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BBA - Molecular Cell Research (v.1773, #11)

Editorial Board (pp. ii).

Biting the hand that feeds: Rpn4-dependent feedback regulation of proteasome function by R. Jürgen Dohmen; Imke Willers; António J. Marques (pp. 1599-1604).
The 26S proteasome of eukaryotic cells mediates ubiquitin-dependent as well as ubiquitin-independent degradation of proteins in many regulatory processes as well as in protein quality control. The proteasome itself is a dynamic complex with varying compositions and interaction partners. Studies in Saccharomyces cerevisiae have revealed that expression of proteasome subunit genes is coordinately controlled by the Rpn4 transcriptional activator. The cellular level of Rpn4 itself is subject to a complex regulation, which, aside of a transcriptional control of its gene, intriguingly involves ubiquitin-dependent as well as ubiquitin-independent control of its stability by the proteasome. A novel study by Ju et al. [D. Ju, H. Yu, X. Wang, Y. Xie, Ubiquitin-mediated degradation of Rpn4 is controlled by a phosphorylation-dependent ubiquitylation signal, Biochim. Biophys. Acta (in press), doi:10.1016/j.bbamcr.2007.04.012] now revealed another level of complexity by showing that phosphorylation of a specific serine residue in Rpn4 is required for its efficient targeting by the Ubr2 ubiquitin ligase.

Keywords: Proteasome; Ubiquitin; Rpn4; Ubr2; Ubc2; Yap1; Pdr3; HSF


Regulation of cyclooxygenase-2 expression by cyclic AMP by Thomas Klein; Pierre Shephard; Hartmut Kleinert; Martin Kömhoff (pp. 1605-1618).
Prostaglandins (PG) regulate many biological processes, among others inflammatory reactions. Cyclooxygenases-1 and -2 (COX-1 and COX-2) catalyse PG synthesis. Since this step is rate limiting, the regulation of COX expression is of critical importance to PG biology. Contrary to COX-1, which is constitutively expressed, COX-2 expression is subject to regulation. For example, COX-2 levels are increased in inflammatory reactions. Many signalling pathways can regulate COX-2 expression, not least those involving receptors for COX products themselves. Analysis of the intracellular signal transducers involved reveals a crucial role for cAMP, albeit as a modulator rather than direct inducer. Indeed, the influence of cAMP on COX-2 expression is complex and dependent on the cell type and cellular environment. This review aims to summarise various topics related to cAMP-dependent COX-2 expression. Firstly, the main aspects of COX-2 regulation are briefly considered. Secondly, the molecular basis for COX-2 gene (post)-transcriptional regulation is reviewed. Lastly, a detailed overview of the effects of cAMP-dependent signalling on COX-2 mRNA and protein expression in various human and rodent cells is provided. There is a large number of marketed, clinical and preclinical concepts promoting the elevation of intracellular cAMP levels for therapeutic purposes (e.g., β2-agoinsts, PG receptor agonists, phosphodiesterase inhibitors). In this respect, the role of cAMP in the regulation of COX-2 expression, especially the human enzyme, is of significant clinical importance.

Keywords: Abbreviations; PG; prostaglandins; COX; cyclooxygenases; cAMP; cyclic adenosine mono-phosphate; AC; adenylate cyclase; AA; arachidonic acid; db-cAMP; di-butyryl-cAMP; LPS; lipopolysaccharide; CRE; cAMP-responsive element; CREB; cAMP-responsive element binding protein; PDE; phosphodiesterasecAMP; Adenylate cyclase; Prostaglandin; cAMP-responsive element; cGMP; Phosphodiesterase


NEDD8: A new ataxin-3 interactor by Anabela Ferro; Ana Luísa Carvalho; Andreia Teixeira-Castro; Carla Almeida; Ricardo J. Tomé; Luísa Cortes; Ana-João Rodrigues; Elsa Logarinho; Jorge Sequeiros; Sandra Macedo-Ribeiro; Patrícia Maciel (pp. 1619-1627).
Machado–Joseph disease (MJD/SCA3) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG tract in the coding portion of the ATXN3 gene. The presence of ubiquitin-positive aggregates of the defective protein in affected neurons is characteristic of this and most of the polyglutamine disorders. Recently, the accumulation of the neural precursor cell expressed developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, in the inclusions of MJD brains was reported. Here, we report a new molecular interaction between wild-type ataxin-3 and NEDD8, using in vitro and in situ approaches. Furthermore, we show that this interaction is not dependent on the ubiquitin-interacting motifs in ataxin-3, since the presence of the Josephin domain is sufficient for the interaction to occur. The conservation of the interaction between the Caenorhabditis elegans ataxin-3 homologue (atx-3) and NEDD8 suggests its biological and functional relevance. Molecular docking studies of the NEDD8 molecule to the Josephin domain of ataxin-3 suggest that NEDD8 interacts with ataxin-3 in a substrate-like mode. In agreement, ataxin-3 displays deneddylase activity against a fluorogenic NEDD8 substrate.

Keywords: Abbreviations; AMC; 7-amido-4-methylcoumarin; Ub; ubiquitin; UIM; ubiquitin-interacting motif; UBL; ubiquitin-like protein; UPS; ubiquitin–proteasome system; Y2H; yeast two-hybrid; SCF; SKP1-Cul-1-F-box protein E3Polyglutamine; UBL; Ubiquitin; E3 ligase; Neurodegeneration; MJD/SCA3


CacyBP/SIP interacts with tubulin in neuroblastoma NB2a cells and induces formation of globular tubulin assemblies by Gabriela Schneider; Krzysztof Nieznanski; Ewa Kilanczyk; Paweł Bieganowski; Jacek Kuznicki; Anna Filipek (pp. 1628-1636).
CacyBP/SIP, originally identified as a S100A6 (calcyclin) target, was later shown to interact with some other members of the S100 family as well as with Siah-1 and Skp1 proteins. Recently, it has been shown that CacyBP/SIP is up-regulated during differentiation of cardiomyocytes. In this work we show that the level of CacyBP/SIP is higher in differentiated neuroblastoma NB2a cells than in undifferentiated ones and that in cells overexpressing CacyBP/SIP the level of GAP-43, a marker of differentiation, was increased. Since the process of differentiation is accompanied by an extensive rearrangement of microtubules, we examined whether CacyBP/SIP interacted with tubulin. By applying cross-linking experiments we found that these two proteins bind directly. The dissociation constant of the tubulin–CacyBP/SIP complex determined by the surface plasmon resonance technique is 1.57×10−7M which suggests that the interaction is tight. The interaction and co-localization of CacyBP/SIP and tubulin was also demonstrated by co-immunoprecipitation, affinity chromatography and immunofluorescence methods. Light scattering measurements and electron microscopy studies revealed that CacyBP/SIP, but not its homologue, Sgt1, increased tubulin oligomerization. Altogether, our results suggest that CacyBP/SIP, via its interaction with tubulin, might contribute to the differentiation of neuroblastoma NB2a cells.

Keywords: Abbreviations; BSA; bovine serum albumin; CacyBP/SIP; calcyclin (S100A6) binding protein and Siah-1 interacting protein; CLIP; cytoplasmic linker protein; DTT; dithiothreitol; EB1; end-binding protein; EDC; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GAPDH; glyceraldehydes 3-phosphate dehydrogenase; GAP-43; growth-associated protein 43; IPTG; isopropyl-1-thio-β-; d; -galactopyranoside; MAPs; microtubule-associated proteins; MCAK; mitotic centromere-associated kinesin; MEM; minimal essential medium; NB2a; mouse neuroblastoma cell line; PBS; phosphate-buffered saline; PAGE; polyacrylamide gel electrophoresis; PMSF; phenylmethylsulfonyl fluoride; SDS; sodium dodecyl sulfate; SNHS; N; -hydroxysulfosuccinimide; SPR; surface plasmon resonance; TPPP/p25; tubulin polymerization promoting proteinCacyBP/SIP; Tubulin; Interaction; Differentiation; Globular assemblies; NB2a cells


HB-EGF induces delayed STAT3 activation via NF-κB mediated IL-6 secretion in vascular smooth muscle cell by Kuy-Sook Lee; Jin-Hee Park; Seahyoung Lee; Hyun-Joung Lim; Hye-Eun Choi; Hyun-Young Park (pp. 1637-1644).
Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family that binds to and activates EGF receptor, and is expressed in a variety of tissues, predominantly in the lung, heart, brain and skeletal muscle. HB-EGF is known to induce vascular smooth muscle cell (VSMC) proliferation by activating PI3K-Akt and MAPK pathway. However, our preliminary data showed that Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway was also involved in HB-EGF induced VSMC proliferation. More interestingly, HB-EGF (10 ng/ml) induced a biphasic activation of STAT3 (early at 5 min and late at 60–120 min). Therefore, we tried to elucidate the underlying mechanism of this delayed STAT3 activation by HB-EGF in VSMCs. First, we examined the effect of HB-EGF on interleukin-6 (IL-6) mRNA expressions, since IL-6 have been implicated in the regulation of STAT3 activation. According to our data, HB-EGF increased transcription of IL-6, cardiotrophin-1 (CT-1), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). The secretion of IL-6 was also increased by HB-EGF. Furthermore, these HB-EGF-mediated up-regulation of IL-6 mRNA expression and secretion were inhibited by NF-κB inhibitor Bay117082 (2.5 μM) treatment suggesting involvement of NF-κB pathway. Again, the late activation of STAT3 by HB-EGF was abolished by both Bay117082 and IL-6 neutralizing antibody (1 μg/ml) indicating IL-6 is a key molecule in the delayed activation of STAT3 by HB-EGF. In addition, IL-6 neutralizing antibody inhibited both HB-EGF conditioned media induced STAT3 activation and HB-EGF induced VSMC proliferation. In conclusion, IL-6 plays an important role in the delayed activation of STAT3 and VSMC proliferation induced by HB-EGF.

Keywords: HB-EGF; Proliferation; STAT3; IL-6; Smooth muscle cell


Alterations of the actin cytoskeleton and increased nitric oxide synthesis are common features in human primary endothelial cell response to changes in gravity by Silvia Versari; Alessandro Villa; Silvia Bradamante; Jeanette A.M. Maier (pp. 1645-1652).
Because endothelial cells are fundamental to the maintenance of the functional integrity of the vascular wall, endothelial modifications in altered gravity conditions might offer some insights into the mechanisms leading to circulatory impairment in astronauts. We cultured human endothelial cells in a dedicated centrifuge (MidiCAR) to generate hypergravity and in two different devices, namely the Rotating Wall Vessel and the Random Positioning Machine, to generate hypogravity. Hypogravity stimulated endothelial growth, did not affect migration, and enhanced nitric oxide production. It also remodeled the actin cytoskeleton and reduced the total amounts of actin. Hypergravity did not affect endothelial growth, markedly stimulated migration, and enhanced nitric oxide synthesis. In addition, hypergravity altered the distribution of actin fibers without, however, affecting the total amounts of actin. A short exposure to hypergravity (8 min) abolished the hypogravity induced growth advantage. Our results indicate that cytoskeletal alterations and increased nitric oxide production represent common denominators in endothelial responses to both hypogravity and hypergravity.

Keywords: Endothelial cell; Hypergravity; Hypogravity; Nitric oxide; Migration; Actin


Arsenic trioxide sensitizes promonocytic leukemia cells to TNFα-induced apoptosis via p38-MAPK-regulated activation of both receptor-mediated and mitochondrial pathways by Donna Amrán; Yolanda Sánchez; Carlos Fernández; Adrián M. Ramos; Elena de Blas; Jacqueline Bréard; Consuelo Calle; Patricio Aller (pp. 1653-1663).
Treatment with the anti-leukemic drug arsenic trioxide (As2O3, 1–4 μM) sensitizes U937 promonocytes and other human myeloid leukemia cell lines (HL60, NB4) to apoptosis induction by TNFα. As2O3 plus TNFα increases TNF receptor type 1 (TNF-R1) expression, decreases c-FLIPL expression, and causes caspase-8 and Bid activation, and apoptosis is reduced by anti-TNF-R1 neutralizing antibody and caspase-8 inhibitor. The treatment also causes Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release from mitochondria, XIAP down-regulation, and caspase-9 and caspase-3 activation. Bcl-2 over-expression inhibits cytochrome c release and apoptosis, and also prevents c-FLIPL down-regulation and caspase-8 activation, but not TNF-R1 over-expression. As2O3 does not affect Akt phosphorylation/activation or intracellular GSH content, nor prevents the TNFα-provoked stimulation of p65-NF-κB translocation to the nucleus and the increase in NF-κB binding activity. Treatments with TNFα alone or with As2O3 plus TNFα cause TNF-R1-mediated p38-MAPK phosphorylation/activation. P38-MAPK-specific inhibitors attenuate the As2O3 plus TNFα-provoked activation of caspase-8/Bid, Bax translocation, cytochrome c release, and apoptosis induction. In conclusion, the sensitization by As2O3 to TNFα-induced apoptosis in promonocytic leukemia cells is an Akt/NF-κB-independent, p38-MAPK-regulated process, which involves the interplay of both the receptor-mediated and mitochondrial executioner pathways.

Keywords: Abbreviations; Ac-DEVD-; p; NA; N; -acetyl-Asp-Glu-Val-Asp-; p; -nitroanilide; Ac-LEHD-; p; NA; N; -acetyl-Leu-Glu-His-Asp-; p; -nitroanilide; APL; acute promyelocytic leukemia; DAPI; 4,6-diamino-2-phenylindole; FITC; fluorescein isothiocyanate; FLIP; Flice inhibitory protein; GSH; reduced glutathione; JNK; Jun N-terminal kinase; mAb; monoclonal antibody; MAPK; mitogen-activated protein kinase; pAb; polyclonal antibody; MEK/ERK; mitogen-induced extracellular kinase/extracellular signal-regulated kinase; PBS; phosphate-buffered saline; PI; propidium iodide; TNFα; tumour necrosis factor alpha; TNF-R; tumour necrosis factor receptor; TRAIL; tumour necrosis factor-related apoptosis-inducing ligand; Z-IETD-Fmk; Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH; 2; F; Z-VAD-Fmk; Z-Val-Ala-Asp(OMe)-CH; 2; FArsenic trioxide; TNFα; Apoptosis; Receptor-mediated pathway; Mitochondrial pathway; Myeloid leukemia cell


The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein β by Martijn Schenning; Claudia M. van Tiel; Karel W.A. Wirtz; Gerry T. Snoek (pp. 1664-1671).
Mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein β (PI-TPβ, SPIβ cells) demonstrate a low rate of proliferation and a high sensitivity towards UV-induced apoptosis when compared with wtNIH3T3 cells. In contrast, SPIβS262A cells overexpressing a mutant PI-TPβ that lacks the protein kinase C-dependent phosphorylation site Ser-262, demonstrate a phenotype comparable with wtNIH3T3 cells. This suggests that the phosphorylation of Ser-262 in PI-TPβ is involved in the regulation of apoptosis. Conditioned medium (CM) from wtNIH3T3 cells contains bioactive factors, presumably arachidonic acid metabolites [H. Bunte, et al., 2006; M. Schenning, et al., 2004] that are able to protect SPIβ cells against UV-induced apoptosis. CM from SPIβ cells lacks this protective activity. However, after heat denaturation CM from SPIβ cells regains a protective activity comparable with that of wtNIH3T3 cells. This indicates that CM from SPIβ cells contains an antagonistic factor interfering with the anti-apoptotic activity present. SPIβS262A cells do not produce the antagonist suggesting that phosphorylation of Ser-262 is required. Moreover, in line with the apparent lack of anti-apoptotic activity, CM from SPIβ cells does not induce the expression of COX-2 or the activation of p42/p44 MAP kinase in SPIβ cells. In contrast, CM from wtNIH3T3 and SPIβS262A cells or heat-treated CM from SPIβ cells does induce these anti-apoptotic markers. Since we have previously shown that some of the arachidonic acid metabolites present in CM from wtNIH3T3 cells are prostaglandin (PG) E2 and PGF2α, we investigated the effect of these PGs on cell survival. Although PGE2 and PGF2α were found to protect wtNIH3T3 and SPIβS262A cells against UV-induced apoptosis, these PGs failed to rescue SPIβ cells. The fact that the concentrations of PGE2 and PGF2α in the CM from SPIβ cells and wtNIH3T3 cells were found to be comparable suggests that the failure of these PGs to protect SPIβ cells could render these cells more apoptosis sensitive. Concomitantly, upon incubation with PGE2 and PGF2α, an increased expression of COX-2 and activation of p42/p44 MAP kinase were observed in wtNIH3T3 and SPIβS262A cells but not in SPIβ cells. Hence, it appears that specific mechanisms of cell survival are impaired in SPIβ cells.

Keywords: Abbreviations; PI-TP; phosphatidylinositol transfer protein; PI; phosphatidylinositol; PC; phosphatidylcholine; SM; sphingomyelin; PKC; protein kinase C; COX-1; cyclooxygenase-1; COX-2; cyclooxygenase-2; DMEM; Dulbecco's modified Eagle medium; CM; conditioned medium; NCS; newborn calf serum; PBS; phosphate buffered saline; DBB; DMEM containing 0.1% bovine serum albumin; PGE; 2; prostaglandin E; 2; PGF; 2α; prostaglandin F; 2α; MAPK; mitogen-activated protein kinase; MKK; MAP kinase kinase; GPCR; G protein-coupled receptorPhospholipid transfer protein; Sphingomyelin metabolism; Eicosanoids; Phosphatidylinositol metabolism


Ubiquitin-mediated degradation of Rpn4 is controlled by a phosphorylation-dependent ubiquitylation signal by Donghong Ju; Haiming Xu; Xiaogang Wang; Youming Xie (pp. 1672-1680).
A ubiquitylation signal of a protein substrate is defined as a short primary sequence or a structural feature recognized by a specific E3. Our previous work has mapped the ubiquitylation signal of Rpn4, the transcription activator for the Saccharomyces cerevisiae proteasome genes, to an N-terminal acidic domain (NAD) consisting of amino acids 211–229. However, the molecular mechanism by which Ubr2, the cognate E3, recognizes NAD remains unclear. Here we show that phosphorylation of either Ser-214 or Ser-220 enhances the binding of NAD to Ubr2. However, phosphorylation of Ser-220 but not Ser-214 plays a predominant role in Rpn4 ubiquitylation and degradation. Interestingly, NAD does not constitute the major Ubr2-binding site of Rpn4 even though it serves as the ubiquitylation signal essential for Rpn4 degradation. By contrast, the stable binding with Ubr2 conferred by other domains of Rpn4 is not required for Rpn4 degradation. Our results indicate that ubiquitin-mediated degradation of Rpn4 is controlled by a phosphorylation-dependent ubiquitylation signal. This study also suggests that binding to E3 may be only a part of the function of a ubiquitylation signal.

Keywords: Ubiquitin; Proteasome; Protein degradation; Protein phosphorylation; Rpn4; Ubr2


Reactive oxygen species (ROS) mediate the effects of leucine on translation regulation and type I collagen production in hepatic stellate cells by María P. Pérez de Obanos; María J. López-Zabalza; Elena Arriazu; Teresa Modol; Jesús Prieto; María T. Herraiz; María J. Iraburu (pp. 1681-1688).
The amino acid leucine causes an increase of collagen α1(I) synthesis in hepatic stellate cells through the activation of translational regulatory mechanisms and PI3K/Akt/mTOR and ERK signaling pathways. The aim of the present study was to evaluate the role played by reactive oxygen species on these effects. Intracellular reactive oxygen species levels were increased in hepatic stellate cells incubated with leucine 5 mM at early time points, and this effect was abolished by pretreatment with the antioxidant glutathione. Preincubation with glutathione also prevented 4E-BP1, eIF4E and Mnk-1 phosphorylation induced by leucine, as well as enhancement of procollagen α1(I) protein levels. Inhibitors for MEK-1 (PD98059), PI3K (wortmannin) or mTOR (rapamycin) did not affect leucine-induced reactive oxygen species production. However, preincubation with glutathione prevented ERK, Akt and mTOR phosphorylation caused by treatment with leucine. The mitochondrial electron chain inhibitor rotenone and the NADPH oxidase inhibitor apocynin prevented reactive oxygen species production caused by leucine. Leucine also induced an increased phosphorylation of IR/IGF-R that was abolished by pretreatment with either rotenone or apocynin. Therefore, leucine exerts on hepatic stellate cells a prooxidant action through NADPH oxidase and mitochondrial Reactive oxygen species production and these effects mediate the activation of IR/IGF-IR and signaling pathways, finally leading to changes in translational regulation of collagen synthesis.

Keywords: Abbreviations; HSC; hepatic stellate cells; ROS; reactive oxygen species; eIF4E; eukaryotic initiation factor 4E; mTOR; mammalian target of rapamycin; PI3K; phosphatidyl inositol 3 kinase; ERK; extracellular-regulated kinase; MAPK; mitogen-activated protein kinase; GSH; glutathione (reduced form); NAC; N-acetylcysteineLeucine; Hepatic stellate cells; Type I collagen; Translational regulation; Oxidative stress


PTPσ binds and dephosphorylates neurotrophin receptors and can suppress NGF-dependent neurite outgrowth from sensory neurons by Clare Faux; Muhamed Hawadle; Jennifer Nixon; Adam Wallace; Simon Lee; Simon Murray; Andrew Stoker (pp. 1689-1700).
Neurotrophin receptors of the Trk family play a vital role in the survival of developing neurons and the process of axonogenesis. The Trk family are receptor protein tyrosine kinases (RTKs) and their signalling in response to neurotrophins is critically dependent upon their ability to transphosphorylate and act as signalling centres for multiple adaptor proteins and distinct, downstream pathways. Such phosphotyrosine signalling also depends upon the appropriate counter-regulation by phosphatases. A large family of receptor-like protein tyrosine phosphatases (RPTPs) are also expressed in developing neurons and in this study we have examined the ability of the phosphatase PTPσ to interact with and regulate Trk proteins in transfected HEK 293T cells. PTPσ can bind differentially to Trk proteins, binding stably in complexes with TrkA and TrkC, but not TrkB. The transmembrane domains of PTPσ and TrkA appear to be sufficient for the direct or indirect interaction between these two receptors. Furthermore, PTPσ is shown to dephosphorylate all three Trk receptors and suppress their phosphorylation in the presence of neurotrophins. In addition, overexpression of PTPσ in primary sensory neurons in culture inhibits neurite outgrowth without affecting the short-term survival of these neurons. PTPσ can thus show differential complex formation with different Trk family members and in neurons can selectively target the neurite-forming signalling pathway driven by TrkA.

Keywords: Receptor protein tyrosine phosphatase σ; Trk receptor tyrosine kinase; Dephosphorylation; Neurite outgrowth; Neurotrophin
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