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  • 1.
    Andrae, Johanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Ehrencrona, Hans
    Gallini, Radiosa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Lal, Mark
    Ding, Hao
    Betsholtz, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Analysis of Mice Lacking the Heparin-Binding Splice Isoform of Platelet-Derived Growth Factor A2013In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 33, no 20, p. 4030-4040Article in journal (Refereed)
    Abstract [en]

    Platelet-derived growth factor A-chain (PDGF-A) exists in two evolutionarily conserved isoforms, PDGF-Along and PDGF-Ashort, generated by alternative RNA splicing. They differ by the presence (in PDGF-Along) or absence (in PDGF-Ashort) of a carboxyterminal heparin/heparan sulfate proteoglycan-binding motif. In mice, similar motifs present in other members of the PDGF and vascular endothelial growth factor (VEGF) families have been functionally analyzed in vivo, but the specific physiological importance of PDGF-A(long) has not been explored previously. Here, we analyzed the absolute and relative expression of the two PDGF-A splice isoforms during early postnatal organ development in the mouse and report on the generation of a Pdgfa allele (Pdgfa(Delta ex6) incapable of producing PDGF-A(long) due to a deletion of the exon 6 splice acceptor site. In situations of limiting PDGF-A signaling through PDGF receptor alpha (PDGFR alpha), or in mice lacking PDGF-C, homozygous carriers of Pdgfa(Delta ex6) showed abnormal development of the lung, intestine, and vertebral column, pinpointing developmental processes where PDGF-A(long) may play a physiological role.

  • 2.
    Arvidsson, Ann-Kristin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Rupp, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Nånberg, Eeva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Downward, Julian
    Signal Transduction Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.
    Rönnstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Wennström, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Schlessinger, Joseph
    Department of Pharmacology, New York University Medical Center, New York New York 10016, USA.
    Heldin, Carl-Henrik
    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.
    Tyr-716 in the platelet-derived growth factor beta-receptor kinase insertis involved in GRB2 binding and Ras activation1994In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 14, no 10, p. 6715-6726Article in journal (Refereed)
    Abstract [en]

    Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.

  • 3. Chernukhin, Igor
    et al.
    Shamsuddin, Shaharum
    Kang, Sung Yun
    Bergström, Rosita
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Animal Development and Genetics.
    Kwon, Yoo-Wook
    Yu, WenQiang
    Whitehead, Joanne
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Animal Development and Genetics.
    Mukhopadhyay, Rituparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Animal Development and Genetics.
    Docquier, France
    Farrar, Dawn
    Morrison, Ian
    Vigneron, Marc
    Wu, Shwu-Yuan
    Chiang, Sheng-Ming
    Loukinov, Dimitri
    Ohlsson, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Animal Development and Genetics.
    Klenova, Elena
    CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide2007In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 27, no 5, p. 1631-1648Article in journal (Refereed)
    Abstract [en]

    CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Serial" chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

  • 4.
    Culver, Carolyn
    et al.
    College of Life Sciences, Wellcome Trust Centre for Gene Regulation and Expression, MSI/WTB/JBC Complex, Dow Street, University of Dundee, Dundee DD1 5EH, Scotland.
    Sundqvist, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Mudie, Sharon
    College of Life Sciences, Wellcome Trust Centre for Gene Regulation and Expression, MSI/WTB/JBC Complex, Dow Street, University of Dundee, Dundee DD1 5EH, Scotland.
    Melvin, Andrew
    College of Life Sciences, Wellcome Trust Centre for Gene Regulation and Expression, MSI/WTB/JBC Complex, Dow Street, University of Dundee, Dundee DD1 5EH, Scotland.
    Xirodimas, Dimitris
    College of Life Sciences, Wellcome Trust Centre for Gene Regulation and Expression, MSI/WTB/JBC Complex, Dow Street, University of Dundee, Dundee DD1 5EH, Scotland.
    Rocha, Sonia
    College of Life Sciences, Wellcome Trust Centre for Gene Regulation and Expression, MSI/WTB/JBC Complex, Dow Street, University of Dundee, Dundee DD1 5EH, Scotland.
    Mechanism of Hypoxia-Induced NF-kappa B2010In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 30, no 20, p. 4901-4921Article in journal (Refereed)
    Abstract [en]

    NF-kappa B activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here we report that under hypoxic conditions, I kappa B kinase (IKK) activity is induced through a calcium/calmodulin-dependent kinase 2 (CaMK2)dependent pathway distinct from that for other common inducers of NF-kappa B. This process still requires IKK and the IKK kinase TAK1, like that for inflammatory inducers of NF-kappa B, but the TAK1-associated proteins TAB1 and TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC (linear ubiquitin chain assembly complex) ubiquitin conjugation system. In contrast to the action of other NF-kappa B inducers, IKK-mediated phosphorylation of I kappa B alpha does not result in its degradation. We show that this results from I kappa B alpha sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48-linked polyubiquitination. Furthermore, inhibition of specific Sumo proteases is sufficient to release RelA from I kappa B alpha and activate NF-kappa B target genes. These results define a novel pathway regulating NF-kappa Bactivation, important to its physiological role in human health and disease.

  • 5.
    Edlund, Sofia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Lee, So Young
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Grimsby, Susanne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Zhang, Shouthing
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Aspenström, Pontus
    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.
    Landström, Maréne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Interaction between Smad7 and beta-catenin: importance for transforming growth factor beta-induced apoptosis2005In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 25, no 4, p. 1475-1488Article in journal (Refereed)
    Abstract [en]

    Members of the transforming growth factor beta (TGF-beta) and Wnt/wingless superfamilies regulate cell fate during development and tissue maintenance. Here we report that Smad7 interacts with beta-catenin and lymphoid enhancer binding factor 1/T-cell-specific factor (LEF1/TCF), transcriptional regulators in Wnt signaling, in a TGF-beta-dependent manner. Smad7 was found to be required for TGF-beta1-induced accumulation of beta-catenin and LEF1 in human prostate cancer (PC-3U) cells as well as in human keratinocytes (HaCaT cells). Moreover, when the endogenous Smad7 was repressed by specific small interfering RNA, TGF-beta-induced increase of activated p38, Akt phosphorylated on Ser473, glycogen synthase kinase 3beta phosphorylated on Ser9 was prevented, as well as the TGF-beta-induced association between beta-catenin and LEF1. Notably, the observed physical association of Smad7 and beta-catenin was found to be important for TGF-beta-induced apoptosis, since suppression of beta-catenin expression by small interfering RNA decreased the apoptotic response to TGF-beta.

  • 6.
    Giandomenico, Valeria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Simonsson, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Grönroos, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Ericsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Coactivator-dependent acetylation stabilizes members of the SREBP family of transcription factors2003In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 23, no 7, p. 2587-2599Article in journal (Refereed)
    Abstract [en]

    Members of the SREBP family of transcription factors control cholesterol and lipid homeostasis and play important roles during adipocyte differentiation. The transcriptional activity of SREBPs is dependent on the coactivators p300 and CBP. We now present evidence that SREBPs are acetylated by the intrinsic acetyltransferase activity of p300 and CBP. In SREBP1a, the acetylated lysine residue resides in the DNA-binding domain of the protein. Coexpression with p300 dramatically increases the expression of both SREBP1a and SREBP2, and this effect is dependent on the acetyltransferase activity of p300, indicating that acetylation of SREBPs regulates their stability. Indeed, acetylation or mutation of the acetylated lysine residue in SREBP1a stabilizes the protein. We demonstrate that the acetylated residue in SREBP1a is also targeted by ubiquitination and that acetylation inhibits this process. Thus, our studies define acetylation-dependent stabilization of transcription factors as a novel mechanism for coactivators to regulate gene expression.

  • 7. Haiko, Paula
    et al.
    Makinen, Taija
    Keskitalo, Salla
    Taipale, Jussi
    Karkkainen, Marika J
    Baldwin, Megan E
    Stacker, Steven A
    Achen, Marc G
    Alitalo, Kari
    Deletion of vascular endothelial growth factor C (VEGF-C) and VEGF-D is not equivalent to VEGF receptor 3 deletion in mouse embryos.2008In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 28, no 15Article in journal (Refereed)
    Abstract [en]

    Lymphatic vessels play an important role in the regulation of tissue fluid balance, immune responses, and fat adsorption and are involved in diseases including lymphedema and tumor metastasis. Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3) is necessary for development of the blood vasculature during early embryogenesis, but later, VEGFR-3 expression becomes restricted to the lymphatic vasculature. We analyzed mice deficient in both of the known VEGFR-3 ligands, VEGF-C and VEGF-D. Unlike the Vegfr3(-/-) embryos, the Vegfc(-/-); Vegfd(-/-) embryos displayed normal blood vasculature after embryonic day 9.5. Deletion of Vegfr3 in the epiblast, using keratin 19 (K19) Cre, resulted in a phenotype identical to that of the Vegfr3(-/-) embryos, suggesting that this phenotype is due to defects in the embryo proper and not in placental development. Interestingly, the Vegfr3(neo) hypomorphic mutant mice carrying the neomycin cassette between exons 1 and 2 showed defective lymphatic development. Overexpression of human or mouse VEGF-D in the skin, under the K14 promoter, rescued the lymphatic hypoplasia of the Vegfc(+/-) mice in the K14-VEGF-D; Vegfc(+/-) compound mice, suggesting that VEGF-D is functionally redundant with VEGF-C in the stimulation of developmental lymphangiogenesis. Our results suggest VEGF-C- and VEGF-D-independent functions for VEGFR-3 in the early embryo.

  • 8.
    Hallböök, Finn
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Ebendal, Ted
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Zoology.
    Persson, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Production and characterization of biologically active recombinant beta nerve growth factor1988In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 8, no 1, p. 452-456Article in journal (Refereed)
    Abstract [en]

    DNA fragments encoding either rat or chicken beta nerve growth factor (NGF) were inserted in the expression vector p91023(B) for transient expression in COS cells. The two NGF constructs produced RNA transcripts and proteins of the predicted sizes. Conditioned media from the transfected cells stimulated neurite outgrowth from cultured chicken embryo sympathetic ganglia. The results show that the rat or chicken NGF gene can direct the synthesis of a biologically active NGF protein after transfection of COS cells.

  • 9. Jaffe, Aron B
    et al.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hall, Alan
    Human CNK1 acts as a scaffold protein, linking Rho and Ras signal transduction pathways2004In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 24, no 4, p. 1736-1746Article in journal (Refereed)
    Abstract [en]

    Rho family GTPases act as molecular switches to control a variety of cellular responses, including cytoskeletal rearrangements, changes in gene expression, and cell transformation. In the active, GTP-bound state, Rho interacts with an ever-growing number of effector molecules, which promote distinct biochemical pathways. Here, we describe the isolation of hCNK1, the human homologue of Drosophila connector enhancer of ksr, as an effector for Rho. hCNK1 contains several protein-protein interaction domains, and Rho interacts with one of these, the PH domain, in a GTP-dependent manner. A mutant hCNK1, which is unable to bind to Rho, or depletion of endogenous hCNK1 by using RNA interference inhibits Rho-induced gene expression via serum response factor but has no apparent effect on Rho-induced stress fiber formation, suggesting that it acts as a specific effector for transcriptional, but not cytoskeletal, activation pathways. Finally, hCNK1 associates with Rhophilin and RalGDS, Rho and Ras effector molecules, respectively, suggesting that it acts as a scaffold protein to mediate cross talk between the two pathways.

  • 10.
    Kowanetz, Marcin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Valcourt, Ulrich
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Bergström, Rosita
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor β and bone morphogenetic protein2004In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 24, no 10, p. 4241-4254Article in journal (Refereed)
    Abstract [en]

    Transforming growth factors beta (TGF-betas) inhibit growth of epithelial cells and induce differentiation changes, such as epithelial-mesenchymal transition (EMT). On the other hand, bone morphogenetic proteins (BMPs) weakly affect epithelial cell growth and do not induce EMT. Smad4 transmits signals from both TGF-beta and BMP pathways. Stimulation of Smad4-deficient epithelial cells with TGF-beta 1 or BMP-7 in the absence or presence of exogenous Smad4, followed by cDNA microarray analysis, revealed 173 mostly Smad4-dependent, TGF-beta-, or BMP-responsive genes. Among 25 genes coregulated by both factors, inhibitors of differentiation Id2 and Id3 showed long-term repression by TGF-beta and sustained induction by BMP. The opposing regulation of Id genes is critical for proliferative and differentiation responses. Hence, ectopic Id2 or Id3 expression renders epithelial cells refractory to growth inhibition and EMT induced by TGF-beta, phenocopying the BMP response. Knockdown of endogenous Id2 or Id3 sensitizes epithelial cells to BMP, leading to robust growth inhibition and induction of transdifferentiation. Thus, Id genes sense Smad signals and create a permissive or refractory nuclear environment that defines decisions of cell fate and proliferation.

  • 11.
    Krampert, Monika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Chirasani, Sridhar Reddy
    Department of Neurology, University of Regensburg, Regensburg, Germany.
    Wachs, Frank-Peter
    Department of Neurology, University of Regensburg, Regensburg, Germany.
    Aigner, Robert
    Department of Neurology, University of Regensburg, Regensburg, Germany.
    Bogdahn, Ulrich
    Department of Neurology, University of Regensburg, Regensburg, Germany.
    Yingling, Jonathan M.
    Eli Lilly, Lilly Research Laboratories, Cancer Research, Indianapolis, Indiana, USA.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Aigner, Ludwig
    Institute of Molecular Regenerative Medicine, Paracelsus Medical University Salzburg, 5020 Salzburg, Austria.
    Heuchel, Rainer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Smad7 Regulates the Adult Neural Stem/Progenitor Cell Pool in a Transforming Growth Factor β- and Bone Morphogenetic Protein-Independent Manner2010In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 30, no 14, p. 3685-3694Article in journal (Refereed)
    Abstract [en]

    Members of the transforming growth factor beta (TGF-beta) family of proteins modulate the proliferation, differentiation, and survival of many different cell types. Neural stem and progenitor cells (NPCs) in the adult brain are inhibited in their proliferation by TGF-beta and by bone morphogenetic proteins (BMPs). Here, we investigated neurogenesis in a hypomorphic mouse model for the TGF-beta and BMP inhibitor Smad7, with the hypothesis that NPC proliferation might be reduced due to increased TGF-beta and BMP signaling. Unexpectedly, we found enhanced NPC proliferation as well as an increased number of label-retaining cells in vivo. The enhanced proliferation potential of mutant cells was retained in vitro in neurosphere cultures. We observed a higher sphere-forming capacity as well as faster growth and cell cycle progression. Use of specific inhibitors revealed that these effects were independent of TGF-beta and BMP signaling. The enhanced proliferation might be at least partially mediated by elevated signaling via epidermal growth factor (EGF) receptor, as mutant cells showed higher expression and activation levels of the EGF receptor. Conversely, an EGF receptor inhibitor reduced the proliferation of these cells. Our data indicate that endogenous Smad7 regulates neural stem/progenitor cell proliferation in a TGF-beta- and BMP-independent manner.

  • 12.
    Kurisaki, Akira
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kurisaki, Keiko
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kowanetz, Marcin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Sugino, Hiromu
    Yoneda, Yoshihiro
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The mechanism of nuclear export of smad3 involves exportin 4 and Ran2006In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 26, no 4, p. 1318-1332Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor beta (TGF-beta) receptors phosphorylate Smad3 and induce its nuclear import so it can regulate gene transcription. Smad3 can return to the cytoplasm to propagate further cycles of signal transduction or to be degraded. We demonstrate that Smad3 is exported by a constitutive mechanism that is insensitive to leptomycin B. The Mad homology 2 (MH2) domain is responsible for Smad3 export, which requires the GTPase Ran. Inactive, GDP-locked RanT24N or nuclear microinjection of Ran GTPase activating protein 1 blocked Smad3 export. Inactivation of the Ran guanine nucleotide exchange factor RCC1 inhibited Smad3 export and led to nuclear accumulation of phosphorylated Smad3. A screen for importin/exportin family members that associate with Smad3 identified exportin 4, which binds a conserved peptide sequence in the MH2 domain of Smad3 in a Ran-dependent manner. Exportin 4 is sufficient for carrying the in vitro nuclear export of Smad3 in cooperation with Ran. Knockdown of endogenous exportin 4 completely abrogates the export of endogenous Smad3. A short peptide representing the minimal interaction domain in Smad3 effectively competes with Smad3 association to exportin 4 and blocks nuclear export of Smad3 in vivo. We thus delineate a novel nuclear export pathway for Smad3.

  • 13.
    Kurisaki, Keiko
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kurisaki, Akira
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Valcourt, Ulrich
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Terentiev, Alexei A
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Pardali, Katerina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    ten Dijke, Peter
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Ericsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Nuclear factor YY1 inhibits transforming growth factor beta- and bone morphogenetic protein-induced cell differentiation2003In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 23, no 13, p. 4494-4510Article in journal (Refereed)
    Abstract [en]

    Smad proteins transduce transforming growth factor beta (TGF-beta) and bone morphogenetic protein (BMP) signals that regulate cell growth and differentiation. We have identified YY1, a transcription factor that positively or negatively regulates transcription of many genes, as a novel Smad-interacting protein. YY1 represses the induction of immediate-early genes to TGF-beta and BMP, such as the plasminogen activator inhibitor 1 gene (PAI-1) and the inhibitor of differentiation/inhibitor of DNA binding 1 gene (Id-1). YY1 inhibits binding of Smads to their cognate DNA elements in vitro and blocks Smad recruitment to the Smad-binding element-rich region of the PAI-1 promoter in vivo. YY1 interacts with the conserved N-terminal Mad homology 1 domain of Smad4 and to a lesser extent with Smad1, Smad2, and Smad3. The YY1 zinc finger domain mediates the association with Smads and is necessary for the repressive effect of YY1 on Smad transcriptional activity. Moreover, downregulation of endogenous YY1 by antisense and small interfering RNA strategies results in enhanced transcriptional responses to TGF-beta or BMP. Ectopic expression of YY1 inhibits, while knockdown of endogenous YY1 enhances, TGF-beta- and BMP-induced cell differentiation. In contrast, overexpression or knockdown of YY1 does not affect growth inhibition induced by TGF-beta or BMP. Accordingly, YY1 does not interfere with the regulation of immediate-early genes involved in the TGF-beta growth-inhibitory response, the cell cycle inhibitors p15 and p21, and the proto-oncogene c-myc. In conclusion, YY1 represses Smad transcriptional activities in a gene-specific manner and thus regulates cell differentiation induced by TGF-beta superfamily pathways.

  • 14. Mandic, Aleksandra
    et al.
    Viktorsson, Kristina
    Molin, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eguchi, Hidetaka
    Hayashi, Shin-Ichi
    Toi, Masakazu
    Hansson, Johan
    Linder, Stig
    Shoshan, Maria C
    Cisplatin induces the proapoptotic conformation of Bak in a deltaMEKK1-dependent manner2001In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 21, no 11, p. 3684-3691Article in journal (Refereed)
    Abstract [en]

    In a panel of four human melanoma cell lines, equitoxic doses of cisplatin induced the proapoptotic conformation of the Bcl-2 family protein Bak prior to the execution phase of apoptosis. Because cisplatin-induced modulation of the related Bax protein was seen in only one cell line, a degree of specificity in the signal to Bak is indicated. Little is known about upstream regulation of Bak activity. In this study, we examined whether the apoptosis-specific pathway mediated by a kinase fragment of MEKK1 (DeltaMEKK1) is involved in the observed Bak modulation. We report that expression of a kinase-inactive fragment of MEKK1 (dominant negative MEKK [dnMEKK]) efficiently blocked cisplatin-induced modulation of Bak and cytochrome c release and consequently also reduced DEVDase activation and nuclear fragmentation. Accordingly, expression of a kinase-active MEKK1 fragment (dominant positive MEKK) was sufficient to induce modulation of Bak in three cell lines and to induce apoptosis in two of these. dnMEKK did not block cisplatin-induced c-Jun N-terminal kinase (JNK) activation, in agreement with a specifically proapoptotic role for the DeltaMEKK1 pathway. Finally, we show that reduction of Bak expression by antisense Bak reduced cisplatin-induced loss of mitochondrial integrity and caspase cleavage activity in breast cancer cell lines. In summary, we have identified Bak as a cisplatin-regulated component downstream in a proapoptotic, JNK-independent DeltaMEKK1 pathway.

  • 15.
    Mohammad, Faizaan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Pandey, Radha Raman
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nagano, Takashi
    Chakalova, Lyubomira
    Mondal, Tanmoy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Fraser, Peter
    Kanduri, Chandrasekhar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kcnq1ot1/Lit1 noncoding RNA mediates transcriptional silencing by targeting to the perinucleolar region2008In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 28, no 11, p. 3713-3728Article in journal (Refereed)
    Abstract [en]

    The Kcnq1ot1 antisense noncoding RNA has been implicated in long-range bidirectional silencing, but the underlying mechanisms remain enigmatic. Here we characterize a domain at the 5' end of the Kcnq1ot1 RNA that carries out transcriptional silencing of linked genes using an episomal vector system. The bidirectional silencing property of Kcnq1ot1 maps to a highly conserved repeat motif within the silencing domain, which directs transcriptional silencing by interaction with chromatin, resulting in histone H3 lysine 9 trimethylation. Intriguingly, the silencing domain is also required to target the episomal vector to the perinucleolar compartment during mid-S phase. Collectively, our data unfold a novel mechanism by which an antisense RNA mediates transcriptional gene silencing of chromosomal domains by targeting them to distinct nuclear compartments known to be rich in heterochromatic machinery.

  • 16.
    Mühlemann, Oliver
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Yue, Bai-Gong
    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.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A novel type of splicing enhancer regulating adenovirus pre-mRNA splicing2000In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 20, no 7, p. 2317-2325Article in journal (Refereed)
    Abstract [en]

    Splicing of the adenovirus IIIa pre-mRNA is subjected to a temporal regulation, such that efficient IIIa 3' splice site usage is confined to the late phase of the infectious cycle. Here we show that IIIa pre-mRNA splicing is activated more than 200-fold in nuclear extracts prepared from late adenovirus-infected cells (Ad-NE) compared to uninfected HeLa cell nuclear extracts (HeLa-NE). In contrast, splicing of the beta-globin pre-mRNA is repressed in Ad-NE. We constructed hybrid pre-mRNAs between IIIa and beta-globin in order to identify the minimal IIIa sequence element conferring enhanced splicing in Ad-NE. Using this approach, we show that the IIIa branch site/pyrimidine tract functions as a Janus element: it blocks splicing in HeLa-NE and functions as a splicing enhancer in Ad-NE. Therefore, we named this sequence the IIIa virus infection-dependent splicing enhancer (3VDE). This element is essential for regulated IIIa pre-mRNA splicing in Ad-NE and sufficient to confer an enhanced splicing phenotype to the beta-globin pre-mRNA in Ad-NE. We further show that the increase in IIIa splicing observed in Ad-NE is not accompanied by a similar increase in U2AF binding to the IIIa pyrimidine tract. This finding suggests that splicing activation by the 3VDE may operate without efficient U2AF interaction with the pre-mRNA. Importantly, this report represents the first description of a splicing enhancer that has evolved to function selectively in the context of a virus infection, a finding that adds a new level at which viruses may subvert the host cell RNA biosynthetic machinery to facilitate their own replication.

  • 17.
    Persson, Camilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Sävenhed, Catrine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Bourdeau, Annie
    Tremblay, Michel L
    Markova, Boyka
    Böhmer, Frank D
    Haj, Fawaz G
    Neel, Benjamin G
    Elson, Ari
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Rönnstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Östman, Arne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellberg, Carina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Site-selective regulation of platelet-derived growth factor beta receptor tyrosine phosphorylation by T-cell protein tyrosine phosphatase2004In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 24, no 5, p. 2190-2201Article in journal (Refereed)
    Abstract [en]

    The platelet-derived growth factor (PDGF) beta receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF beta receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF beta receptor, we compared PDGF beta receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF beta receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase Cgamma1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase Cgamma1 activity and migratory hyperresponsiveness to PDGF. PDGF beta receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTPepsilon ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF beta receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors.

  • 18.
    Popova, Svetlana N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Barczyk, Malgorzata
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tiger, Carl-Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Beertsen, Wouter
    Zigrino, Paola
    Aszodi, Attila
    Miosge, Nicolai
    Forsberg, Erik
    Gullberg, Donald
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    alpha 11 beta 1 integrin-dependent regulation of periodontal ligament function in the erupting mouse incisor2007In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 27, no 12, p. 4306-4316Article in journal (Refereed)
    Abstract [en]

    The fibroblast integrin alpha 11 beta 1 is a key receptor for fibrillar collagens. To study the potential function of alpha 11. in vivo, we generated a null allele of the alpha 11 gene. Integrin alpha 1(-/-) mice are viable and fertile but display dwarfism with increased mortality, most probably due to severely defective incisors. Mutant incisors are characterized by disorganized periodontal ligaments, whereas molar ligaments appear normal. The primary defect in the incisor ligament leads to halted tooth eruption. alpha 11 beta 1-defective embryonic fibroblasts displayed severe defects in vitro, characterized by (i) greatly reduced cell adhesion and spreading on collagen 1, (ii) reduced ability to retract collagen lattices, and (iii) reduced cell proliferation. Analysis of matrix metalloproteinase in vitro and in vivo revealed disturbed MMP13 and MMP14 synthesis in alpha 11(-/-) cells. We show that alpha 11 beta 1 is the major receptor for collagen I on mouse embryonic fibroblasts and suggest that alpha 11 beta 1 integrin is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate the forces needed for axial tooth movement. Our data show a unique role for alpha 11 beta 1 integrin during tooth eruption.

  • 19. Ruediger, R
    et al.
    Roeckel, D
    Fait, J
    Bergqvist, Anders
    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 Biochemistry and Microbiology.
    Magnusson, G
    Walter, G
    Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.1992In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 12, no 11, p. 4872-82Article in journal (Refereed)
    Abstract [en]

    Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.

  • 20.
    Ruusala, Aino
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    The atypical Rho GTPase Wrch1 collaborates with the nonreceptor tyrosine kinases Pyk2 and Src in regulating cytoskeletal dynamics2008In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 28, no 5, p. 1802-14Article in journal (Refereed)
    Abstract [en]

    The Cdc42-like GTPase Wnt responsive Cdc42 homolog 1 (Wrch1) has several atypical features; it has an N-terminal proline-rich extension that confers binding to SH3 domains, and it harbors an extremely high intrinsic nucleotide exchange activity, which overrides the normal GTPase activity. As a result, Wrch1 resides mainly in the active, GTP-loaded conformation under normal cellular conditions. We have previously shown that ectopic expression of Wrch1 in fibroblasts resulted in an altered cell morphology visible as a formation of filopodia, a loss of stress fibers, and a reduction in focal adhesions. Here, we show that Wrch1 binds to the nonreceptor tyrosine kinase Pyk2. The interaction required Wrch1 to be in a GTP conformation and also required an intact N-terminal proline-rich extension as well as an intact effector loop. Wrch1 requires Pyk2 in imposing the cytoskeletal effects, seen as the formation of filopodia, since treatment of cells with a Pyk2-specific small interfering RNA abrogated this response. Interestingly, we found that the presence and activity of Src were needed for the formation of a Wrch1-Pyk2 complex as well as for the Wrch1-induced formation of filopodia. We propose a model in which Pyk2 and Src function to coordinate the Wrch1-dependent effects on cytoskeletal dynamics.

  • 21. Wang, Nancy
    et al.
    Söderbom, Fredrik
    Center for Molecular Genetics, Department of Biology, University of California—San Diego, La Jolla, California .
    Anjard, Christophe
    Shaulsky, Gad
    Loomis, William F
    SDF-2 induction of terminal differentiation in Dictyostelium discoideum is mediated by the membrane-spanning sensor kinase DhkA.1999In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 19, no 7, p. 4750-4756Article in journal (Refereed)
    Abstract [en]

    SDF-2 is a peptide released by prestalk cells during culmination that stimulates prespore cells to encapsulate. Genetic evidence indicates that the response is dependent on the dhkA gene. This gene encodes a member of the histidine kinase family of genes that functions in two-component signal transduction pathways. The sequence of the N-terminal half of DhkA predicts two hydrophobic domains separated by a 310-amino-acid loop that could bind a ligand. By inserting MYC6 epitopes into DhkA, we were able to show that the loop is extracellular while the catalytic domain is cytoplasmic. Cells expressing the MYC epitope in the extracellular domain of DhkA were found to respond only if induced with 100-fold-higher levels of SDF-2 than required to induce dhkA+ cells; however, they could be induced to sporulate by addition of antibodies specific to the MYC epitope. To examine the enzymatic activity of DhkA, we purified the catalytic domain following expression in bacteria and observed incorporation of labelled phosphate from ATP consistent with histidine autophosphorylation. Site-directed mutagenesis of histidine1395 to glutamine in the catalytic domain blocked autophosphorylation. Furthermore, genetic analyses showed that histidine1395 and the relay aspartate2075 of DhkA are both critical to its function but that another histidine kinase, DhkB, can partially compensate for the lack of DhkA activity. Sporulation is drastically reduced in double mutants lacking both DhkA and DhkB. Suppressor studies indicate that the cyclic AMP (cAMP) phosphodiesterase RegA and the cAMP-dependent protein kinase PKA act downstream of DhkA.

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