BBA - Molecular Cell Research (v.1783, #12)

BRCA1 is regulated by Chk2 in response to spindle damage by Corinne Chabalier-Taste; Carine Racca; Christine Dozier; Florence Larminat (2223-2233).
Inherited mutations of the breast cancer susceptibility gene 1 (BRCA1) confer an increased risk for breast, ovarian and prostate cancer. BRCA1 has been involved in regulation of cell cycle progression, DNA damage signaling and repair, maintenance of genome integrity, ubiquitination and regulation of transcription. Aside from its essential functions in the DNA damage response BRCA1 has been also involved in the cellular response to microtubule damage. Emerging evidence indicates that BRCA1 regulates the duplication and the function of centrosomes, participates in mitotic spindle assembly and is required in the spindle checkpoint. Given BRCA1 distinct functions in microtubule-dependent pathways, we hypothesized that BRCA1 might be regulated following microtubule damage. In the present study, we report the novel finding that BRCA1 is phosphorylated by the checkpoint kinase Chk2 on the previously identified site Ser988 following anti-mitotic treatment in human cancer cells. Ser988-phosphorylated BRCA1 accumulates at centrosomes in response to microtubule damage but Ser988 is not essential for BRCA1 localization at the microtubule-organizing centers. We further demonstrate that the Ser988 phosphorylation is important for the inhibiting microtubule nucleation activity of BRCA1 and for BRCA1 function in cell survival following microtubule damage. These findings reveal a striking outcome of BRCA1 phosphorylation by Chk2 on its role in microtubule-dependent pathways and suggest a fine cross-talk between DNA damage and spindle damage responses.
Keywords: BRCA1; Chk2; Microtubule nucleation; Paclitaxel; Spindle damage response;

Membrane progestin receptors α and γ in renal epithelium by Julie Lemale; May Bloch-Faure; Adrien Grimont; Boutaïna El Abida; Martine Imbert-Teboul; Gilles Crambert (2234-2240).
Sex hormones have broader effects than regulating reproductive functions. Recent identification of membrane progestin receptors expressed in kidney prompted us to investigate their putative involvement in the renal effects of this hormone. We first focused our investigations on mPRα and γ by analyzing three parameters 1/ their distribution along the mouse nephron and their subcellular location in native kidney, 2/ the ability of progesterone to stimulate ERK pathway and/or Ca2+ release from internal stores in native kidney structures and 3/ the cellular localization of mPRα and its molecular determinants in heterologous expression system. We observed that 1/ mPRα expression is restricted to proximal tubules of both male and female mice whereas mPRγ exhibits a much broader expression all along the nephron except the glomerulus, 2/ mPRα and γ are not localized at the plasma membrane in native kidney, 3/ this expression does not permit either progesterone-induced ERK phosphorylation or Ca2+ release and 4/ in HEK transfected cells, mPRα localizes in the endoplasmic reticulum (ER) due to a C-terminal ER retention motif (− KXX). Therefore, we have characterized mPRs in kidney but their role in renal physiology remains to be elucidated.
Keywords: Sex hormone; Nephron; Progesterone; ER exit; Di-basic retention motif;

ERK and p38 pathways regulate amino acid signalling by Eduard Casas-Terradellas; Irantzu Tato; Ramon Bartrons; Francesc Ventura; Jose Luis Rosa (2241-2254).
The ribosomal protein S6 kinase 1 (S6K1) is emerging as a common downstream target of signalling by hormones and nutrients such as insulin and amino acids. Here, we have investigated how amino acids signal through the S6K1 pathway. First, we found that a commercial anti-phospho-Thr389-S6K1 antibody detects an 80–90 kDa protein that is rapidly phosphorylated in response to amino acids. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI-3 kinase inhibitors, and knockdown experiments showed that this protein was not S6K1. Looking for candidate targets of this phosphorylation, we found that amino acids stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. In turn, these phosphorylations required the activity of either p38 or ERK MAP kinases, which could compensate for each other. Moreover, we show that these MAP kinases are also needed for the amino acid-induced phosphorylation of S6K1 at Thr421/Ser424, as well as for that of S6K1 substrate, the S6 ribosomal protein. Consistent with these results, concomitant inhibition of p38 and ERK pathways also antagonised the well-known effects of amino acids on the process of autophagy. Altogether, these findings demonstrate a previously unknown role for MAP kinases in amino acid signalling.
Keywords: Amino acid; ERK; MSK; p38; RSK; S6K1;

Tau binds both subunits of calcineurin, and binding is impaired by calmodulin by Da-yu Yu; Li Tong; Gao-jie Song; Wei-lin Lin; Lai-qun Zhang; Wei Bai; He Gong; Yan-xia Yin; Qun Wei (2255-2261).
Calcineurin, an important protein Ser/Thr phosphatase which acts on tau in vivo, is a heterodimer of a catalytic subunit, calcineurin A, and a regulatory subunit, calcineurin B, and is unique in being regulated by calmodulin. Here, we find that both subunits of calcineurin bind tau, and calmodulin interferes with the association between calcineurin and tau. The domains of both subunits of calcineurin and tau involved in binding are mapped. We also investigate the functional consequences of the interactions between both subunits of calcineurin, tau and calmodulin, and reveal the interactions affect dephosphorylation of tau by calcineurin and contribute to the balance of phosphorylation and dephosphorylation of tau in vivo. Our findings may be of potential significance in neuronal physiology and also in neurodegenerative disorders. They shed some light on how the interactions might control the phosphorylation state of tau under physiological conditions, and provide new insights into the treatment of tauopathies such as Alzheimer's disease.
Keywords: Tau; Calcineurin A subunit; Calcineurin B subunit; Calmodulin; Protein interaction; Tauopathy;

Plasma membrane diffusion of g protein-coupled receptor oligomers by Stephen J. Briddon; Jorge Gandía; Olavo B. Amaral; Sergi Ferré; Carme Lluís; Rafael Franco; Stephen J. Hill; Francisco Ciruela (2262-2268).
G protein-coupled receptors are known to form homo-and heteromers at the plasma membrane, but the molecular properties of these oligomers are relatively unknown. Here, we show a method that allows the diffusion of G protein-coupled receptors oligomers in the plasma membrane to be monitored in single cells by combining Bimolecular Fluorescence Complementation and Fluorescence Correlation Spectroscopy. With this approach we have measured, for the first time, the membrane diffusional characteristics of adenosine A1 and A2A receptor homo-and heterodimers in Chinese Hamster Ovary cells. Interestingly, both homodimers display similar diffusion co-efficients (D) when expressed in living cells (D  = 5.0 and 4.8 × 10− 9 cm2/s, respectively) but the heterodimer formed by these receptors exhibit a significantly faster plasma membrane diffusion co-efficent (D  = 5.6 × 10− 9 cm2/s) when compared to the adenosine A1 receptor tagged with the full-length yellow fluorescent protein (D  = 4.0 × 10− 9 cm2/s). Overall, these results demonstrate differences in plasma membrane diffusion between adenosine receptor homo-and heterodimers, providing new insights into the molecular plasticity of G protein-coupled receptor oligomerization.
Keywords: G protein-coupled receptor; Receptor oligomerization; Adenosine receptor; Membrane diffusion;

Bovine serum amine oxidase and spm potentiate docetaxel and interferon-α effects in inducing apoptosis on human cancer cells through the generation of oxidative stress by M. Marra; A. Lombardi; E. Agostinelli; G. Giuberti; S. Zappavigna; G. Tempera; G. Vitale; M. Bifulco; A. Abbruzzese; M. Caraglia (2269-2278).
It was previously demonstrated that bovine serum amine-oxidase (BSAO) and SPM (SPM) addition to cancer cells induces cell growth inhibition and over-run the multi-drug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites. In this study, it is reported that BSAO/SPM enzymatic system antagonizes the survival pathway induced by either docetaxel (DTX) or interferon alpha (IFNα) in human epidermoid cancer KB cells. The combination of BSAO/SPM with either DTX or IFNα had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent and were paralleled by the enhancement of intracellular O2−, nitric oxide levels and of lipo-oxidation. The scavenger moiety N-acetyl-cysteine antagonized the effects on apoptosis and cell growth inhibition induced by the combination suggesting a role of the oxidative products of SPM. These effects occurred together with a decrease of the physiological scavenger MnSOD and an increase of both p38 kinase activity and DNA damage. The results suggest that DTX and IFNα could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM through the induction of a survival ras-dependent pathway and the consequent elevation of the intracellular polyamine pool. These data allow the design of new therapeutic strategy based on the use of this combination in human neoplasms.
Keywords: Bovine serum amine oxidase; Polyamines; Docetaxel; IFNα; Oxidative stress;

Retinoids regulate TGFβ signaling at the level of Smad2 phosphorylation and nuclear accumulation by Loretta L. Hoover; Elizabeth G. Burton; Megan L. O'Neill; Bonnie A. Brooks; Shilpa Sreedharan; Nineveh A. Dawson; Steven W. Kubalak (2279-2286).
Indirect regulation of transforming growth factor (TGF)-β signaling by retinoids occurs on a long-term timescale, secondary to transcriptional events. Studies by our group show loss of retinoid X receptor (RXR) alpha results in increased TGFβ2 in the midgestational heart, which may play a role in the cardiac defects seen in this model [S.W. Kubalak, D.R. Hutson, K.K. Scott and R.A. Shannon, Elevated transforming growth factor beta2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor alpha knockout embryos, Development 129 (2002) 733–746.]. Acute and direct interactions between retinoid and TGFβ signaling, however, are not clearly understood. Treatment of dispersed hearts and NIH3T3 cells for 1 h with TGFβ and retinoids (dual treatment) resulted in increased phosphorylated Smad2 and Smad3 when compared to treatment with TGFβ alone. Of all dual treatments, those with the RXR agonist Bexarotene, resulted in the highest level of phosphorylated Smad2, a 7-fold increase over TGFβ2 alone. Additionally, during dual treatment phosphorylation of Smad2 occurs via the TGFβ type I receptor but not by increased activation of the receptor. As loss of RXRα results in increased levels of Smad2 phosphorylation in response to TGFβ treatment and since nuclear accumulation of phosphorylated Smad2 is decreased during dual treatment, we propose that RXRα directly regulates the activities of Smad2. These data show retinoid signaling influences the TGFβ pathway in an acute and direct manner that has been unappreciated until now.
Keywords: Smad; Retinoic acid; Transforming growth factor-β; Retinoid X receptor; Phosphorylation; Nuclear accumulation;

Importin-α is essential for classical nucleocytoplasmic transport of nuclear proteins. Here, we report that importin-α is cleaved by caspases during apoptosis, generating importin-α lacking an IBB domain. This truncated importin-α binds tightly to the MCM replication licensing factor and, thus, prevents its binding to chromatin and downregulates DNA synthesis. Together, our data reveal for the first time that a dying cell effectively salvages limited supplies of cellular energy to ensure an orderly process of its own demise by simultaneously downregulating nucleocytoplasmic protein transport and DNA synthesis. Strikingly, cells can achieve this multi-task process by simply cleaving-off a key nuclear import protein.
Keywords: Importin; IBB domain; Protein transport; Caspase; DNA replication; Apoptosis;

Role of the proteolytic hierarchy between cathepsin L, cathepsin D and caspase-3 in regulation of cellular susceptibility to apoptosis and autophagy by Xin Zheng; Fei Chu; Bernard L. Mirkin; Thangirala Sudha; Shaker A. Mousa; Abdelhadi Rebbaa (2294-2300).
The present investigation was undertaken to measure the relative abilities of pro-death versus pro-survival proteases in degrading each other and to determine how this might influence cellular susceptibility to death. For this, we first carried out in vitro experiments in which recombinant pro-death proteases (caspase-3 or cathepsin D) were incubated with the pro-survival protease (cathepsin L) in their respective optimal conditions and determined the effects of these reactions on enzyme integrity and activity. The results indicated that cathepsin L was able to degrade cathepsin D, which in turn cleaves caspase-3, however the later enzyme was unable to degrade any of the cathepsins. The consequences of this proteolytic sequence on cellular ability to undergo apoptosis or other types of cell death were studied in cells subjected to treatment with a specific inhibitor of cathepsin L or the corresponding siRNA. Both treatments resulted in suppression of cellular proliferation and the induction of a cell death with no detectable caspase-3 activation or DNA fragmentation, however, it was associated with increased accumulation of cathepsin D, cellular vaculolization, expression of the mannose-6-phosphate receptor, and the autophagy marker LC3-II, all of which are believed to be associated with autophagy. Genetic manipulations leading either to the gain or loss of cathepsin D expression implicated this enzyme as a key player in the switch from apoptosis to autophagy. Overall, these findings suggest that a hierarchy between pro-survival and pro-death proteases may have important consequences on cell fate.
Keywords: Cathepsin L; Cathepsin D; LC3; Apoptosis; Autophagy;

Accessory subunit Ac45 controls the V-ATPase in the regulated secretory pathway by Eric J.R. Jansen; Wim J.J.M. Scheenen; Theo G.M. Hafmans; Gerard J.M. Martens (2301-2310).
The vacuolar (H+)-ATPase (V-ATPase) is crucial for multiple processes within the eukaryotic cell, including membrane transport and neurotransmitter secretion. How the V-ATPase is regulated, e.g. by an accessory subunit, remains elusive. Here we explored the role of the neuroendocrine V-ATPase accessory subunit Ac45 via its transgenic expression specifically in the Xenopus intermediate pituitary melanotrope cell model. The Ac45-transgene product did not affect the levels of the prohormone proopiomelanocortin nor of V-ATPase subunits, but rather caused an accumulation of the V-ATPase at the plasma membrane. Furthermore, a higher abundance of secretory granules, protrusions of the plasma membrane and an increased Ca2+-dependent secretion efficiency were observed in the Ac45-transgenic cells. We conclude that in neuroendocrine cells Ac45 guides the V-ATPase through the secretory pathway, thereby regulating the V-ATPase-mediated process of Ca2+-dependent peptide secretion.
Keywords: Xenopus transgenesis; Ca2+-dependent peptide secretion; Intermediate pituitary melanotrope cells; Proton pump trafficking; Secretory capacity; Plasma membrane protrusions;

SRC-dependent signalling regulates actin ruffle formation induced by glycerophosphoinositol 4-phosphate by Beatrice Maria Filippi; Stefania Mariggiò; Teodoro Pulvirenti; Daniela Corda (2311-2322).
The glycerophosphoinositols are diffusible phosphoinositide metabolites reported to modulate actin dynamics and tumour cell spreading. In particular, the membrane permeant glycerophosphoinositol 4-phosphate (GroPIns4P) has been shown to act at the level of the small GTPase Rac1, to induce the rapid formation of membrane ruffles. Here, we have investigated the signalling cascade involved in this process, and show that it is initiated by the activation of Src kinase. In NIH3T3 cells, exogenous addition of GroPIns4P induces activation and translocation of Rac1 and its exchange factor TIAM1 to the plasma membrane; in addition, in in-vitro assays, GroPIns4P favours the formation of a protein complex that includes Rac1 and TIAM1. Neither of these processes involves direct actions of GroPIns4P on these proteins. Thus, through the use of specific inhibitors of tyrosine kinases and phospholipase C (and by direct evaluation of kinase activities and inositol 1,4,5-trisphosphate production), we show that GroPIns4P activates Src, and as a consequence, phospholipase Cγ and Ca2+/calmodulin kinase II, the last of which directly phosphorylates TIAM1 and leads to TIAM1/Rac1-dependent ruffle formation.
Keywords: Glycerophosphoinositol; Phosphoinositide; Actin cytoskeleton; Signalling; Tyrosine kinase;

Integrin-dependent interaction of epithelial tumor cells with extracellular matrix (ECM) is critical for their migration, but also for hematogenous dissemination. Elevated expression and activity of Src family kinases (SFKs) in colon cancer cells is often required in the disease progression. In this work, we highlighted how focal adhesion kinase (FAK) and SFKs interacted and we analyzed how PI3K/Akt and MAPK/Erk1/2 signaling pathways were activated in early stages of colon cancer cell adhesion. During the first hour, integrin engagement triggered FAK-Y397 phosphorylation and a fraction of FAK was located in lipid rafts/caveolae domains where it interacted with Fyn. The FAK-Y861 and/or -Y925 phosphorylations led to a subsequently FAK translocation out of lipid domains. In parallel, a PI3K/Akt pathway dependent of lipid microdomain integrity was activated. In contrast, the MAPK/Erk1/2 signaling triggered by adhesion increased during at least 4 h and was independent of cholesterol disturbing. Thus, FAK/Fyn interaction in lipid microdomains and a Akt-1 activation occurred at the same time during early contact with ECM suggesting a specific signaling dependent of lipid rafts/caveolae domains.
Keywords: Akt-1; Early adhesion; FAK; Lipid domain; SFK; Signal transduction;

Extracellular superoxide released from mitochondria mediates mast cell death by advanced glycation end products by Tetsuro Yoshimaru; Yoshihiro Suzuki; Toshio Inoue; Shigeru Nishida; Chisei Ra (2332-2343).
Advanced glycation end products (AGEs) accumulate during aging and to higher extents under pathological conditions such as diabetes. Since we previously showed that mast cells expressed the AGE-binding protein, receptor for AGEs (RAGE) on their cell surface, we examined whether AGE affected mast cell survival. Herein, we demonstrate that mast cells undergo apoptosis in response to AGE. Glycated albumin (GA), an AGE, but not stimulation with the high-affinity IgE receptor (FcɛRI), can induce mast cell death, as measured by annexin V/propidium iodide double-staining. GA (≥ 0.1 mg/ml) exhibited this pro-apoptotic activity in a concentration-dependent manner. GA and FcɛRI stimulation increased the cytosolic Ca2+ levels to a similar extent, whereas GA, but not FcɛRI stimulation, caused mitochondrial Ca2+ overload and membrane potential collapse, resulting in mitochondrial integrity disruption, cytochrome c release and caspase-3/7 activation. In addition, GA, but not FcɛRI stimulation, induced extracellular release of superoxide from mitochondria, and this release played a key role in the disruption of Ca2+ homeostasis. Knockdown of RAGE expression using small interfering RNA abolished GA-induced apoptosis, mitochondrial Ca2+ overload, and superoxide release, demonstrating that RAGE mediates the GA-induced mitochondrial death pathway. AGE-induced mast cell apoptosis may contribute to the immunocompromised and inflammatory conditions.
Keywords: Superoxide; Mitochondria; Cell death; Mast cell;

Calreticulin regulates insulin receptor expression and its downstream PI3 Kinase/Akt signalling pathway by Shahrzad Jalali; Maliheh Aghasi; Behzad Yeganeh; Nasrin Mesaeli (2344-2351).
Defects in insulin signalling and glucose metabolism are associated with the development of diabetes. Insulin signalling is initiated by the binding of insulin to its receptor and triggering cascades of events including activation of PI3kinase/Akt signalling pathway. Calreticulin (CRT) is a calcium binding chaperone molecule located in the endoplasmic reticulum. Targeted deletion of CRT in mice is embryonic lethal as a result of developmental and metabolic abnormalities. Rescued CRT null mice develop severe hypoglycemia the reason for which is not known. In addition, ventricular cardiomyocytes isolated from CRT null (crt−/−) mice have increased glycogen deposits. Therefore, the aim of this study was to investigate the changes in the glucose uptake and insulin signalling pathway (mainly PI3 kinase/Akt) in the absence of CRT. Here we show a significant increase in the glucose uptake by the crt−/− cells. This increase was accompanied by a significant increase in both insulin receptor β expression, Insulin receptor substrate-1 phosphorylation, GLUT-1 expression and in insulin stimulated Akt phosphorylation and kinase activity in the crt−/− cells. Intriguingly, the increased expression of insulin receptor β in the crt−/− was due to decreased levels of p53 protein. The current study is the first evidence for the up-regulation of insulin receptor density and activity in the absence of CRT function.
Keywords: Calreticulin; Insulin receptor; Akt; Glucose uptake; PI3 kinase; p53;

Modulation of Mrps12/Sarsm promoter activity in response to mitochondrial stress by Ernesto Zanotto; Veikka Lehtonen; Howard T. Jacobs (2352-2362).
Transcription from the bidirectional promoter of two mouse genes encoding components of the mitochondrial translational apparatus, mitoribosomal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase (Sarsm), was shown previously to be dependent upon an array of four CCAAT boxes, interacting with the transcription factor NF-Y. Here we report that the homologous human promoter is governed by a CCAAT box array acting in an essentially similar manner. Analysis of the transcriptional response of both the human and mouse promoters to various mitochondrially acting toxins, including inhibitors of mitochondrial protein synthesis, and agents that bring about uncoupling or respiratory chain inhibition, produced either of two distinct outcomes, depending on the cell type and the conditions used. In mouse C2C12 myoblasts, human HEK293 cells or U2OS osteosarcoma cells, plus HeLa cells at high drug doses or mouse 3T3 fibroblasts subjected to prolonged drug exposure, a dose-dependent, bidirectional suppression of transcription was observed. In 3T3 cells subjected only to pre-treatment with the drugs, bidirectional Mrps12/Sarsm promoter activity was strongly stimulated. A similar, though weaker stimulation was observed at lower drug doses in HeLa cells. Reporter studies using mutated variants of the mouse promoter construct indicated that the stimulation of promoter activity in response to mitochondrial OXPHOS stress in 3T3 cells was independent of the CCAAT box array and of putative binding sites for NRF-2, AP-1 and other transcription factors, whereas transcriptional downregulation under prolonged mitochondrial stress was CCAAT box-dependent. Promoter stimulation was correlated with mitochondrial ROS production, which may be a crucial component in its signalling.
Keywords: Mitochondria; OXPHOS; Mitochondrial protein synthesis; NF-Y; CCAAT box; NRF-2; Promoter; Reactive oxygen species;

The budding yeast RasGEF Cdc25 reveals an unexpected nuclear localization by Renata Tisi; Fiorella Belotti; Chiara Paiardi; Francesco Brunetti; Enzo Martegani (2363-2374).
The mechanisms regulating the activity of Saccharomyces cerevisiae Ras-GEF Cdc25 are still largely unknown. While the catalytical function of the C-terminal domain has been thoroughly studied, only recently a role of negative control on the protein activity has been suggested for the dispensable N-terminal domain. In order to investigate Cdc25 localization and the role of its different domains, several fusion proteins were constructed using the full length Cdc25 or different fragments of the protein with the green fluorescent protein. Unexpectedly, even if only slightly overexpressed, the full protein was not located in the cell plasma membrane, but accumulates inside the cell and also into the nucleus. Moreover, the endogenous Cdc25, tagged with HA, was also found in purified nuclear extracts. The fusions spanning aa 353–875, aa 876–1100 or aa 353–1100 localize heavily in the nucleus, concentrating in the nuclear peripheral area, in a region distinct from the nucleolus. This could be related to the presence of two predicted nuclear localization signals (NLS) in positions 547 and 806, but also to the contribution of another region, spanning residues 876–1100. This localization is likely to be physiological, since the fusion proteins can be efficiently exported and then imported back into the nucleus.
Keywords: Saccharomyces cerevisiae; Nucleus; Ras;

A single Sec61-complex functions as a protein-conducting channel by Kai-Uwe Kalies; Vivica Stokes; Enno Hartmann (2375-2383).
During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61α-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.
Keywords: Protein-conducting channel; Sec61; Ribosome binding; ER-membrane; Cotranslational protein translocation;

During vertebrate segmentation, oscillatory activation of Notch signaling is important in the clock that regulates the timing of somitogenesis. In mice, the cyclic activation of NOTCH1 requires the periodic expression of Lunatic fringe (Lfng). For LFNG to play a role in the segmentation clock, its cyclic transcription must be coupled with post-translational mechanisms that confer a short protein half-life. LFNG protein is cleaved and released into the extracellular space, and here we examine the hypothesis that this secretion contributes to a short LFNG intracellular half-life, facilitating rapid oscillations within the segmentation clock. We localize N-terminal protein sequences that control the secretory behavior of fringe proteins and find that LFNG processing is promoted by specific proprotein convertases including furin and SPC6. Mutations that alter LFNG processing increase its intracellular half-life without preventing its secretion. These mutations do not affect the specificity of LFNG function in the Notch pathway, thus regulation of protein half-life affects the duration of LFNG activity without altering its function. Finally, the embryonic expression pattern of Spc6 suggests a role in terminating LFNG activity during somite patterning. These results have important implications for the mechanisms that contribute to the tight control of Notch signaling during vertebrate segmentation.
Keywords: Notch; Somitogenesis; Segmentation clock; Lunatic fringe; Glycosyltransferase;

Repression of CFTR activity in human MMNK-1 cholangiocytes induces sulfotransferase 1E1 expression in co-cultured HepG2 hepatocytes by Dongning He; Teresa W. Wilborn; Josie L. Falany; Li Li; Charles N. Falany (2391-2397).
Mouse models of cystic fibrosis (CF) indicate that sulfotransferase (SULT) 1E1 is significantly induced in livers of many mice lacking cystic fibrosis transmembrane receptor (CFTR) activity. Increased SULT1E1 activity results in the alteration of estrogen-regulated protein expression in the livers of these mice. In this study, human MMNK-1 cholangiocytes with repressed CFTR function were used to induce SULT1E1 expression in human HepG2 hepatocytes to investigate whether SULT1E1 can be increased in human CF liver. CFTR expression was inhibited in MMNK-1 cholangiocytes using CFTR-siRNA, then the MMNK-1 and HepG2 cells were co-cultured in a membrane-separated Transwell system. Expression of SULT1E1 and selected estrogen-regulated proteins were then assayed in the HepG2 cells. Results demonstrate that inhibition of CFTR expression in MMNK-1 cells results in the induction of SULT1E1 message and activity in HepG2 cells in the Transwell system. The expression of estrogen-regulated proteins including insulin-like growth factor (IGF)-1, glutathione-S-transferase (GST) P1 and carbonic anhydrase (CA) II expression are repressed in the HepG2 cells cultured with the CFTR-siRNA-MMNK-1 cells apparently in response to the increased sulfation of β-estradiol. Thus, we have shown that co-culture of HepG2 hepatocytes with MMNK-1 cholangiocytes with siRNA repressed CFTR expression results in the selective induction of SULT1E1 in the HepG2 cells. Loss of CFTR function in cholangiocytes may have a paracrine regulatory effect on hepatocytes via the induction of SULT1E1 and the increased sulfation of β-estradiol. Experiments are presently underway in our laboratory to elucidate the identity of these paracrine regulatory factors.
Keywords: Sulfotransferase; SULT1E1; Cystic fibrosis; Cystic fibrosis transmembrane conductance regulator; Hepatocyte; Cholangiocyte;

Chromatin structure influences the sensitivity of DNA to γ-radiation by Martin Falk; Emilie Lukášová; Stanislav Kozubek (2398-2414).
For the first time, DNA double-strand breaks (DSBs) were directly visualized in functionally and structurally different chromatin domains of human cells. The results show that genetically inactive condensed chromatin is much less susceptible to DSB induction by γ-rays than expressed, decondensed domains. Higher sensitivity of open chromatin for DNA damage was accompanied by more efficient DSB repair. These findings follow from comparing DSB induction and repair in two 11 Mbp-long chromatin regions, one with clusters of highly expressed genes and the other, gene-poor, containing mainly genes having only low transcriptional activity. The same conclusions result from experiments with whole chromosome territories, differing in gene density and consequently in chromatin condensation. It follows from our further results that this lower sensitivity of DNA to the damage by ionizing radiation in heterochromatin is not caused by the simple chromatin condensation but very probably by the presence of a higher amount of proteins compared to genetically active and decondensed chromatin. In addition, our results show that some agents potentially used for cell killing in cancer therapy (TSA, hypotonic and hypertonic) influence cell survival of irradiated cells via changes in chromatin structure and efficiency of DSB repair in different ways.
Keywords: Chromatin structure; DNA damage; Double-strand break (DSB); DNA repair; Experimentally changed radiosensitivity; Apoptosis;

Sun1 forms immobile macromolecular assemblies at the nuclear envelope by Wenshu Lu; Josef Gotzmann; Lucia Sironi; Verena-Maren Jaeger; Maria Schneider; Yvonne Lüke; Mathias Uhlén; Cristina Al-Khalili Szigyarto; Andreas Brachner; Jan Ellenberg; Roland Foisner; Angelika A. Noegel; Iakowos Karakesisoglou (2415-2426).
SUN-domain proteins form a novel and conserved family of inner nuclear membrane (INM) proteins, which establish physical connections between the nucleoplasm and the cytoskeleton. In the current study, we provide evidence that within the nuclear envelope (NE) Sun1 proteins form highly immobile oligomeric complexes in interphase cells. By performing inverse fluorescence recovery after photobleaching analysis, we demonstrate in vivo that both perinuclear and nucleoplasmic Sun1 segments are essential for maintenance of Sun1 immobility at the NE. Our data in particular underline the self-association properties of the C-terminal coiled-coil Sun1 segment, the ability of which to form dimers and tetramers is demonstrated. Furthermore, the Sun1 tertiary structure involves interchain disulfide bonds that might contribute to higher homo-oligomer formation, although the overall dynamics of the Sun1 C-terminus remains unaffected when the cysteins involved are mutated. While a major Sun1 pool colocalizes with nuclear pore complex proteins, a large fraction of the Sun1 protein assemblies colocalize with immunoreactive foci of Sun2, another SUN-domain paralogue at the NE. We demonstrate that the Sun1 coiled-coil domain permits these heterophilic associations with Sun2. Sun1 therefore provides a non-dynamic platform for the formation of different macromolecular assemblies at the INM. Our data support a model in which SUN-protein-containing multi-variate complexes may provide versatile outer nuclear membrane attachment sites for cytoskeletal filaments.
Keywords: SUN-domain; Sun1; Sun2; Nuclear envelope; Nesprin; KASH-domain; iFRAP;