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  • 1.
    Antoun, Ayman
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andersson, Kerstin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Tenson, Tanel
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis2003In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, no 20, p. 5593-5601Article in journal (Refereed)
    Abstract [en]

    The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2–GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2.

  • 2.
    Antoun, Ayman
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael Y
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Lovmar, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    How initiation factors tune the rate of initiation of protein synthesis in bacteria.2006In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 25, no 11, p. 2539-50Article in journal (Refereed)
    Abstract [en]

    The kinetics of initiator transfer RNA ( tRNA) interaction with the messenger RNA ( mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency ( k(cat)/K-M) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.

  • 3. Arand, Michael
    et al.
    Hallberg, B Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Zou, Jinyu
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Oesch, Franz
    van der Werf, Mariette
    de Bont, Jan A M
    Jones, T Alwyn
    Mowbray, Sherry L
    Department of Molecular Biosciences, Swedish University of Agricultural Science.
    Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site.2003In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, no 11, p. 2583-2592Article in journal (Refereed)
  • 4.
    Blume-Jensen, Peter
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Siegbahn, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Zsebo, Krisztina M.
    AMGEN Inc., AMGEN Center, Thousand Oaks, CA 91320, USA.
    Westermark, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Pathology.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Activation of the human c-kit product by ligand-induced dimerization mediates circular actin reorganization and chemotaxis1991In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 10, no 13, p. 4121-4128Article in journal (Refereed)
    Abstract [en]

    The proto-oncogene c-kit is allelic with the murine white spotting (W) locus and encodes a transmembrane protein tyrosine kinase that is structurally related to the receptors for platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1). Recently the ligand for the c-kit product, stem cell factor (SCF), was identified in both transmembrane and soluble forms. In order to examine the mechanism for receptor activation by SCF and biological properties of the activated c-kit product, we transfected the wild-type human c-kit cDNA into porcine aortic endothelial cells. We found that the receptor was down-regulated and transmitted a mitogenic signal in response to stimulation with soluble SCF. We also demonstrate that SCF induces dimerization of the c-kit product in intact cells, and that dimerization of the receptor is correlated with activation of its kinase. Activation of the c-kit product by SCF was found to induce circular actin reorganization indistinguishable from that mediated by the PDGF beta-receptor in response to PDGF-BB. Furthermore, soluble SCF was a potent chemotactic agent for cells expressing the c-kit product, a property which might be of importance during embryonic development.

  • 5. Cabeen, Matthew T.
    et al.
    Charbon, Godefroid
    Vollmer, Waldemar
    Born, Petra
    Ausmees, Nora
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Weibel, B
    Jacobs-Wagner, Christine
    Bacterial cell curvature through mechanical control of cell growth2009In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 28, no 9, p. 1208-1219Article in journal (Refereed)
    Abstract [en]

    The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament-like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology. The EMBO Journal (2009) 28, 1208-1219. doi: 10.1038/emboj.2009.61; Published online 12 March 2009

  • 6. Cevher, Murat A.
    et al.
    Zhang, Xiaokan
    Fernandez, Sully
    Kim, Sergey
    Baquero, Jorge
    Nilsson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Lee, Sean
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kleiman, Frida E.
    Nuclear deadenylation/polyadenylation factors regulate 3 ' processing in response to DNA damage2010In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 29, no 10, p. 1674-1687Article in journal (Refereed)
    Abstract [en]

    We previously showed that mRNA 3' end cleavage reaction in cell extracts is strongly but transiently inhibited under DNA-damaging conditions. The cleavage stimulation factor-50 (CstF-50) has a role in this response, providing a link between transcription-coupled RNA processing and DNA repair. In this study, we show that CstF-50 interacts with nuclear poly(A)-specific ribonuclease (PARN) using in vitro and in extracts of UV-exposed cells. The CstF-50/PARN complex formation has a role in the inhibition of 3' cleavage and activation of deadenylation upon DNA damage. Extending these results, we found that the tumour suppressor BARD1, which is involved in the UV-induced inhibition of 3' cleavage, strongly activates deadenylation by PARN in the presence of CstF-50, and that CstF-50/BARD1 can revert the cap-binding protein-80 (CBP80)mediated inhibition of PARN activity. We also provide evidence that PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA-damaging conditions. We speculate that the interplay between polyadenylation, deadenylation and tumour-suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.

  • 7. Djupedal, Ingela
    et al.
    Kos-Braun, C
    Mosher, A
    Söderholm, Niklas
    Simmer, Femke
    Hardcastle, J
    Fender, Aurelie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Heidrich, Nadja
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kagansky, Alexander
    Bayne, Elizabeth
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Baulcombe, C
    Allshire, C
    Ekwall, Karl
    Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA2009In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 28, no 24, p. 3832-3844Article in journal (Refereed)
    Abstract [en]

    The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 50-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1D cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity. The EMBO Journal (2009) 28, 3832-3844. doi: 10.1038/emboj.2009.351; Published online 26 November 2009

  • 8.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Schneider, G
    Schnackerz, K D
    Lindqvist, Y
    Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 4, p. 650-660Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidine dehydrogenase catalyzes the first step in pyrimidine degradation: the NADPH-dependent reduction of uracil and thymine to the corresponding 5,6-dihydropyrimidines. Its controlled inhibition has become an adjunct target for cancer therapy, since the enzyme is also responsible for the rapid breakdown of the chemotherapeutic drug 5-fluorouracil. The crystal structure of the homodimeric pig liver enzyme (2x 111 kDa) determined at 1.9 A resolution reveals a highly modular subunit organization, consisting of five domains with different folds. Dihydropyrimidine dehydrogenase contains two FAD, two FMN and eight [4Fe-4S] clusters, arranged in two electron transfer chains that pass the dimer interface twice. Two of the Fe-S clusters show a hitherto unobserved coordination involving a glutamine residue. The ternary complex of an inactive mutant of the enzyme with bound NADPH and 5-fluorouracil reveals the architecture of the substrate-binding sites and residues responsible for recognition and binding of the drug.

  • 9.
    Estmer Nilsson, Camilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Petersen-Mahrt, Svend
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Durot, C
    Shtrichman, R
    Krainer, AR
    Kleinberger, T
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The adenovirus E4-ORF4 splicing enhancer protein interacts with a subsetof phosphorylated SR proteins2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, p. 864-871Article in journal (Refereed)
    Abstract [en]

    SR proteins purified from uninfected HeLa cells inhibit adenovirus IIIa pre-mRNA splicing by binding to the intronic IIIa repressor element (3RE). In contrast, SR proteins purified from late adenovirus-infected cells are functionally inactivated as splicing repressor proteins by a virus-induced dephosphorylation. We have shown that the adenovirus E4-ORF4 protein, which binds the cellular protein phos phatase 2A (PP2A) and activates IIIa splicing in vitro and in vivo, induces SR protein dephosphorylation. Here we show that E4-ORF4 interacts with only a subset of SR proteins present in HeLa cells. Thus, E4-ORF4 interacts efficiently with SF2/ASF and SRp30c, but not with other SR proteins. Interestingly, E4-ORF4 interacts with SF2/ASF through the latter's RNA recognition motifs. Furthermore, E4-ORF4 interacts preferentially with the hyperphosphorylated form of SR proteins found in uninfected HeLa cells. E4-ORF4 mutant proteins that fail to bind strongly to PP2A or SF2/ASF do not relieve the repressive effect of HeLa SR proteins on IIIa pre-mRNA splicing in transient transfection experiments, suggesting that an interaction between all three proteins is required for E4-ORF4-induced SR protein dephosphorylation

  • 10. Forrest, D
    et al.
    Hallböök, F
    Persson, H
    Vennström, B
    Distinct functions for thyroid hormone receptors alpha and beta in brain development indicated by differential expression of receptor genes.1991In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 10, no 2, p. 269-75Article in journal (Refereed)
    Abstract [en]

    Thyroid hormones are essential for correct brain development, and since vertebrates express two thyroid hormone receptor genes (TR alpha and beta), we investigated TR gene expression during chick brain ontogenesis. In situ hybridization analyses showed that TR alpha mRNA was widely expressed from early embryonic stages, whereas TR beta was sharply induced after embryonic day 19 (E19), coinciding with the known hormone-sensitive period. Differential expression of TR mRNAs was striking in the cerebellum: TR beta mRNA was induced in white matter and granule cells after the migratory phase, suggesting a main TR beta function in late, hormone-dependent glial and neuronal maturation. In contrast, TR alpha mRNA was expressed in the earlier proliferating and migrating granule cells, and in the more mature granular and Purkinje cell layers after hatching, indicating a role for TR alpha in both immature and mature neural cells. Surprisingly, both TR genes were expressed in early cerebellar outgrowth at E9, before known hormone requirements, with TR beta mRNA restricted to the ventricular epithelium of the metencephalon and TR alpha expressed in migrating cells and the early granular layer. The results implicate TRs with distinct functions in the early embryonic brain as well as in the late phase of hormone requirement.

  • 11. Frick, Inga-Maria
    et al.
    Åkesson, Per
    Herwald, Heiko
    Mörgelin, Matthias
    Malmsten, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Nägler, Dorit K.
    Björck, Lars
    The contact system - a novel branch of innate immunity generating antibacterial peptides2006In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 25, no 23, p. 5569-5578Article in journal (Refereed)
    Abstract [en]

    Activation of the contact system has two classical consequences: initiation of the intrinsic pathway of coagulation, and cleavage of high molecular weight kininogen (HK) leading to the release of bradykinin, a potent proinflammatory peptide. In human plasma, activation of the contact system at the surface of significant bacterial pathogens was found to result in further HK processing and bacterial killing. A fragment comprising the D3 domain of HK is generated, and within this fragment a sequence of 26 amino acids is mainly responsible for the antibacterial activity. A synthetic peptide covering this sequence kills several bacterial species, also at physiological salt concentration, as effectively as the classical human antibacterial peptide LL-37. Moreover, in an animal model of infection, inhibition of the contact system promotes bacterial dissemination and growth. These data identify a novel and important role for the contact system in the defence against invasive bacterial infection.

  • 12.
    Ghosh, Anirban
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Wäneskog, Marcus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elkhalifa, Dina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Larsson, Disa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations2018In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 9, article id UNSP e98026Article in journal (Refereed)
    Abstract [en]

    Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing cells is increased only when there are many cells of the same genotype. This may be one of the mechanisms of how CDI systems increase the fitness of their hosts.

  • 13. Groth, Anja
    et al.
    Lukas, Jiri
    Nigg, Erich A.
    Silljé, Herman H. W.
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Bartek, Jiri
    Hansen, Klaus
    Human Tousled like kinases are targeted by an ATM- and Chk1-dependent DNA damage checkpoint2003In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, no 7, p. 1676-1687Article in journal (Refereed)
    Abstract [en]

    All eukaryotes respond to DNA damage by modulation of diverse cellular processes to preserve genomic integrity and ensure survival. Here we identify mammalian Tousled like kinases (Tlks) as a novel target of the DNA damage checkpoint. During S-phase progression, when Tlks are maximally active, generation of DNA double-strand breaks (DSBs) leads to rapid and transient inhibition of Tlk activity. Experiments with chemical inhibitors, genetic models and gene targeting through RNA interference demonstrate that this response to DSBs requires ATM and Chk1 function. Chk1 phosphorylates Tlk1 on serine 695 (S695) in vitro, and this UCN-01- and caffeine-sensitive site is phosphorylated in vivo in response to DNA damage. Substitution of S695 to alanine impaired efficient downregulation of Tlk1 after DNA damage. These findings identify an unprecedented functional co- operation between ATM and Chk1 in propagation of a checkpoint response during S phase and suggest that, through transient inhibition of Tlk kinases, the ATM-Chk1-Tlk pathway may regulate processes involved in chromatin assembly.

  • 14.
    Haegerling, Rene
    et al.
    Max Planck Inst Mol Biomed, Mammalian Cell Signaling Lab, Munster, Germany.
    Hoppe, Esther
    Max Planck Inst Mol Biomed, Mammalian Cell Signaling Lab, Munster, Germany;Univ Munster, EIMI, Munster, Germany.
    Dierkes, Cathrin
    Max Planck Inst Mol Biomed, Mammalian Cell Signaling Lab, Munster, Germany.
    Stehling, Martin
    Max Planck Inst Mol Biomed, Flow Cytometry Unit, Munster, Germany.
    Mäkinen, Taija
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Butz, Stefan
    Max Planck Inst Mol Biomed, Dept Vasc Cell Biol, Munster, Germany.
    Vestweber, Dietmar
    Max Planck Inst Mol Biomed, Dept Vasc Cell Biol, Munster, Germany;DFG Cluster Excellence 1003 CiM Cells Mot, Munster, Germany.
    Kiefer, Friedemann
    Max Planck Inst Mol Biomed, Mammalian Cell Signaling Lab, Munster, Germany;Univ Munster, EIMI, Munster, Germany;DFG Cluster Excellence 1003 CiM Cells Mot, Munster, Germany.
    Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds2018In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 22, article id e98271Article in journal (Refereed)
    Abstract [en]

    Endothelial cells line blood and lymphatic vessels and form intercellular junctions, which preserve vessel structure and integrity. The vascular endothelial cadherin, VE-cadherin, mediates endothelial adhesion and is indispensible for blood vessel development and permeability regulation. However, its requirement for lymphatic vessels has not been addressed. During development, VE-cadherin deletion in lymphatic endothelial cells resulted in abortive lymphangiogenesis, edema, and prenatal death. Unexpectedly, inducible postnatal or adult deletion elicited vessel bed-specific responses. Mature dermal lymph vessels resisted VE-cadherin loss and maintained button junctions, which was associated with an upregulation of junctional molecules. Very different, mesenteric lymphatic collectors deteriorated and formed a strongly hyperplastic layer of lymphatic endothelial cells on the mesothelium. This massive hyperproliferation may have been favored by high mesenteric VEGF-C expression and was associated with VEGFR-3 phosphorylation and upregulation of the transcriptional activator TAZ. Finally, intestinal lacteals fragmented into cysts or became highly distended possibly as a consequence of the mesenteric defects. Taken together, we demonstrate here the importance of VE-cadherin for lymphatic vessel development and maintenance, which is however remarkably vessel bed-specific.

  • 15.
    Hansen, Klaus
    et al.
    Ludwiginstitutet för Cancerforskning.
    Johnell, Matilda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Siegbahn, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Rorsman, Charlotte
    Ludwiginstitutet för Cancerforskning.
    Engström, Ulla
    Ludwiginstitutet för Cancerforskning.
    Wernstedt, Christer
    Ludwiginstitutet för Cancerforskning.
    Heldin, Carl-Henrik
    Ludwiginstitutet för Cancerforskning.
    Rönnstrand, Lars
    Ludwiginstitutet för Cancerforskning.
    Mutation of a Src phosphorylation site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis1996In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 15, no 19, p. 5299-5313Article in journal (Refereed)
    Abstract [en]

    Ligand induced activation of the beta-receptor for platelet-derived growth factor (PDGF) leads to activation of Src family tyrosine kinases. We have explored the possibility that the receptor itself is a substrate for Src. We show that Tyr934 in the kinase domain of the PDGF receptor is phosphorylated by Src. Cell lines expressing a beta-receptor mutant, in which Tyr934 was replaced with a phenyalanine residue, showed reduced mitogenic signaling in response to PDGF-BB. In contrast, the mutant receptor mediated increased signals for chemotaxis and actin reorganization. Whereas the motility responses of cells expressing wild-type beta-receptors were attenuated by inhibition of phosphatidylinositol 3'-kinase, those of cells expressing the mutant receptor were only slightly influenced. In contrast, PDGF-BB-induced chemotaxis of the cells with the mutant receptor was attenuated by inhibition of protein kinase C, whereas the chemotaxis of cells expressing the wild-type beta-receptor was less affected. Moreover, the PDGF-BB-stimulated tyrosine phosphorylation of phospholipase C-gamma was increased in the mutant receptor cells compared with wild-type receptor cells. In conclusion, the characteristics of the Y934F mutant suggest that the phosphorylation of Tyr934 by Src negatively modulates a signal transduction pathway leading to motility responses which involves phospholipase C-gamma, and shifts the response to increased mitogenicity.

  • 16. Hinke, Simon A.
    et al.
    Navedo, Manuel F.
    Ulman, Allison
    Whiting, Jennifer L.
    Nygren, Patrick J.
    Tian, Geng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Jimenez-Caliani, Antonio J.
    Langeberg, Lorene K.
    Cirulli, Vincenzo
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Dell'Acqua, Mark L.
    Santana, L. Fernando
    Scott, John D.
    Anchored phosphatases modulate glucose homeostasis2012In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 20, p. 3991-4004Article in journal (Refereed)
    Abstract [en]

    Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic beta-cells involves ion channels and mobilization of Ca2+ and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-beta-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca2+ currents, and attenuates cytoplasmic accumulation of Ca2+ and cAMP in beta-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.

  • 17.
    Hoekzema, Mirthe
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Romilly, Cedric
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Holmqvist, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation2019In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, article id e101199Article in journal (Refereed)
    Abstract [en]

    Small RNAs post-transcriptionally regulate many processes inbacteria. Base-pairing of sRNAs near ribosome-binding sites inmRNAs inhibits translation, often requiring the RNA chaperoneHfq. In the canonical model, Hfq simultaneously binds sRNAs andmRNA targets to accelerate pairing. Here, we show that theEscher-ichia colisRNAs OmrA and OmrB inhibit translation of the diguany-late cyclase DgcM (previously: YdaM), a player in biofilmregulation. In OmrA/B repression ofdgcM, Hfq is not required as anRNA interaction platform, but rather unfolds an inhibitory RNAstructure that impedes OmrA/B binding. This restructuring involvesdistal face binding of Hfq and is supported by RNA structuremapping. A corresponding mutant protein cannot support inhibi-tionin vitroandin vivo; proximal and rim mutations have negligi-ble effects. Strikingly, OmrA/B-dependent translational inhibitionin vitrois restored, in complete absence of Hfq, by a deoxyoligori-bonucleotide that base-pairs to the biochemically mapped Hfq siteindgcMmRNA. We suggest that Hfq-dependent RNA structureremodeling can promote sRNA access, which represents a mecha-nism distinct from an interaction platform model.

  • 18.
    Holmqvist, Erik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Reimegård, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sterk, Maaike
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Grantcharova, Nina
    Römling, Ute
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Wagner, Gerhart Eduard Heinrich
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis2010In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 29, no 11, p. 1840-1850Article in journal (Refereed)
    Abstract [en]

    Escherichia coli produces proteinaceous surface structures called curli that are involved in adhesion and biofilm formation. CsgD is the transcriptional activator of curli genes. We show here that csgD expression is, in part, controlled post-transcriptionally by two redundant small RNAs (sRNAs), OmrA and OmrB. Their overexpression results in curli deficiency, in accordance with the inhibition of chromosomally encoded, FLAG-tagged CsgD. Downregulation of csgD occurs by a direct antisense interaction within the csgD 5'-UTR, far upstream of the ribosome-binding site (RBS). OmrA/B downregulate plasmid-borne csgD-gfp fusions in vivo, and inhibit CsgD translation in vitro. The RNA chaperone Hfq is required for normal csgD mRNA and OmrA/B levels in the cell, and enhances sRNA-dependent inhibition of csgD translation in vitro. Translational inhibition involves two phylogenetically conserved secondary structure modules that are supported by chemical and enzymatic probing. The 5'-most element is necessary and sufficient for regulation, the one downstream comprises the RBS and affects translational efficiency. OmrA/B are two antisense RNAs that regulate a transcription factor to alter a morphotype and group behaviour.

  • 19.
    Huels, David J.
    et al.
    Canc Res UK Beatson Inst, Glasgow, Lanark, Scotland..
    Ridgway, Rachel A.
    Canc Res UK Beatson Inst, Glasgow, Lanark, Scotland..
    Radulescu, Sorina
    Canc Res UK Beatson Inst, Glasgow, Lanark, Scotland..
    Leushacke, Marc
    ASTAR, Inst Med Biol, Singapore, Singapore..
    Campbell, Andrew D.
    Canc Res UK Beatson Inst, Glasgow, Lanark, Scotland..
    Biswas, Sujata
    Univ Oxford, Wellcome Trust Ctr Human Genet, Gastrointestinal Stem Cell Biol Lab, Oxford, England.;John Radcliffe Hosp, Nuffield Dept Clin Med, Div Expt Med, Translat Gastroenterol Unit, Oxford OX3 9DU, Headington, England..
    Leedham, Simon
    Univ Oxford, Wellcome Trust Ctr Human Genet, Gastrointestinal Stem Cell Biol Lab, Oxford, England.;John Radcliffe Hosp, Nuffield Dept Clin Med, Div Expt Med, Translat Gastroenterol Unit, Oxford OX3 9DU, Headington, England..
    Serra, Stefano
    Univ Hlth Network, Toronto Med Labs, Dept Pathol, Toronto, ON, Canada..
    Chetty, Runjan
    Univ Hlth Network, Toronto Med Labs, Dept Pathol, Toronto, ON, Canada..
    Moreaux, Guenievre
    Canc Res UK Beatson Inst, Glasgow, Lanark, Scotland..
    Parry, Lee
    Cardiff Univ, European Canc Stem Cell Res Inst, Cardiff CF10 3AX, S Glam, Wales..
    Matthews, James
    Cardiff Univ, European Canc Stem Cell Res Inst, Cardiff CF10 3AX, S Glam, Wales..
    Song, Fei
    Univ Giessen, Inst Physiol, D-35390 Giessen, Germany..
    Hedley, Ann
    Canc Res UK Beatson Inst, Glasgow, Lanark, Scotland..
    Kalna, Gabriela
    Canc Res UK Beatson Inst, Glasgow, Lanark, Scotland..
    Ceteci, Fatih
    Canc Res UK Beatson Inst, Glasgow, Lanark, Scotland..
    Reed, Karen R.
    Cardiff Univ, European Canc Stem Cell Res Inst, Cardiff CF10 3AX, S Glam, Wales..
    Meniel, Valerie S.
    Cardiff Univ, European Canc Stem Cell Res Inst, Cardiff CF10 3AX, S Glam, Wales..
    Maguire, Aoife
    St James Hosp, Trinity Coll Dublin, Dept Histopathol, Dublin 8, Ireland..
    Doyle, Brendan
    Canc Res UK Beatson Inst, Glasgow, Lanark, Scotland.;St James Hosp, Trinity Coll Dublin, Dept Histopathol, Dublin 8, Ireland..
    Soderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Barker, Nick
    ASTAR, Inst Med Biol, Singapore, Singapore..
    Watson, Alastair
    Univ E Anglia, Norwich Med Sch, Norwich NR4 7TJ, Norfolk, England..
    Larue, Lionel
    Inst Curie, CNRS UMR3347, INSERM, U1021,Equipe Labellisee Ligue Natl Canc, F-91405 Orsay, France..
    Clarke, Alan R.
    Cardiff Univ, European Canc Stem Cell Res Inst, Cardiff CF10 3AX, S Glam, Wales..
    Sansom, Owen J.
    Canc Res UK Beatson Inst, Glasgow, Lanark, Scotland..
    E-cadherin can limit the transforming properties of activating beta-catenin mutations2015In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 34, no 18, p. 2321-2333Article in journal (Refereed)
    Abstract [en]

    Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in beta-catenin (CTNNB1). We have compared the dynamics and the potency of beta-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of beta-catenin took much longer to achieve Wnt deregulation and acquire a crypt-progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of beta-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of beta-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin: beta-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of beta-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of beta-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated beta-catenin.

  • 20. Ibáñez, C F
    et al.
    Hallböök, F
    Ebendal, T
    Persson, H
    Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.1990In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 9, no 5, p. 1477-83Article in journal (Refereed)
    Abstract [en]

    Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site-directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15-60% of receptor binding and 40-80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability.

  • 21.
    Idevall Hagren, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Yale Univ, Sch Med, Dept Cell Biol, New Haven, CT 06510 USA.;Yale Univ, Sch Med, Howard Hughes Med Inst, New Haven, CT 06510 USA.;Yale Univ, Sch Med, Program Cellular Neurosci Neurodegenerat & Repair, New Haven, CT 06510 USA..
    Lue, Alice
    Yale Univ, Sch Med, Dept Cell Biol, New Haven, CT 06510 USA.;Yale Univ, Sch Med, Howard Hughes Med Inst, New Haven, CT 06510 USA.;Yale Univ, Sch Med, Program Cellular Neurosci Neurodegenerat & Repair, New Haven, CT 06510 USA..
    Xie, Beichen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    De Camilli, Pietro
    Yale Univ, Sch Med, Dept Cell Biol, New Haven, CT 06510 USA.;Yale Univ, Sch Med, Howard Hughes Med Inst, New Haven, CT 06510 USA.;Yale Univ, Sch Med, Program Cellular Neurosci Neurodegenerat & Repair, New Haven, CT 06510 USA..
    Triggered Ca2+ influx is required for extended synaptotagmin 1-induced ER-plasma membrane tethering2015In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 34, no 17, p. 2291-2305Article in journal (Refereed)
    Abstract [en]

    The extended synaptotagmins (E-Syts) are ER proteins that act as Ca2+-regulated tethers between the ER and the plasma membrane (PM) and have a putative role in lipid transport between the two membranes. Ca2+ regulation of their tethering function, as well as the interplay of their different domains in such function, remains poorly understood. By exposing semi-intact cells to buffers of variable Ca2+ concentrations, we found that binding of E-Syt1 to the PI(4,5)P-2-rich PM critically requires its C2C and C2E domains and that the EC50 of such binding is in the low micromolar Ca2+ range. Accordingly, E-Syt1 accumulation at ER-PM contact sites occurred only upon experimental manipulations known to achieve these levels of Ca2+ via its influx from the extracellular medium, such as store-operated Ca2+ entry in fibroblasts and membrane depolarization in -cells. We also show that in spite of their very different physiological functions, membrane tethering by E-Syt1 (ER to PM) and by synaptotagmin (secretory vesicles to PM) undergo a similar regulation by plasma membrane lipids and cytosolic Ca2+.

  • 22. Ikonen, E
    et al.
    Baumann, M
    Grön, K
    Syvänen, Ann-Christine
    Enomaa, N
    Halila, R
    Aula, P
    Peltonen, L
    Aspartylglucosaminuria: cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease1991In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 10, no 1, p. 51-58Article in journal (Refereed)
    Abstract [en]

    We have isolated a 2.1 kb cDNA which encodes human aspartylglucosaminidase (AGA, E.C. 3.5.1.26). The activity of this lysosomal enzyme is deficient in aspartylglucosaminuria (AGU), a recessively inherited lysosomal accumulation disease resulting in severe mental retardation. The polypeptide chain deduced from the AGA cDNA consists of 346 amino acids, has two potential N-glycosylation sites and 11 cysteine residues. Transient expression of this cDNA in COS-1 cells resulted in increased expression of immunoprecipitable AGA protein. Direct sequencing of amplified AGA cDNA from an AGU patient revealed a G----C transition resulting in the substitution of cysteine 163 with serine. This mutation was subsequently found in all the 20 analyzed Finnish AGU patients, in the heterozygous form in all 53 carriers and in none of 67 control individuals, suggesting that it represents the major AGU causing mutation enriched in this isolated population. Since the mutation produces a change in the predicted flexibility of the AGA polypeptide chain and removes an intramolecular S-S bridge, it most probably explains the deficient enzyme activity found in cells and tissues of AGU patients.

  • 23.
    Ikushima, Hiroaki
    et al.
    University of Tokyo.
    Komuro, Akiyoshi
    University of Tokyo.
    Isogaya, Kazunobu
    University of Tokyo.
    Shinozaki, Masahiko
    University of Tokyo.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Miyazawa, Keiji
    University of Tokyo.
    Miyazono, Kohei
    University of Tokyo.
    An Id-like molecule, HHM, is a synexpression group-restricted regulator of TGF-β signalling2008In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 27, no 22, p. 2955-2965Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor (TGF)-β induces various cellular responses principally through Smad-dependent transcriptional regulation. Activated Smad complexes cooperate with transcription factors in regulating a group of target genes. The target genes controlled by the same Smad-cofactor complexes are denoted a synexpression group. We found that an Id-like helix-loop-helix protein, human homologue of Maid (HHM), is a synexpression group-restricted regulator of TGF-β signalling. HHM suppressed TGF-β-induced growth inhibition and cell migration but not epithelial–mesenchymal transition. In addition, HHM inhibited TGF-β-induced expression of plasminogen activator inhibitor-type 1 (PAI-1), PDGF-B, and p21WAF, but not Snail. We identified a basic-helix-loop-helix protein, Olig1, as one of the Smad-binding transcription factors affected by HHM. Olig1 interacted with Smad2/3 in response to TGF-β stimulation, and was involved in transcriptional activation of PAI-1 and PDGF-B. HHM, but not Id proteins, inhibited TGF-β signalling-dependent association of Olig1 with Smad2/3 through physical interaction with Olig1. HHM thus appears to regulate a subset of TGF-β target genes including the Olig1-Smad synexpression group. HHM is the first example of a cellular response-selective regulator of TGF-β signalling with clearly determined mechanisms.

  • 24.
    Irobi, Edward
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Aguda, Adeleke, H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Guerin, Christophe
    Yin, Helen, L.
    Burtnick, Leslie, D.
    Blanchoin, Laurent
    Robinson, Robert, C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Structural basis of actin sequestration by thymosin β4: implications for WH2 proteins2004In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 23, no 18, p. 3599-608Article in journal (Refereed)
    Abstract [en]

    The WH2 (Wiscott-Aldridge syndrome protein homology domain 2) repeat is an actin interacting motif found in monomer sequestering and filament assembly proteins. We have stabilized the prototypical WH2 family member, thymosin-beta4 (Tbeta4), with respect to actin, by creating a hybrid between gelsolin domain 1 and the C-terminal half of Tbeta4 (G1-Tbeta4). This hybrid protein sequesters actin monomers, severs actin filaments and acts as a leaky barbed end cap. Here, we present the structure of the G1-Tbeta4:actin complex at 2 A resolution. The structure reveals that Tbeta4 sequesters by capping both ends of the actin monomer, and that exchange of actin between Tbeta4 and profilin is mediated by a minor overlap in binding sites. The structure implies that multiple WH2 motif-containing proteins will associate longitudinally with actin filaments. Finally, we discuss the role of the WH2 motif in arp2/3 activation.

  • 25.
    Itoh, Fumiko
    et al.
    Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
    Asao, Hironobu
    Department of Microbiology and Immunology, Tohoku University of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
    Sugamura, Kazuo
    Department of Microbiology and Immunology, Tohoku University of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    ten Dijke, Peter
    Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
    Itoh, Susumu
    Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
    Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 15, p. 4132-4142Article in journal (Refereed)
    Abstract [en]

    Inhibitory Smads, i.e. Smad6 and Smad7, are potent antagonists of the BMP-Smad pathway by interacting with activated bone morphogenetic protein (BMP) type I receptors and thereby preventing the activation of receptor-regulated Smads, or by competing with activated R-Smads for heteromeric complex formation with Smad4. The molecular mechanisms that underlie the regulation of I-Smad activity have remained elusive. Here we report the identification of a cytoplasmic protein, previously termed associated molecule with the SH3 domain of STAM (AMSH), as a direct binding partner for Smad6. AMSH interacts with Smad6, but not with R- and Co-Smads, upon BMP receptor activation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co-localization of Smad6 with AMSH in the cytoplasm. Ectopic expression of AMSH prolongs BMP-induced Smad1 phosphorylation, and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. The data strongly suggest that the molecular mechanism by which AMSH exerts its action is by inhibiting the binding of Smad6 to activated type I receptors or activated R-Smads.

  • 26. Johansson, Cecilia
    et al.
    Somberg, Monika
    Li, Xiaoze
    Backström Winquist, Ellenor
    Fay, Joanna
    Ryan, Fergus
    Pim, David
    Banks, Lawrence
    Schwartz, Stefan
    HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation.2012In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 14, p. 3212-27Article in journal (Refereed)
    Abstract [en]

    We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.

  • 27.
    Kanamoto, Takashi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Functional proteomics of transforming growth factor-beta1-stimulated Mv1Lu epithelial cells: Rad51 as a target of TGFbeta1-dependent regulation of DNA repair2002In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 21, no 5, p. 1219-1230Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor-beta (TGFbeta) conveys regulatory signals through multiple intracellular pathways, subsequently affecting various cellular functions. To identify new targets for TGFbeta, we studied the changes in the proteome of Mv1Lu lung epithelial cells in response to TGFbeta1 treatment. Thirty-eight non-abundant protein spots, affected by TGFbeta1, were selected, and proteins were identified by peptide mass-fingerprinting (PMF). Among them, proteins involved in regulation of immune response, apoptosis, regulation of TGFbeta signalling, metabolism and DNA repair were identified. Twenty-eight of the 38 proteins are new targets for TGFbeta1, thus suggesting novel ways of integration of TGFbeta signalling in intracellular regulatory processes. We show that TGFbeta1-dependent decrease in expression of one of the new targets, Rad51, correlates with a decrease in DNA repair efficiency. This was evaluated by formation of nuclear Rad51-containing DNA repair complexes in response to DNA damage, by single cell gel electrophoresis and by cell survival assay. The TGFbeta1-dependent inhibition of DNA repair was reversed by ectopic overexpression of Rad51. Therefore, TGFbeta can promote DNA instability through down-regulation of Rad51 and inhibition of DNA repair.

  • 28.
    Li, Wen
    et al.
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
    Agirrezabala, Xabier
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
    Lei, Jianlin
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
    Bouakaz, Lamine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Brunelle, Julie L
    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
    Ortiz-Meoz, Rodrigo F
    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
    Green, Rachel
    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Frank, Joachim
    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
    Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM2008In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 27, no 24, p. 3322-3331Article in journal (Refereed)
    Abstract [en]

    The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNA(Phe), Trp-tRNA(Trp), or Leu-tRNA(LeuI). The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same 'loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon-anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection.

  • 29.
    Lovmar, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Nilsson, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lukk, Eliisa
    Institute of Technology, University of Tartu, Tartu, Estonia.
    Vimberg, Vladimir
    Institute of Technology, University of Tartu, Tartu, Estonia.
    Tenson, Tanel
    Institute of Technology, University of Tartu, Tartu, Estonia.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Erythromycin resistance by L4/L22 mutations and resistance masking by drug efflux pump deficiency2009In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 28, no 6, p. 736-744Article in journal (Refereed)
    Abstract [en]

    We characterized the effects of classical erythromycin resistance mutations in ribosomal proteins L4 and L22 of the large ribosomal subunit on the kinetics of erythromycin binding. Our data are consistent with a mechanism in which the macrolide erythromycin enters and exits the ribosome through the nascent peptide exit tunnel, and suggest that these mutations both impair passive transport through the tunnel and distort the erythromycin-binding site. The growth-inhibitory action of erythromycin was characterized for bacterial populations with wild-type and L22-mutated ribosomes in drug efflux pump deficient and proficient backgrounds. The L22 mutation conferred reduced erythromycin susceptibility in the drug efflux pump proficient, but not deficient, background. This 'masking' of drug resistance by pump deficiency was reproduced by modelling with input data from our biochemical experiments. We discuss the general principles behind the phenomenon of drug resistance 'masking', and highlight its potential importance for slowing down the evolution of drug resistance among pathogens.

  • 30. Mäkinen, T
    et al.
    Veikkola, T
    Mustjoki, S
    Karpanen, T
    Catimel, B
    Nice, E C
    Wise, L
    Mercer, A
    Kowalski, H
    Kerjaschki, D
    Stacker, S A
    Achen, M G
    Alitalo, K
    Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3.2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 17Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth factor receptor-3 (VEGFR-3/Flt4) binds two known members of the VEGF ligand family, VEGF-C and VEGF-D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR-3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR-3. We show that VEGFR-3 stimulation alone protects the lymphatic endothelial cells from serum deprivation-induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C-dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin-sensitive induction of Akt phosphorylation. These results define the critical role of VEGF-C/VEGFR-3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.

  • 31. Nemeth, Attila
    et al.
    Guibert, Sylvain
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology.
    Tiwari, Vijay Kumar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology.
    Ohlsson, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology.
    Laengst, Gernot
    Epigenetic regulation of TTF-I-mediated promoter-terminator interactions of rRNA genes2008In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 27, no 8, p. 1255-1265Article in journal (Refereed)
    Abstract [en]

    Ribosomal RNA synthesis is the eukaryotic cell's main transcriptional activity, but little is known about the chromatin domain organization and epigenetics of actively transcribed rRNA genes. Here, we show epigenetic and spatial organization of mouse rRNA genes at the molecular level. TTF-I-binding sites subdivide the rRNA transcription unit into functional chromatin domains and sharply delimit transcription factor occupancy. H2A. Z-containing nucleosomes occupy the spacer promoter next to a newly characterized TTF-I-binding site. The spacer and the promoter proximal TTF-I-binding sites demarcate the enhancer. DNA from both the enhancer and the coding region is hypomethylated in actively transcribed repeats. 3C analysis revealed an interaction between promoter and terminator regions, which brings the beginning and end of active rRNA genes into close contact. Reporter assays show that TTF-I mediates this interaction, thereby linking topology and epigenetic regulation of the rRNA genes.

  • 32.
    Nilsson, Ingrid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Bahram, Fuad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Li, Xiujuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gualandi, Laura
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Koch, Sina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Anisimov, Andrey
    Kholová, Ivana
    Pytowski, Bronislaw
    Baldwin, Megan
    Ylä-Herttuala, Seppo
    Alitalo, Kari
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts2010In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 29, no 8, p. 1377-1388Article in journal (Refereed)
    Abstract [en]

    The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/-3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/-3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.

  • 33.
    Pavlov, Michael Y
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Antoun, Ayman
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lovmar, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting2008In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 27, no 12, p. 1706-1717Article in journal (Refereed)
    Abstract [en]

    We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine-Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G.

  • 34.
    Pavlov, Michael Y.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Zorzet, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits2011In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 30, no 2, p. 289-301Article in journal (Refereed)
    Abstract [en]

    We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNA(i)). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNA(i), implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNA(i) and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10(-4) for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases.

     

  • 35.
    Petersen-Mahrt, Svend K
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Estmer, Camilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Öhrmalm, Christina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Matthews, DA
    Russell, WC
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The splicing factor-associated protein, p32, regulates RNA splicing byinhibiting ASF/SF2 RNA binding and phosphorylation1999In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 18, no 4, p. 1014-1024Article in journal (Refereed)
    Abstract [en]

    The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.

  • 36. Petrova, Tatiana V
    et al.
    Mäkinen, Taija
    Mäkelä, Tomi P
    Saarela, Janna
    Virtanen, Ismo
    Ferrell, Robert E
    Finegold, David N
    Kerjaschki, Dontscho
    Ylä-Herttuala, Seppo
    Alitalo, Kari
    Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor.2002In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 21, no 17Article in journal (Refereed)
    Abstract [en]

    Lymphatic vessels are essential for fluid homeostasis, immune surveillance and fat adsorption, and also serve as a major route for tumor metastasis in many types of cancer. We found that isolated human primary lymphatic and blood vascular endothelial cells (LECs and BECs, respectively) show interesting differences in gene expression relevant for their distinct functions in vivo. Although these phenotypes are stable in vitro and in vivo, overexpression of the homeobox transcription factor Prox-1 in the BECs was capable of inducing LEC-specific gene transcription in the BECs, and, surprisingly, Prox-1 suppressed the expression of approximately 40% of the BEC-specific genes. Prox-1 did not have global effects on the expression of LEC-specific genes in other cell types, except that it up-regulated cyclin E1 and E2 mRNAs and activated the cyclin e promoter in various cell types. These data suggest that Prox-1 acts as a cell proliferation inducer and a fate determination factor for the LECs. Furthermore, the data provide insights into the phenotypic diversity of endothelial cells and into the possibility of transcriptional reprogramming of differentiated endothelial cells.

  • 37.
    Rahmanto, Aldwin Suryo
    et al.
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.
    Savov, Vasil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Brunner, Andrä
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.
    Bolin, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Weishaupt, Holger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Malyukova, Alena
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.
    Rosén, Gabriela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Čančer, Matko
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Hutter, Sonja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. German Canc Consortium DKTK, Heidelberg, Germany; German Canc Res Ctr, Heidelberg, Germany.
    Sundström, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Kawauchi, Daisuke
    German Canc Consortium DKTK, Heidelberg, Germany; German Canc Res Ctr, Heidelberg, Germany.
    Jones, David T.W.
    German Canc Consortium DKTK, Heidelberg, Germany; German Canc Res Ctr, Heidelberg, Germany.
    Spruck, Charles
    Sanford Burnham Prebys Med Discovery Inst, Ctr Canc, Tumor Initiat & Maintenance Program, La Jolla, CA USA.
    Taylor, Michael D.
    Hosp Sick Children, Arthur & Sonia Labatt Brain Tumour Res Ctr, Toronto, ON, Canada.
    Cho, Yoon Jae
    Stanford Univ, Sch Med, Dept Neurol & Neurol Sci, Stanford, CA USA.
    Pfister, Stefan M.
    German Canc Consortium DKTK, Heidelberg, Germany; German Canc Res Ctr, Heidelberg, Germany; German Canc Res Ctr, Heidelberg, Germany.
    Kool, Marcel
    German Canc Consortium DKTK, Heidelberg, Germany; German Canc Res Ctr, Heidelberg, Germany.
    Korshunov, Andrey
    German Canc Consortium DKTK, Heidelberg, Germany; German Canc Res Ctr, Heidelberg, Germany; Univ Heidelberg Hosp, Dept Neuropathol, Heidelberg, Germany.
    Swartling, Fredrik J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Sangfelt, Olle
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.
    FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma2016In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 35, no 20, p. 2192-2212Article in journal (Refereed)
    Abstract [en]

    SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW7 alpha. Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation-resistant SOX9 mutant reveals activation of pro-metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.

  • 38.
    Shimokawa, Noriaki
    et al.
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany.
    Haglund, Kaisa
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany.
    Hölter, Sabine M
    Institute of Developmental Genetics, Helmholtz Zentrum Mu¨nchen, German Research Center for Environmental Health, Neuherberg, Germany.
    Grabbe, Caroline
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany.
    Kirkin, Vladimir
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany.
    Koibuchi, Noriyuki
    Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan.
    Schultz, Christian
    Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany.
    Rozman, Jan
    Molecular Nutritional Medicine, Else-Kroner-Fresenius Center, Technische Universitat Munchen, Freising-Weihenstephan, Germany.
    Hoeller, Daniela
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany.
    Qiu, Chun-Hong
    Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan.
    Londoño, Marina B
    Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan.
    Ikezawa, Jun
    Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan.
    Jedlicka, Peter
    Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany.
    Stein, Birgit
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany.
    Schwarzacher, Stephan W
    Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany.
    Wolfer, David P
    Institute of Anatomy, University of Zurich, and Institute of Human Movement Sciences and Sport, ETH Zurich, Zurich, Switzerland.
    Ehrhardt, Nicole
    Molecular Nutritional Medicine, Else-Kroner-Fresenius Center, Technische Universitat Munchen, Freising-Weihenstephan, Germany.
    Heuchel, Rainer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Nezis, Ioannis
    Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.
    Brech, Andreas
    Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.
    Schmidt, Mirko H H
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany.
    Fuchs, Helmut
    Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany.
    Gailus-Durner, Valerie
    Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany.
    Klingenspor, Martin
    Molecular Nutritional Medicine, Else-Kroner-Fresenius Center, Technische Universitat Munchen, Freising-Weihenstephan, Germany.
    Bogler, Oliver
    Departments of Neurosurgery, Neuro-Oncology and Brain Tumor Center, University of Texas MD Anderson Cancer Center, Houston, TX, USA.
    Wurst, Wolfgang
    Institute of Developmental Genetics, Helmholtz Zentrum Mu¨nchen, German Research Center for Environmental Health, Neuherberg, Germany.
    Deller, Thomas
    Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany.
    de Angelis, Martin Hrabé
    Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany.
    Dikic, Ivan
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany.
    CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice2010In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 29, no 14, p. 2421-2432Article in journal (Refereed)
    Abstract [en]

    Despite extensive investigations of Cbl-interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85(Deltaex2)) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85(Deltaex2) animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post-synaptic compartment of striatal neurons in which it co-clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85(Deltaex2) mice.

  • 39.
    Urner, Sofia
    et al.
    Heinrich Heine Univ Dusseldorf, Inst Metab Physiol, Dusseldorf, Germany.
    Planas-Paz, Lara
    Heinrich Heine Univ Dusseldorf, Inst Metab Physiol, Dusseldorf, Germany.
    Hilger, Laura Sophie
    Heinrich Heine Univ Dusseldorf, Inst Metab Physiol, Dusseldorf, Germany.
    Henning, Carina
    Heinrich Heine Univ Dusseldorf, Inst Metab Physiol, Dusseldorf, Germany.
    Branopolski, Anna
    Heinrich Heine Univ Dusseldorf, Inst Metab Physiol, Dusseldorf, Germany;Heinrich Heine Univ Dusseldorf, Med Fac, Div Cardiol Pulmonol & Vasc Med, Dusseldorf, Germany.
    Kelly-Goss, Molly
    Univ Virginia, Dept Biomed Engn, Charlottesville, VA USA.
    Stanczuk, Lukas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Pitter, Bettina
    Ludwig Maximilians Univ Munchen, Univ Hosp, Walter Brendel Ctr Expt Med, Munich, Germany.
    Montanez, Eloi
    Ludwig Maximilians Univ Munchen, Univ Hosp, Walter Brendel Ctr Expt Med, Munich, Germany.
    Peirce, Shayn M.
    Univ Virginia, Dept Biomed Engn, Charlottesville, VA USA.
    Mäkinen, Taija
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Lammert, Eckhard
    Heinrich Heine Univ Dusseldorf, Inst Metab Physiol, Dusseldorf, Germany;Heine Univ Dusseldorf, Leibniz Ctr Diabet Res Heinrich, German Diabet Ctr DDZ, Inst Beta Cell Biol, Dusseldorf, Germany;German Ctr Diabet Res DZD eV, Neuherberg, Germany.
    Identification of ILK as a critical regulator of VEGFR3 signalling and lymphatic vascular growth2019In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 38, no 2, article id e99322Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth factor receptor-3 (VEGFR3) signalling promotes lymphangiogenesis. While there are many reported mechanisms of VEGFR3 activation, there is little understanding of how VEGFR3 signalling is attenuated to prevent lymphatic vascular overgrowth and ensure proper lymph vessel development. Here, we show that endothelial cell-specific depletion of integrin-linked kinase (ILK) in mouse embryos hyper-activates VEGFR3 signalling and leads to overgrowth of the jugular lymph sacs/primordial thoracic ducts, oedema and embryonic lethality. Lymphatic endothelial cell (LEC)-specific deletion of Ilk in adult mice initiates lymphatic vascular expansion in different organs, including cornea, skin and myocardium. Knockdown of ILK in human LECs triggers VEGFR3 tyrosine phosphorylation and proliferation. ILK is further found to impede interactions between VEGFR3 and beta 1 integrin in vitro and in vivo, and endothelial cell-specific deletion of an Itgb1 allele rescues the excessive lymphatic vascular growth observed upon ILK depletion. Finally, mechanical stimulation disrupts the assembly of ILK and beta 1 integrin, releasing the integrin to enable its interaction with VEGFR3. Our data suggest that ILK facilitates mechanically regulated VEGFR3 signalling via controlling its interaction with beta 1 integrin and thus ensures proper development of lymphatic vessels.

  • 40. Veikkola, T
    et al.
    Jussila, L
    Makinen, T
    Karpanen, T
    Jeltsch, M
    Petrova, T V
    Kubo, H
    Thurston, G
    McDonald, D M
    Achen, M G
    Stacker, S A
    Alitalo, K
    Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice.2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 6Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.

  • 41.
    Åström, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Åström, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease1991In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 10, no 10, p. 3067-3071Article in journal (Refereed)
    Abstract [en]

    We have identified a 3' exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3' end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3' exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3' end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.

1 - 41 of 41
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