Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
BBA - Molecular Cell Research (v.1773, #9)
Adaptive modification and flexibility of the proteasome system in response to proteasome inhibition by Cord Naujokat; Dominik Fuchs; Carsten Berges (pp. 1389-1397).
The highly conserved ubiquitin–proteasome system is the principal machinery for extralysosomal protein degradation in eukaryotic cells. The 26S proteasome, a large multicatalytic multisubunit protease that processes cell proteins by limited and controlled proteolysis, constitutes the central proteolytic component of the ubiquitin–proteasome system. By processing cell proteins essential for development, differentiation, proliferation, cell cycling, apoptosis, gene transcription, signal transduction, senescence, and inflammatory and stress response, the 26S proteasome plays a key role in the regulation and maintenance of basic cellular processes. Various synthetic and biologic inhibitors with different inhibitory profiles towards the proteolytic activities of the 26S proteasome have been identified recently. Such proteasome inhibitors induce apoptosis and cell cycle arrest preferentially in neoplastic cells. Based on these findings proteasome inhibitors became useful in cancer therapy. However, under the pressure of continuous proteasome inhibition, eukaryotic cells can develop complex adaptive mechanisms to subvert the lethal attack of proteasome inhibitors. Such mechanisms include the adaptive modification of the proteasome system with increased expression, enhanced proteolytic activity and altered subcomplex assembly and subunit composition of proteasomes as well as the expression of a giant oligomeric protease complex, tripeptidyl peptidase II, which partially compensates for impaired proteasome function. Here we review the adaptive mechanisms developed by eukaryotic cells in response to proteasome inhibition. These mechanisms reveal enormous flexibility of the proteasome system and may have implications in cancer biology and therapy.
Keywords: Proteasome; Proteasome inhibition; Proteasome inhibitor; Tripeptidyl peptidase II; Adaptive modification; Apoptosis resistance
Identification and characterization of two putative nuclear localization signals (NLS) in the DNA-binding protein NUCKS by Kirsten Grundt; Ingvild V. Haga; Henrik S. Huitfeldt; Anne Carine Østvold (pp. 1398-1406).
Immunofluorescence analyses show that the vertebrate specific and DNA-binding protein NUCKS is distributed throughout the cytoplasm in mitotic cells and targeted to the reforming nuclei in late telophase of the cell cycle. Computer analysis of the primary structure of NUCKS revealed the presence of two regions of highly charged, basic residues, which were identified as potential nuclear localization signals (NLSs). One of these signals (NLS1) is highly conserved between the species investigated, and fits to the description of being a classical bipartite NLS. The other amino acid motif (NLS2) is less conserved and does not constitute a classical bipartite NLS consensus sequence. We have shown that each of the two putative NLSs is capable of translocating green fluorescent protein (GFP) into the nucleus. The highly conserved NLS1 is monopartite, resembling the signals of c-Myc and RanBP3. Surprisingly, a natural occurring splice variant of NUCKS lacking 40 amino acids including NLS1, is not capable of translocating a corresponding NUCKS–GFP fusion protein into the nucleus, indicating that NLS1 is the main nuclear localization signal in NUCKS. This is also confirmed by site-directed mutagenesis of the full-length protein. By GFP-immunoprecipitation and GST-pull down experiments, we show that NUCKS binds to importin α3 and importin α5 in vitro, suggesting that the nuclear targeting of NUCKS follows a receptor-mediated and energy-dependent import mechanism.
Keywords: NUCKS; Nuclear transport; NLS; Importin; Cell cycle
Tyrosine phosphorylation of β2-chimaerin by Src-family kinase negatively regulates its Rac-specific GAP activity by Masahiro Kai; Satoshi Yasuda; Shin-ichi Imai; Hideo Kanoh; Fumio Sakane (pp. 1407-1415).
β2-Chimaerin, an intracellular receptor for the second messenger diacylglycerol and phorbol esters, is a GTPase-activating protein (GAP) specific for Rac. β2-Chimaerin negatively controls many Rac-dependent pathophysiological events including tumor development. However, the regulatory mechanism of β2-chimaerin remains largely unknown. Here we report that β2-chimaerin is tyrosine-phosphorylated by Src-family kinases (SFKs) upon cell stimulation with epidermal growth factor (EGF). Mutational analysis identified Tyr-21 in the N-terminal regulatory region as a major phosphorylation site. Intriguingly, the addition of SFK inhibitor and the replacement of Tyr-21 with Phe (Y21F) markedly enhanced Rac-GAP activity of β2-chimaerin in EGF-treated cells. Moreover, the Y21F mutant inhibited integrin-dependent cell spreading, in which Rac1 plays a critical role, more strongly than wild-type β2-chimaerin. These results suggest Tyr-21 phosphorylation as a novel, SFK-dependent mechanism that negatively regulates β2-chimaerin Rac-GAP activity.
Keywords: GTPase-activating protein; Diacylglycerol; Src kinase; Tyrosine phosphorylation; Epidermal growth factor; Cell spreading
Characterization of the activity of human MAP kinase-interacting kinase Mnk1b by Ana O’Loghlen; Víctor M. González; Teresa Jurado; Matilde Salinas; M. Elena Martín (pp. 1416-1427).
Human mitogen-activated protein (MAP) kinase interacting kinase 1b (Mnk1b) is a splice variant of human Mnk1a, which has been identified in our laboratory [A. O’Loghlen, V.M. Gonzalez, D. Pineiro, M.I. Perez-Morgado, M. Salinas, M.E. Martin, Identification and molecular characterization of Mnk1b, a splice variant of human MAP kinase-interacting kinase Mnk1, Exp. Cell Res. 299 (2004) 343–355]. Mnk1b has much higher basal eIF4E kinase activity than Mnk1a. Because Mnk1b presents different features in its C-terminus with respect to Mnk1a, we have studied in this paper the potential role of these structural differences in determining the higher basal eIF4E kinase activity as well as the subcellular localization of Mnk1b. In this paper, we demonstrate that phosphorylation of the Thr209 and Thr214 in the activation loop of Mnk1b is necessary for its activation. However, the different kinase activity between Mnk1a and Mnk1b is independent of the phosphorylation status of the activation loop residues. By deletion of the C-terminal tail in Mnk1a, we confirmed that the absence of this sequence is not responsible for the higher eIF4E kinase activity present in Mnk1b. Moreover, our findings support a crucial role of the 12 amino acids, particularly the Ala344, in the C-terminal specific region of Mnk1b (Mnk1bSR), on the kinase activity of the protein.
Keywords: Eukaryotic initiation factor 4E; Kinase activity; MAP kinase-interacting kinase 1; Translation regulation
The peptidyl prolyl cis/trans isomerase Pin1 downregulates the Inhibitor of Apoptosis Protein Survivin by P. Dourlen; K. Ando; M. Hamdane; S. Begard; L. Buée; M.C. Galas (pp. 1428-1437).
The peptidyl prolyl cis–trans isomerase Pin1 and the Inhibitor of Apoptosis Protein (IAP) Survivin are two major proteins involved in cancer. They both modulate apoptosis, mitosis, centrosome duplication and neuronal development but until now no functional relationship has been reported between these two proteins. We tested Pin1-induced regulation of Survivin in neuroblastoma cells. Pin1 overexpression in SY5Y neuroblastoma cells decreased Survivin levels. Immunocytochemical studies indicated that they partially co-localized in interphase and mitotic cells. Co-immunoprecipitation further demonstrates the existence of a Pin1/Survivin complex. Pin1-induced effect on Survivin was confirmed in COS cells. RT-PCR and mutagenesis experiments suggested that this Pin1-induced decrease of Survivin occurred at the protein level. Survivin downregulation depended on the binding ability of Pin1 but was not related to the single Thr–Pro site, suggesting an indirect relationship into a protein complex. Finally, this functional regulation of Survivin by Pin1 is reciprocal since Pin1 silencing led to an increase in Survivin levels. The characterization of this functional relationship between Pin1 and Survivin might help to better understand mitosis control and cancer mechanisms.
Keywords: Cell cycle; Mitosis; Neuroblastoma; WW domain; PPIase; Cancer
Sonic hedgehog promotes proliferation and differentiation of adult muscle cells: Involvement of MAPK/ERK and PI3K/Akt pathways by Dafna Elia; Dorit Madhala; Eti Ardon; Ram Reshef; Orna Halevy (pp. 1438-1446).
Sonic hedgehog (Shh) has been reported to act as a mitogen and survival factor for muscle satellite cells. However, its role in their differentiation remains ambiguous. Here, we provide evidence that Shh promotes the proliferation and differentiation of primary cultures of chicken adult myoblasts (also termed satellite cells) and mouse myogenic C2 cells. These effects are reversed by cyclopamine, a specific chemical inhibitor of the Shh pathway. In addition, we show that Shh and its downstream molecules are expressed in adult myoblast cultures and localize adjacent to Pax7 in muscle sections. These gene expressions are regulated during postnatal muscle growth in chicks. Most importantly, we report that Shh induces MAPK/ERK and phosphoinositide 3-kinase (PI3K)-dependent Akt phosphorylation and that activation of both signaling pathways is essential for Shh's signaling in muscle cells. However, the effect of Shh on Akt phosphorylation is more robust than that on MAPK/ERK, and data suggest that Shh influences these pathways in a manner similar to IGF-I. By exploiting specific chemical inhibitors of the MAPK/ERK and PI3K/Akt signaling pathways, UO126 and Ly294002, respectively, we demonstrate that Shh-induced Akt phosphorylation, but not that of MAPK/ERK, is required for its promotive effects on muscle cell proliferation and differentiation. Taken together, we suggest that Shh acts in an autocrinic manner in adult myoblasts, and provide first evidence of a role for PI3K/Akt in Shh signaling during myoblast differentiation.
Keywords: Sonic hedgehog; Satellite cells; Skeletal muscle; Differentiation; MAPK; Akt; Signal transduction
Alien inhibits E2F1 gene expression and cell proliferation by Stephan P. Tenbaum; Maria Papaioannou; Christina A. Reeb; Frauke Goeman; Niko Escher; Robert Kob; Ferdinand von Eggeling; Christian Melle; Aria Baniahmad (pp. 1447-1454).
Recently, using a proteomic approach we have identified the corepressor Alien as a novel interacting factor of the cell cycle regulator E2F1. Unclear was whether this interaction influences cell proliferation and endogenous E2F1 target gene expression. Here, we show by chromatin immunoprecipitation (ChIP) that Alien is recruited in vivo to the E2F binding sites present in the E2F1 gene promoter, inhibits the transactivation of E2F1 and represses endogenous E2F1 gene expression. Interestingly, using synchronized cells to assess the expression of Alien profile during cell cycle the levels of endogenous Alien are increased during G1, G1/S and G2 phase. Furthermore, stable transfection of Alien leads to reduction of cell proliferation. Thus, the data suggest that Alien acts as a corepressor for E2F1 and is involved in cell cycle regulation.
Keywords: Abbreviations; ChIP; chromatin immunoprecipitation; CoIP; co-immunoprecipitation; IB; immunoblot; IP; immunoprecipitation; MS; Mass spectrometry; pRB; retinoblastoma protein; T3; thyroid hormone; TR; thyroid hormone receptor; tTA; Tet activator; VDR; vitamin D3 receptorCorepressor; Gene repression; Transcription; E2F1; Cell cycle
The transcription factors Stat5a/b are not required for islet development but modulate pancreatic β-cell physiology upon aging by Ji-Yeon Lee; Oksana Gavrilova; Behrous Davani; Risu Na; Gertraud W. Robinson; Lothar Hennighausen (pp. 1455-1461).
In insulinoma cell lines proliferation and insulin gene transcription are stimulated by growth hormone and prolactin, which convey their signals through the transcription factors Stat5a and 5b (referred to as Stat5). However, the contribution of Stat5 to the physiology of β-cells in vivo could not be assessed directly since Stat5-null mice die perinataly. To explore the physiological role of Stat5 in the mouse, the corresponding gene locus targeted with loxP sites was inactivated in β-cells using two lines of Cre recombinase expressing transgenic mice. While the RIP-Cre transgene is active in pancreatic β-cells and the hypothalamus, the Pdx1-Cre transgene is active in precursor cells of the endocrine and exocrine pancreas. Mice carrying two floxed Stat5alleles and a RIP-Cre transgene developed mild obesity, were hyperglycemic and exhibited impaired glucose tolerance. Since RIP-Cre transgenic mice by themselves display some glucose intolerance, the significance of these data is unclear. In contrast, mice, in which the Stat5 locus had been deleted with the Pdx1-Cre transgene, developed functional islets and were glucose tolerant. Mild glucose intolerance occurred with age. We conclude that Stat5 is not essential for islet development but may modulate β-cell function.
Keywords: Abbreviations; Stat; Signal transducer and activator of transcription; JAK; Janus kinase; GH; growth hormone; PRL; prolactin; PL; placental lactogen; GHR; GH receptor; PRLR; PRL receptor; FFA; free fatty acidStat5; Rip-Cre; Pdx1-Cre; Islets of Langerhans; β-cells; Glucose homeostasis
Up-regulation of VEGF expression by NGF that enhances reparative angiogenesis during thymic regeneration in adult rat by Hyun-Joo Park; Mi Na Kim; Jong-Gab Kim; Yun-Hee Bae; Moon-Kyoung Bae; Hee-Jun Wee; Tae-Woo Kim; Bong-Seon Kim; Jae-Bong Kim; Soo-Kyung Bae; Sik Yoon (pp. 1462-1472).
Angiogenesis is important for adult tissue regeneration as well as normal development. Vascular endothelial growth factor (VEGF) is a unique potent angiogenic factor, and plays an essential role in regulating angiogenesis during embryonic development, normal tissue growth, and tissue regeneration. Recent evidence shows that nerve growth factor (NGF) also plays a role as an angiogenic regulator as well as a well-known neurotrophic factor. The aim of this study was to investigate whether thymus regeneration accompanies reparative angiogenesis and also to evaluate whether the thymic expression of VEGF is regulated by NGF in vivo and in vitro. Here, we show that high VEGF mRNA and protein levels are concomitant with reparative angiogenesis that occurs dramatically during regeneration following acute involution induced by cyclophosphamide (CY) in the rat thymus. Fluorescent thymus angiography using FITC-dextran showed that thymic regeneration is associated with a much denser capillary network compared with normal control thymus. Furthermore, the expressions of NGF and TrkA were highly increased during thymic regeneration. We also show that NGF mediates thymic epithelial induction of VEGF expression in vitro and in vivo. Taken together, our results suggest that NGF-mediated VEGF up-regulation in thymic epithelial cells may contribute to reparative angiogenesis during thymic regeneration in adult.
Keywords: Angiogenesis; Thymic Regeneration; Thymic Epithelial Cell; VEGF; NGF
Oxidative stress-induced expression and modulation of Phosphatase of Regenerating Liver-1 (PRL-1) in mammalian retina by Ling Yu; Una Kelly; Jessica N. Ebright; Goldis Malek; Peter Saloupis; Dennis W. Rickman; Brian S. McKay; Vadim Y. Arshavsky; Catherine Bowes Rickman (pp. 1473-1482).
The phosphatase of regenerating liver-1, PRL-1, gene was detected in a screen for foveal cone photoreceptor-associated genes. It encodes a small protein tyrosine phosphatase that was previously immunolocalized to the photoreceptors in primate retina. Here we report that in cones and cone-derived cultured cells both PRL-1 activity and PRL-1 gene expression are modulated under oxidative stress. Oxidation reversibly inhibited the phosphatase activity of PRL-1 due to the formation of an intramolecular disulfide bridge between Cys104 within the active site and another conserved Cys, Cys49. This modulation was observed in vitro, in cell culture and in isolated retinas exposed to hydrogen peroxide. The same treatment caused a rapid increase in PRL-1 expression levels in cultured cells which could be blocked by the protein translation inhibitor, cycloheximide. Increased PRL-1 expression was also observed in living rats subjected to constant light exposure inducing photooxidative stress. We further demonstrated that both oxidation and overexpression of PRL-1 upon oxidative stress are greatly enhanced by inhibition of the glutathione system responsible for cellular redox regulation. These findings suggest that PRL-1 is a molecular component of the photoreceptor's response to oxidative stress acting upstream of the glutathione system.
Keywords: Abbreviations; PRL-1; phosphatase of regenerating liver; PTP; protein tyrosine phosphatase; H; 2; O; 2; hydrogen peroxide; Cys; cysteine; Ser; serine; Thr; threonine; DiFMUP; 6, 8-difluoro-4-methylumbelliferyl phosphate; Na; 3; VO; 4; sodium orthovanadate; NaF; sodium fluoride; BSA; bovine serum albumin; MALDI-TOF-MS; matrix assisted laser desorption ionization-time of flight-mass spectrometry; AMS; 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; NEM; N; -ethylmaleimide; BSO; buthionine sulfoximine; OS; outer segment; PIP; 3; phosphatidylinositol-3,4,5-triphosphate; GSH; glutathione; Trx; thioredoxinPhosphatase; Oxidation/reduction; Cysteine; Retina; Photoreceptor; Light damage
Ionomycin-induced apoptosis of thymocytes is independent of Nur77 NBRE or NurRE binding, but is accompanied by Nur77 mitochondrial targeting by Izabela Stasik; Andrzej Rapak; Wojciech Kalas; Ewa Ziolo; Leon Strzadala (pp. 1483-1490).
The induction of thymocyte apoptosis through the Nur77-mediated intrinsic pathway can be of physiological importance in the clonal deletion of autoreactive thymocytes during negative selection in the thymus and/or in thymocytes undergoing oncogenic transformation. Ionomycin treatment induces endogenous Nur77 expression as well as apoptosis and cytochrome c release in thymocytes. Here it is shown for the first time that in normal thymocytes undergoing apoptosis, ionomycin induces translocation of endogenous Nur77 not only to the nucleus, but also to mitochondria. Immunosuppressant FK506 inhibits Nur77 NBRE and NurRE binding activity but has no effect on thymocytes apoptosis, the subcellular localization of Nur77, or cytochrome c release. This indicates that thymocytes can undergo apoptosis through the intrinsic Nur77-mediated mitochondrial pathway and that the transactivation activity of Nur77 monomers or dimers is not necessary for thymocyte apoptosis.
Keywords: Thymocytes; Apoptosis; Nur77; Mitochondrial translocation; DNA-binding