKA-phosphorylated SF2 but decreased the level of SF2. While SF2 regulates myelogenous cell leukemia-1 (Mcl-1, anti-apoptotic factor), RA treatment reduced the level of Mcl-1L (full-length Mcl-1 long) and increased the level of Mcl-1S (Mcl-1 short; a short splicing variant of the Mcl-1). Furthermore, treatment with a PKA inhibitor reversed these effects on Mcl-1 and inhibited RA-induced cell differentiation. In contrast, treatment with a Mcl-1L inhibitor enhanced RA-induced cell differentiation. These results indicate that RA activates PKA in the nucleus, increases phosphorylation of SF2, raises levels of Mcl-1S and lowers levels of Mcl-1L, resulting in the induction of differentiation. RA-modified PKA may play an important role in inducing cell differentiation and suppressing cell proliferation.

Primary nitrate responses mediated by calcium signalling and diverse protein phosphorylation.

Nitrate, the major source of inorganic nitrogen for plants, is a critical signal controlling nutrient transport and assimilation and adaptive growth responses throughout the plant. Understanding how plants perceive nitrate and how this perception is transduced into responses that optimize growth are important for the rational improvement of crop productivity and for mitigating pollution from the use of fertilizers. This review highlights recent findings that reveal key roles of cytosolic–nuclear calcium signalling and dynamic protein phosphorylation via diverse mechanisms in the primary nitrate response (PNR). Nitrate-triggered calcium signatures as well as the critical functions of subgroup III calcium-sensor protein kinases, a specific protein phosphatase 2C, and RNA polymerase II C-terminal domain phosphatase-like 3 are discussed. Moreover, genome-wide meta-analysis of nitrate-regulated genes encoding candidate protein kinases and phosphatases for modulating critical phosphorylation events in the PNR are elaborated. We also consider how phosphoproteomics approaches can contribute to the identification of putative regulatory protein kinases in the PNR. Exploring and integrating experimental strategies, new methodologies, and comprehensive datasets will further advance our understanding of the molecular and cellular mechanisms underlying the complex regulatory processes in the PNR.

Regulation of GSK3 cellular location by FRAT modulates mTORC1-dependent cell growth and sensitivity to rapamycin

The mTORC1 pathway regulates cell growth and proliferation by properly coupling critical processes such as gene expression, protein translation, and metabolism to the availability of growth factors and hormones, nutrients, cellular energetics, oxygen status, and cell stress. Although multiple cytoplasmic substrates of mTORC1 have been identified, how mTORC1 signals within the nucleus remains incompletely understood. Here, we report a mechanism by which mTORC1 modulates the phosphorylation of multiple nuclear events. We observed a significant nuclear enrichment of GSK3 when mTORC1 was suppressed, which promotes phosphorylation of several proteins such as GTF2F1 and FOXK1. Importantly, nuclear localization of GSK3 is sufficient to suppress cell proliferation. Additionally, expression of a nuclear exporter of GSK3, FRAT, restricts the nuclear localization of GSK3, represses nuclear protein phosphorylation, and prevents rapamycin-induced cytostasis. Finally, we observe a correlation between rapamycin resistance and FRAT expression in multiple-cancer cell lines. Resistance to Food and Drug Administration (FDA)-approved rapamycin analogs (rapalogs) is observed in many tumor settings, but the underling mechanisms remain incompletely understood. Given that FRAT expression levels are frequently elevated in various cancers, our observations provide a potential biomarker and strategy for overcoming rapamycin resistance.

Abstract 308: Role of Snf1-related Kinase as a Regulator of Chromatin Modifications and Dna-damage Response in Heart Injury

Introduction: Altered DNA damage response (DDR) can result in phenotypic changes that share features with age-associated pathological conditions, including metabolic and cardiovascular abnormalities. Snf1-related kinase (SNRK) is a serine/threonine kinase that belongs to the AMPK family. Our recent data demonstrates that SNRK expression is upregulated in heart failure (HF) and low energy states and that SNRK improves cardiac mitochondrial efficiency and cell survival. Here, we assessed the hypothesis that in response to stress, SNRK translocates to the nucleus where it modulates nuclear processes to enhance DNA repair and ultimately reduces cardiomyocyte death. Results: We first showed that the nuclear distribution of endogenous SNRK in cardiomyoblast is enhanced under low nutrient and oxidative stress. Next, we performed global phosphoproteomics analysis on cells with overexpression of wild type SNRK and mutant kinase-dead SNRK (D158A). We found significant increase of several phosphopeptides involved in nuclear processes, in particular in the regulation of chromatin remodeling and DDR. SNRK downregulation resulted in an increase in nucleus volume and heterochromatin content, with increased nuclear dysmorphia when subjected to oxidative stress. Despite having lower expression of DDR signal effectors p53 and Mdm2, these cells accumulated higher levels of pH2AX (DDR marker) and displayed higher cellular death after oxidative stress, consistent with increased DNA damage. Etoposide treatment in SNRK knockdown cells failed to induce p53 levels despite pH2AX accumulation. Additionally, pharmacological stabilization of p53 protein through nutlin-3A treatment did not increase p53 in SNRK knockdown cells. These findings suggest that SNRK regulates p53 expression, thereby acting as a new modulator of DDR. Conclusions: Our results demonstrate increased nuclear localization of SNRK under stress conditions, and that SNRK regulates phosphorylation of nuclear proteins involved in chromatin remodelling and DDR. Thus, SNRK may induce its pro-survival cardiac functions through regulation of chromatin architecture and enhancing DDR. Those novel findings can contribute to development of new therapies against HF.

A putative direct repeat element plays a dual role in the induction and repression of insect vitellogenin-1 gene expression.

Juvenile hormones (JH) regulate wide-ranging physiological and developmental processes in insects. However, molecular mechanisms underlying JH signaling remain to be determined. Vitellogenin (Vg) is primarily an egg-yolk protein, but recently proposed to serve many functions in insects. In the female American cockroach (Periplaneta americana), vitellogenin (Vg) genes are activated by JH III and suppressed by 20-hydroxyecdysone (20E) via cis-regulatory elements in a dose-dependent manner. In the present study, the upstream promoter region (935 bp) of Vg1 was cloned to elucidate the action of these hormones. A luciferase reporter assay identified an 81 bp region in the promoter region of Vg1 (-120 to -39 bp) that we found to be critical for JH III activation and 20E suppression. This 81 bp region contains a direct repeat separated by a 2-nucleotide spacer-designated Vg1HRE- that is similar to the Drosophila ecdysone response element direct repeat 4. Moreover, nuclear proteins isolated from nymphs, males, females, and Sf9 cells successfully bound to Vg1HRE, while binding was outcompeted by a 100-fold excess of cold probe or dephosphorylated nuclear protein extracts. In addition, binding was outcompeted by other ecdysone and JH response elements with similar half-site sequences (direct repeats) but to varying extents. Ultimately, we postulate that JH III indirectly activates Vg expression by interfering with or inhibiting the phosphorylation of nuclear proteins bound to Vg1HRE. Involvement of JH III in both induction of Vg1 and control of nuclear proteins binding to Vg1HRE suggest the latter to play an important role in JH signaling.

The Driving Force: Nuclear Mechanotransduction in Cellular Function, Fate, and Disease.

Cellular behavior is continuously affected by microenvironmental forces through the process of mechanotransduction, in which mechanical stimuli are rapidly converted to biochemical responses. Mounting evidence suggests that the nucleus itself is a mechanoresponsive element, reacting to cytoskeletal forces and mediating downstream biochemical responses. The nucleus responds through a host of mechanisms, including partial unfolding, conformational changes, and phosphorylation of nuclear envelope proteins; modulation of nuclear import/export; and altered chromatin organization, resulting in transcriptional changes. It is unclear which of these events present direct mechanotransduction processes and which are downstream of other mechanotransduction pathways. We critically review and discuss the current evidence for nuclear mechanotransduction, particularly in the context of stem cell fate, a largely unexplored topic, and in disease, where an improved understanding of nuclear mechanotransduction is beginning to open new treatment avenues. Finally, we discuss innovative technological developments that will allow outstanding questions in the rapidly growing field of nuclear mechanotransduction to be answered.

TrkB-ICD Fragment, Originating From BDNF Receptor Cleavage, Is Translocated to Cell Nucleus and Phosphorylates Nuclear and Axonal Proteins.

The signaling of brain-derived neurotrophic factor (BDNF) has been suggested to be impaired in Alzheimer’s disease (AD), which may compromise the function of BDNF upon neuronal activity and survival. Accordingly, decreased levels of BDNF and its tropomyosin-receptor kinase B-full-length (TrkB-FL) have been detected in human brain samples of AD patients. We have previously found that neuronal exposure to amyloid-β (Aβ) peptide, a hallmark of AD, leads to calpain overactivation and subsequent TrkB-FL cleavage leading to decreased levels of TrkB-FL and the generation of two new fragments: a membrane-bound truncated receptor (TrkB-T′) and an intracellular fragment (TrkB-ICD). Importantly, we identified this TrkB-FL cleavage and TrkB-ICD presence in human brain samples, which indicates that this molecular mechanism contributes to the loss of BDNF signaling in humans. The exact role of this TrkB-ICD fragment is, however, unknown. Here, we used a human neuroglioma cell line and rat cortical primary neuronal cultures to track TrkB-ICD intracellularly. Our data show that TrkB-ICD is a relatively stable fragment that accumulates in the nucleus over time, through a phosphorylation-dependent process. We also found that TrkB-ICD has tyrosine kinase activity, inducing the phosphorylation of nuclear and axonal proteins. These findings suggest that TrkB-ICD may lead to a dysregulation of the activity of several proteins, including proteins in the nucleus, to where TrkB-ICD migrates. Since TrkB-ICD is formed by Aβ peptide-induced cleavage of TrkB-FL, the present data highlights a new mechanism that may have a role in AD pathophysiology.

Antimetastatic effect of the pharmacological inhibition of serine/arginine-rich protein kinases (SRPK) in murine melanoma

The Serine/arginine-rich protein kinases (SRPK) are involved in pre-mRNA splicing control through the phosphorylation of the SR protein family of splicing factors. Over the last years, several studies have shown the relevance of SRPK for human cancers and their potential as promising drug targets. In this context, we have previously selected three trifluoromethyl arylamides (named here as SRVIC24, SRVIC30 and SRVIC36) with improved in vitro antileukemia effect and ability of impairing the cellular activity of SRPK. Given the increasing amount of reports on the implication of these kinases in metastatic cancers, in this study, we have evaluated the antimetastatic effect of these compounds and the known SRPK inhibitor (SRPIN340) on a murine model of metastatic melanoma. The compounds were able to impact the melanoma cell metastatic behavior by decreasing migration, invasion, adhesion, and colony formation in in vitro assays. Also, they presented antimetastatic in vivo activity, without apparent signs of systemic toxicity after treatments, as revealed by the histology of organs and analysis of key serum biochemical markers. Moreover, the effect of the treatments on SRPK1 nuclear translocation and SR protein phosphorylation was observed. Finally, molecular docking studies were carried out to gain structural information on the SRPK-compound complexes. Together, these data suggest that SRPK pharmacological inhibition should be considered as an interesting therapeutic strategy against metastatic cancers.

Analyzing the influence of kinase inhibitors on DNA repair by differential proteomics of chromatin-interacting proteins and nuclear phospho-proteins.

The combination of radiotherapy and pharmacological inhibition of cellular signal transduction pathways offers promising strategies for enhanced cancer cell inactivation. However, the molecular effects of kinase inhibitors especially on DNA damage detection and repair after X-irradiation have to be understood to facilitate the development of efficient and personalized treatment regimens. Therefore, we applied differential proteomics for analyzing inhibitor-induced changes in either chromatin-bound or phosphorylated nuclear proteins. The effect of the multi kinase inhibitor sorafenib on DNA repair, chromatin binding and phosphorylation of nuclear proteins was analyzed in UT-SCC 42B head and neck cancer cells using metabolic labeling based differential proteomics (SILAC). Sorafenib significantly inhibited DNA repair but failed to significantly affect chromatin interactions of 90 quantified proteins. In contrast, analyzing nuclear phospho-proteins following sorafenib treatment, we detected quantitative changes in 9 out of 59 proteins, including DNA-repair proteins. In conclusion, the analysis of nuclear phospho-proteins by differential proteomics is an effective tool for determining the molecular effects of kinase inhibitors on X-irradiated cells. Analyzing chromatin binding might be less promising.

Protein kinase A activation by retinoic acid in the nuclei of HL60 cells

BackgroundActivation of protein kinase A (PKA) occurs during retinoic acid (RA)-induced granulocytic differentiation of human promyelocytic leukemia HL60 cells. It is known that the RIIα regulatory subunit of PKA, is modified by RA (retinoylated) in the early stages of differentiation. We have investigated the effects of RA on PKA during cell differentiation in order to understand the potential significance of this process in the retinoylation of RIIα subunits. MethodsImmunoblotting, immunoprecipitation, confocal microscopy, PCR, and PKA activity assays were employed for characterizing the effects of RA on PKA. ResultsWe found that RA induces intracellular mobility of RIIα and the activation of PKA in HL60 cells. Increases in RIIα levels were observed in RA-treated HL60 cells. RA treatment altered intracellular localization of the PKA subunits, RIIα and Cα, and increased their protein levels in the nuclei as detected by both immunoblotting and immunostaining analyses. Coincident with the increase in nuclear Cα, RA-treated HL60 cells showed increases in both the protein phosphorylation activity of PKA and the levels of phosphorylated proteins in nuclear fractions as compared to control cells. In addition, RIIα protein was stabilized in RA-treated HL60 cells as compared to control cells. ConclusionsThese results suggest that RA stabilizes RIIα protein and activates PKA in the nucleus, with a resultant increase in the phosphorylation of nuclear proteins. General significanceOur evidence suggests that retinoylation of PKA might contribute to its stabilization and activation and that this could potentially participate in RA's ability to induce granulocytic differentiation of HL60 cells.

LECT2 association with macrophage-mediated killing of Helicobacter pylori by activating NF-κB and nitric oxide production.

Helicobacter pylori employs unique methods to colonize the stomach, which induces chronic inflammation. It is also able to avoid eradication by macrophages and other immune cells. Leukocyte cell-derived chemotaxin 2 (LECT2), a multi-functional cytokine involved in many pathological conditions, has recently been shown to activate macrophages via the CD209a receptor. Therefore, we aimed to investigate the effects of LECT2 on H. pylori-infected macrophages. Macrophages were treated with recombinant LECT2, and both their ability to kill H. pylori and produce nitric oxide were analyzed. Western blot was performed to determine nuclear translocation and protein phosphorylation of p65, a subunit of nuclear factor (NF)-κB. Transfection experiments were performed to analyze the signaling pathway of LECT2 in macrophages. We found that treatment with LECT2 enhanced H. pylori killing and nitric oxide production in macrophages. In addition, DNA-binding activity and nuclear translocation of p65 were up-regulated by LECT2 treatment. Furthermore, we found that NF-κB activation by LECT2 was mediated by Raf-1 in macrophages, and Raf-1 phosphorylation was specifically altered in response to LECT2. Moreover, LECT2 induced Ser28 phosphorylation in the intracellular domain of CD209a. CD209a Ser28 phosphorylation was required for LECT2-induced Raf-1 and NF-κB activation in RAW264.7 macrophages. Our study showed that the effects of LECT2 on H. pylori killing and nitric oxide production were dependent on CD209a phosphorylation, Raf-1, and NF-κB activation. Together, these results demonstrate for the first time that exposure to LECT2 can modulate specific intracellular mechanisms downstream of CD209a to enhance H. pylori killing and nitric oxide production in macrophages.

Proteomic Analysis of Phosphoproteins in the Rice Nucleus During the Early Stage of Seed Germination.

The early stage of seed germination is the first step in the plant life cycle without visible morphological change. To investigate the mechanism controlling the early stage of rice seed germination, we performed gel-and label-free nuclear phosphoproteomics. A total of 3467 phosphopeptides belonging to 102 nuclear phosphoproteins from rice embryos were identified. Protein-synthesis-related proteins were mainly phosphorylated. During the first 24 h following imbibition, 115 nuclear phosphoproteins were identified, and significant changes in the phosphorylation level over time were observed in 29 phosphoproteins. Cluster analysis indicated that nucleotide-binding proteins and zinc finger CCCH- and BED-type proteins increased in abundance during the first 12 h of imbibition and then decreased. The in silico protein-protein interactions for 29 nuclear phosphoproteins indicated that the Sas10/Utp3 protein, which functions in snoRNA binding and gene silencing, was the center of the phosphoprotein network in nuclei. The germination rate of seeds was significantly slowed with phosphatase inhibitor treatment. The mRNA expression of the zinc finger CCCH-type protein did not change, and the zinc finger BED-type protein was upregulated in rice embryos during the early stage of germination with phosphatase inhibitor treatment. These results suggest that the phosphorylation and dephosphorylation of nuclear proteins are involved in rice seed germination. Furthermore, transcription factors such as zinc finger CCCH- and BED-type proteins might play a key role through nuclear phosphoproteins, and Sas10/Utp3 protein might interact with nuclear phosphoproteins in rice embryos to mediate the early stage of seed germination.

Role for NUP62 depletion and PYK2 redistribution in dendritic retraction resulting from chronic stress.

Genetic evidence suggests cell-type-specific functions for certain nucleoporins, and gene expression profiling has revealed that nucleoporin p62 (NUP62) transcripts are decreased in the prefrontal cortex of major depressives. Chronic stress, which can precipitate depression, induces changes in the architecture and plasticity of apical dendrites that are particularly evident in the CA3 region of the hippocampus. Genetically targeted translating ribosome affinity purification revealed a selective reduction in translated Nup62 transcripts in CA3 of chronically stressed mice, and the Nup62 protein content of nuclei extracted from whole hippocampus was found to be decreased in chronically stressed rats. In cultured cells, phosphorylation of a FAK/proline-rich tyrosine kinase 2 (PYK2) consensus site in the alpha-helical domain of NUP62 (human Y422) is shown to be associated with shedding of NUP62 from the nuclear pore complex (NPC) and/or retention of NUP62 in the cytoplasm. Increased levels of phospho-Y425 Nup62 were observed in cytoplasmic fractions of hippocampi from chronically stressed rats, and immunofluorescence microscopy revealed redistribution of activated Pyk2 to the perinuclear region of stressed pyramidal neurons. Depletion of Nup62 from cultured embryonic day 18 rat hippocampal and cortical neurons resulted in simplification and retraction of dendritic arbors, without disruption of axon initial segment integrity. Thus, at least two types of mechanisms--one affecting expression and the other association with the NPC--could contribute to loss of NUP62 from CA3 pyramidal neurons during chronic stress. Their combined actions may account for the enhanced responsiveness of CA3 apical dendrites to chronic stress and may either be pathogenic or serve to protect CA3 neurons from permanent damage.

Effect of testosterone on markers of mitochondrial oxidative phosphorylation and lipid metabolism in muscle of aging men with subnormal bioavailable testosterone

Recent studies have indicated that serum testosterone in aging men is associated with insulin sensitivity and expression of genes involved in oxidative phosphorylation (OxPhos), and that testosterone treatment increases lipid oxidation. Herein, we investigated the effect of testosterone therapy on regulators of mitochondrial biogenesis and markers of OxPhos and lipid metabolism in the skeletal muscle of aging men with subnormal bioavailable testosterone levels. Skeletal muscle biopsies were obtained before and after treatment with either testosterone gel (n=12) or placebo (n=13) for 6 months. Insulin sensitivity and substrate oxidation were assessed by euglycemic-hyperinsulinemic clamp and indirect calorimetry. Muscle mRNA levels and protein abundance and phosphorylation of enzymes involved in mitochondrial biogenesis, OxPhos, and lipid metabolism were examined by quantitative real-time PCR and western blotting. Despite an increase in lipid oxidation (P<0.05), testosterone therapy had no effect on insulin sensitivity or mRNA levels of genes involved in mitochondrial biogenesis (PPARGC1A, PRKAA2, and PRKAG3), OxPhos (NDUFS1, ETFA, SDHA, UQCRC1, and COX5B), or lipid metabolism (ACADVL, CD36, CPT1B, HADH, and PDK4). Consistently, protein abundance of OxPhos subunits encoded by both nuclear (SDHA and UQCRC1) and mitochondrial DNA (ND6) and protein abundance and phosphorylation of AMP-activated protein kinase and p38 MAPK were unaffected by testosterone therapy. The beneficial effect of testosterone treatment on lipid oxidation is not explained by increased abundance or phosphorylation-dependent activity of enzymes known to regulate mitochondrial biogenesis or markers of OxPhos and lipid metabolism in the skeletal muscle of aging men with subnormal bioavailable testosterone levels.

The Akt-SRPK-SR Axis Constitutes a Major Pathway in Transducing EGF Signaling to Regulate Alternative Splicing in the Nucleus

Pre-mRNA splicing is regulated by developmental and environmental cues, but little is known about how specific signals are transduced in mammalian cells to regulate this critical gene expression step. Here, we report massive reprogramming of alternative splicing in response to EGF signaling. By blocking individual branches in EGF signaling, we found that Akt activation plays a major role, while other branches, such as the JAK/STAT and ERK pathways, make minor contributions to EGF-induced splicing. Activated Akt next branches to SR protein-specific kinases, rather than mTOR, by inducing SRPK autophosphorylation that switches the splicing kinases from Hsp70- to Hsp90-containing complexes. This leads to enhanced SRPK nuclear translocation and SR protein phosphorylation. These findings reveal a major signal transduction pathway for regulated splicing and place SRPKs in a central position in the pathway, consistent with their reputed roles in a large number of human cancers.

Lysine residue at position 22 of the AID protein regulates its class switch activity.

BackgroundActivation induced deaminase (AID) mediates class switch recombination and somatic hypermutation of immunoglobulin (Ig) genes in germinal centre B cells. In order to regulate its specific activity and as a means to keep off-target mutations low, several mechanisms have evolved, including binding to specific cofactors, phosphorylation and destabilization of nuclear AID protein. Although ubiquitination at lysine residues of AID is recognized as an essential step in initiating degradation of nuclear AID, any functional relevance of lysine modifications has remained elusive.Methodology/Principal FindingsHere, we report functional implications of lysine modifications of the human AID protein by generating a panel of lysine to arginine mutants of AID and assessment of their catalytic class switch activity. We found that only mutation of Lys22 to Arg resulted in a significant reduction of class switching to IgG1 in transfected primary mouse B cells. This decrease in activity was neither reflected in reduced hypermutation of Ig genes in AID-mutant transfected DT40 B cell lines nor recapitulated in bacterial deamination assays, pointing to involvement of post-translational modification of Lys22 for AID activity in B cells.Conclusions/SignificanceOur results imply that lysine modification may represent a novel level of AID regulation and that Lys22 is important for effective AID activity.

Nicotinamide inhibits nuclear factor-kappa B translocation after transient focal cerebral ischemia

We explored the putative anti-inflammatory effects of nicotinamide against experimental stroke. Prospective laboratory study. Research laboratory in a university teaching hospital. Adult male Sprague-Dawley rats (250-300 g). The antioxidant, radical scavenging, and anti-inflammatory actions of nicotinamide were evaluated using a panel of acellular assays and lipopolysaccharide-stimulated RAW 264.7 and BV2 cells. Animals were subjected to transient middle cerebral artery occlusion for 90 mins. Nicotinamide (500 mg/kg) or vehicle was given intravenously at reperfusion onset. Nicotinamide effectively inhibited nuclear factor-κB translocation and binding activity as well as the production of tumor necrosis factor-α, nitrite/nitrate, and interleukin-6 in the lipopolysaccharide-stimulated RAW 264.7 and BV2 cells (p < .05, respectively) but exhibited weak antioxidant and radical-scavenging actions. Relative to controls, nicotinamide-treated animals had significant reductions in neutrophil and macrophage/activated microglial infiltration in the ischemic brain by 53% and 77% (p < .05, respectively). Additionally, nicotinamide significantly attenuated phosphorylation of nuclear factor-κB's inhibitory protein, nuclear factor-κB translocation and binding activity, and the synthesis of inducible nitric oxide in the ischemic brain (p < .05, respectively). Consequently, nicotinamide effectively reduced brain infarction and improved neurobehavioral outcome by 43% and 50% (p < .05, respectively). Nicotinamide effectively attenuated postischemic nuclear factor-kappa]B activation and exhibited robust anti-inflammatory actions against ischemic stroke.

Sumoylated protein tyrosine phosphatase 1B localizes to the inner nuclear membrane and regulates the tyrosine phosphorylation of emerin

Protein tyrosine phosphatase (PTP)1B is an abundant non-transmembrane enzyme that plays a major role in regulating insulin and leptin signaling. Recently, we reported that PTP1B is inhibited by sumoylation, and that sumoylated PTP1B accumulates in a perinuclear distribution, consistent with its known localization in the endoplasmic reticulum (ER) and the contiguous outer nuclear membrane. Here, we report that, in addition to its localization at the ER, PTP1B also is found at the inner nuclear membrane, where it is heavily sumoylated. We also find that PTP1B interacts with emerin, an inner nuclear membrane protein that is known to be tyrosine phosphorylated, and that PTP1B expression levels are inversely correlated with tyrosine phosphorylation levels of emerin. PTP1B sumoylation greatly increases as cells approach mitosis, corresponding to the stage where tyrosine phosphorylation of emerin is maximal. In addition, expression of a non-sumoylatable mutant of PTP1B greatly reduced levels of emerin tyrosine phosphorylation. These results suggest that PTP1B regulates the tyrosine phosphorylation of a key inner nuclear membrane protein in a sumoylation- and cell-cycle-dependent manner.

Type 2 histone deacetylases play a major role in the control of elicitor-induced cell death in tobacco

The cell death which characterizes the onset of the Hypersensitive Response (HR) is a very important weapon evolved by plants to block pathogen development. By the use of numerous plant/avirulent pathogen or plant/elicitor models, we have now obtained detailed signalling pathways allowing, after pathogen or elicitor perception, the control of the expression of specific sets of genes that contribute to cell death. However, our knowledge of the molecular actors involved in this process still remains limited. This is particularly true when regarding what happen in the nucleus. We recently reported that nuclear post-translational protein modifications are major processes that control cell death. Using the tobacco / cryptogein model, we showed that type 2 histone deacetylase activities, which act as negative regulators of cell death, depend on their phosphorylation status. In the present paper, we integrated all these results to propose a model depicting the putative nuclear signalling pathways controlling the establishment of cell death in tobacco in response to the cryptogein elicitor. This model highlights the role of the nuclear protein acetylation and phosphorylation in the establishment of plant defences.