TY - THES A1 - Wenzel, Jens T1 - Regulation of TLR-induced macrophage responses by cytoskeleton-associated phosphoproteins T1 - Regulation der Antwort von Makrophagen auf TLR-Stimulation durch Zytoskelett-assoziierte Phosphoproteine N2 - Toll-like receptors (TLR) are pattern recognition receptors (PRR) by which macrophages (MØ) sense pathogen-associated molecular patterns (PAMPs). The recognition of lipopolysaccharide (LPS), the PAMP of gram negative bacteria, by TLR4 triggers signaling cascades and leads to the pro-inflammatory activation of the cells. A recent quantitative and kinetic analysis of the phosphoproteome of LPS-activated primary macrophages highlighted the cytoskeleton as a cell compartment with an enriched protein phosphorylation. In total 44 cytoskeleton-associated proteins were regulated by this post-translational modification and thus might be involved in the control and regulation of key macrophage functions like spreading, motility and phagocytosis. To investigate the control of cytoskeleton-associated cell functions by TLR4 activation, we first developed a method to quantitatively measure the spreading response of bone marrow MØ after stimulation with LPS. Fluorescence microscopy was used for cell imaging and visualisation of the MØ contact area. In collaboration with the Fraunhofer Institute Erlangen, we developed and validated a software tool for the semi-automated segmentation and quantitation of MØ fluorescence microscopy data, which allowed fast, robust and objective image analysis. Using this method, we observed that LPS caused time-dependent spreading, which was detectable after 1-2 h and maximal after 24 h. Next, the impact of genetic or pharmacological inhibition of known TLR signaling components was investigated. Deficiency in the adapter protein MYD88 strongly reduced spreading activity at the late time points, but had no impact early after LPS-stimulation. A similar effect was observed upon pharmacological inhibition of ERK1/2 signaling, indicating that ERK1/2 mediates MYD88-dependent MØ spreading. In contrast, MØ lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8-24 h after stimulation. The genetic deletion of the MAPK phosphatases DUSP1 and DUSP16 resulted in impaired late spreading, corroborating the essential role for functional MAPK signaling in TLR4-driven MØ spreading. To identify the contribution of other cytoskeletal phosphoproteins to MØ spreading, siRNA knockdown of selected candidate genes in primary murine MØ was employed and combined with automated quantitative image analysis. These experiments revealed a functional role for the Myosins MYO1e and MYO1f in MØ spreading. These motor proteins are strongly phosphorylated in LPS-activated MØ. Because of their ability to simultaneously bind to actin filaments and cell membrane or other proteins, we investigated their role in phagocytosis, cytokine production and antigen presentation. Phagocytosis and killing of bacteria were not affected in Myo1e-/- macrophages. However, MYO1e plays a role in chemokine secretion and antigen presentation processes. MCP1 (CCL2) release was selectively increased in Myo1e-deficient MØ and dendritic cells (DC), while cytokine secretion was unaffected. Furthermore, macrophages and DCs lacking MYO1e showed lower levels of MHC-II on the cell surface. However, mRNA levels of CCL2 and of MHC-II were unaltered. These data suggest a role for MYO1e in the transport of selected chemokines and of MHC-II molecules to the cell surface. MHC-II-restricted antigen presentation assays revealed an impaired capacity of macrophages and DC lacking MYO1e to stimulate antigen-specific T cells, suggesting that the reduced MHC-II expression is functionally relevant. Taken together, in this study first a quantitative image analysis method was developed which allows the unbiased, robust and efficient investigation of the macrophage spreading response. Combination of this method with siRNA knockdown of selected cytoskeleton-associated phosphoproteins led to the identification of MYO1e and MYO1f as regulators of macrophage spreading. Furthermore, we identified MYO1e in MØ and DC to be essential for the intracellular transport of CCL2 and MHC-II to the cell surface and for optimal stimulation of antigen-specific CD4 T cells. N2 - Toll-like Rezeptoren (TLR) sind Mustererkennungsrezeptoren (PRR) durch die Makrophagen (MØ) pathogen-assoziierte molekulare Muster (PAMPs) erkennen. Die Erkennung von Lipopolysacchariden (LPS), dem PAMP gramnegativer Bakterien, durch TLR4 löst Signalkaskaden aus, die zu einer pro-inflammatorischen Aktivierung der Zellen führen. Eine quantitative und kinetische Analyse des Phosphoproteoms LPS-aktivierter primärer Makrophagen identifizierte das Zytoskelett als ein Zellkompartiment mit gesteigerter Proteinphosphorylierung. Insgesamt wurden 44 Zytoskelett-assoziierte Proteine identifiziert, die durch diese post-translationale Modifikation reguliert wurden und demzufolge an der Regulation wichtiger Zellfunktionen von Makrophagen wie Spreading, Motilität und Phagozytose beteiligt sein könnten. Um die Kontrolle Zytoskelett-vermittelter Zellfunktionen nach TLR4 Aktivierung zu untersuchen, entwickelten wir zunächst eine Methode zur quantitativen Messung der Spreadingantwort von Knochenmarksmakrophagen nach LPS Stimulation. Die Visualisierung der Zellen sowie ihrer Kontaktfläche erfolgte hierbei mittels Fluoreszenzmikroskopie. Für eine schnelle, robuste und objektive Analyse der Fluoreszenzaufnahmen entwickelten und validierten wir in Kollaboration mit dem Fraunhofer Institut in Erlangen eine Software zur halbautomatischen Segmentierung und Quantifizierung der Kontaktfläche. Unter Verwendung dieser Methode konnte eine zeitabhängige LPS-induzierte Zunahme der Zellkontaktfläche beobachtet werden, die nach 1-2 Stunden detektierbar war und ein Maximum nach 24 Stunden erreichte. Durch den Einsatz pharmakologischer Inhibitoren sowie genetisch veränderter Zellen wurde anschließend der Einfluss bekannter TLR4-Signalwegkomponenten untersucht. Die genetische Defizienz des Adapterproteins MYD88 führte hierbei zu einer stark reduzierten Spreadingaktivität der Zellen während der späten LPS Stimulationsphase, wohingegen das initiale Spreading nicht beeinflusst wurde. Ein vergleichbarer Effekt konnte unter Verwendung eines pharmakologischen Inhibitors zur Hemmung des ERK1/2 Signalweges identifiziert werden. Diese Beobachtungen deuten darauf hin, dass ERK1/2 für die Weiterleitung des MYD88 vermittelten Spreading notwendig ist. Im Gegensatz dazu wurde in p38-defizienten Makrophagen ein beeinträchtigtes initiales Spreading beobachtet, wohingegen das späte Spreading nach 8 – 24 Stunden nicht beeinflusst war. Die genetische Deletion der MAPK Phosphatasen DUSP1 und DUSP16 resultierte ebenfalls in einer Minderung des späten Spreadings, ebenfalls ein Hinweis auf die essentielle Rolle funktioneller MAPK Signalwege. Um die Beteiligung weiter Zytoskelett-Phosphoproteine am Zellspreading zu identifizieren, wurde die Expression ausgewählter Kandidatengene in primären Makrophagen mittels spezifischer siRNA unterdrückt und das Zellspreading mit Hilfe der entwickelten Software quantifiziert. Diese Versuche zeigten eine funktionelle Rolle der Myosine MYO1e und MYO1f. Diese Motorproteine weisen ebenfalls eine starke Phosphorylierung nach LPS Stimulation auf. Aufgrund ihrer Eigenschaft simultan mit Aktinfilamenten und Zellmembranen sowie anderen Proteinen zu interagieren, untersuchten wir ihre Rolle während der Phagozytose, Zytokinfreisetzung und Antigenpräsentation. Obwohl Myo1e defiziente Makrophagen keine Beeinträchtigung der Phagozytose oder Abtötung von Bakterien aufwiesen, spielte das Motorprotein eine wichtige Rolle in der Chemokinfreisetzung und Antigenpräsentation. Interessanterweise war die Sekretion des Chemokins MCP1 (CCL2) in Myo1e-defizienten Makrophagen und dendritischen Zellen (DC) selektiv erhöht, während die Zytokinfreisetzung unbeeinträchtigt war. Des Weiteren wiesen Myo1e KO Makrophagen und DC eine reduzierte MHC-II Oberflächen-Expression auf, obwohl die MHC-II als auch die CCL2 Transkription auf mRNA Ebene nicht beeinflusst war. Diese Daten legen nahe, dass MYO1e während des Transports bestimmter Chemokine, sowie von MHC-II zur Zelloberfläche eine wichtige Rolle spielt. Zudem zeigten Myo1e KO Makrophagen und DC in einem MHC-II-abhängigen Antigenpräsentationsassay eine abgeschwächte Fähigkeit zur Antigen-spezifischen T-Zell Aktivierung, was die funktionelle Relevanz der reduzierten Expression von MHC-II nahelegt. Zusammenfassend wurde in dieser Studie zunächst eine Methode zur quantitativen Bildanalyse entwickelt, welche eine unvoreingenommene, robuste und effiziente Untersuchung des Spreadings von Makrophagen erlaubte. Die Kombination dieser Methode mit dem spezifischen siRNA Knockdown ausgewählter Zytoskelett-assoziierter Phosphoproteine führte zur Identifizierung von MYO1e und MYO1f als wichtige Regulatoren dieser Zellfunktion. Darüber hinaus konnte in Makrophagen und DC eine essentielle Rolle für MYO1e im intrazellulären Transport von CCL2 und MHC-II an die Zelloberfläche identifiziert werden, sowie dessen Notwendigkeit für eine vollständige Aktivierung antigen-spezifischer CD4 T Zellen. KW - Toll-like-Rezeptoren KW - Makrophage KW - Phosphoproteine KW - Zellskelett KW - macrophage KW - cytoskeleton KW - phosphorylation KW - TLR4 Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-98843 ER - TY - JOUR A1 - Weigand, Isabel A1 - Ronchi, Cristina L. A1 - Vanselow, Jens T. A1 - Bathon, Kerstin A1 - Lenz, Kerstin A1 - Herterich, Sabine A1 - Schlosser, Andreas A1 - Kroiss, Matthias A1 - Fassnacht, Martin A1 - Calebiro, Davide A1 - Sbiera, Silviu T1 - PKA Cα subunit mutation triggers caspase-dependent RIIβ subunit degradation via Ser\(^{114}\) phosphorylation JF - Science Advances N2 - Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing’s syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser\(^{114}\) phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing’s syndrome. KW - mutation triggers KW - phosphorylation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270445 VL - 7 IS - 8 ER - TY - JOUR A1 - Szabó, Áron A1 - Papin, Christian A1 - Zorn, Daniela A1 - Ponien, Prishila A1 - Weber, Frank A1 - Raabe, Thomas A1 - Rouyer, François T1 - The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator JF - PLoS Biology N2 - Phosphorylation is a pivotal regulatory mechanism for protein stability and activity in circadian clocks regardless of their evolutionary origin. It determines the speed and strength of molecular oscillations by acting on transcriptional activators and their repressors, which form negative feedback loops. In Drosophila, the CK2 kinase phosphorylates and destabilizes the PERIOD (PER) and TIMELESS (TIM) proteins, which inhibit CLOCK (CLK) transcriptional activity. Here we show that CK2 also targets the CLK activator directly. Downregulating the activity of the catalytic alpha subunit of CK2 induces CLK degradation, even in the absence of PER and TIM. Unexpectedly, the regulatory beta subunit of the CK2 holoenzyme is not required for the regulation of CLK stability. In addition, downregulation of \(CK2\alpha\) activity decreases CLK phosphorylation and increases per and tim transcription. These results indicate that CK2 inhibits CLK degradation while reducing its activity. Since the CK1 kinase promotes CLK degradation, we suggest that CLK stability and transcriptional activity result from counteracting effects of CK1 and CK2. KW - negative feedback loop KW - PER-TIM complex KW - posttranslational regulation KW - transcription factor KW - in-vivo KW - behavioral rhythms KW - proteins period KW - beta-subunit KW - phosphorylation KW - gene KW - CT, circadian time KW - LD, light:dark KW - DD, constant darkness Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127234 SN - 1545-7885 VL - 11 IS - 8 ER - TY - JOUR A1 - Subramanian, Hariharan A1 - Döring, Frank A1 - Kollert, Sina A1 - Rukoyatkina, Natalia A1 - Sturm, Julia A1 - Gambaryan, Stepan A1 - Stellzig-Eisenhauer, Angelika A1 - Meyer-Marcotty, Philipp A1 - Eigenthaler, Martin A1 - Wischmeyer, Erhard T1 - PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling JF - PLoS One N2 - Aim Primary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet. Materials and Methods In HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in Xenopus laevis oocytes. Results In HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished. Conclusions PTH1R mutations affect intracellular PTH-regulated signaling in vitro. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement. KW - phosphorylation KW - xenopus oocytes KW - calcium signaling KW - intracellular receptors KW - mutation KW - teeth KW - tooth eruption KW - transfection Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147967 VL - 11 IS - 11 ER - TY - JOUR A1 - Stölting, Miriam A1 - Wiesner, Christiane A1 - van Vliet, Vanessa A1 - Butt, Elke A1 - Pavenstädt, Hermann A1 - Linder, Stefan A1 - Kremerskothen, Joachim T1 - Lasp-1 Regulates Podosome Function JF - PLoS One N2 - Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins. Lasp-1 is a ubiquitously expressed, actin-binding protein that is known to regulate cytoskeleton architecture and cell migration. This multidomain protein is predominantely present at focal adhesions, however, a second pool of Lasp-1 molecules is also found at lamellipodia and vesicle-like microdomains in the cytosol. In this report, we show that Lasp-1 is a novel component and regulator of podosomes. Immunofluorescence studies reveal a localization of Lasp-1 in the podosome ring structure, where it colocalizes with zyxin and vinculin. Life cell imaging experiments demonstrate that Lasp-1 is recruited in early steps of podosome assembly. A siRNA-mediated Lasp-1 knockdown in human macrophages affects podosome dynamics as well as their matrix degradation capacity. In summary, our data indicate that Lasp-1 is a novel component of podosomes and is involved in the regulation of podosomal function. KW - discrete KW - smooth muscle cells KW - microdomains KW - actin cytoskeleton KW - endothelial cells KW - epithelial cells KW - cancer cells KW - phosphorylation KW - invadopodia KW - dependent protein-kinase KW - camp signaling pathway Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134315 VL - 7 IS - 4 ER - TY - THES A1 - Sibilski, Claudia T1 - Identification and characterization of the novel mKSR1 phosphorylation site Tyr728 and its role in MAPK signaling T1 - Identifizierung und Charakterisierung der neuartigen mKSR1-Phosphorylierungsstelle Tyr728 und deren Rolle in der MAPK-Signalkaskade N2 - In mammals, KSR1 functions as an essential scaffold that coordinates the assembly of RAF/MEK/ERK complexes and regulates intracellular signal transduction upon extracellular stimulation. Aberrant activation of the equivalent MAPK signaling pathway has been implicated in multiple human cancers and some developmental disorders. The mechanism of KSR1 regulation is highly complex and involves several phosphorylation/dephosphorylation steps. In the present study, a number of novel in vivo phosphorylation sites were detected in mKSR1 by use of mass spectrometry analysis. Among others, Tyr728 was identified as a unique regulatory residue phosphorylated by LCK, a Src kinase family member. To understand how phosphorylation of Tyr728 may regulate the function of KSR1 in signal transduction and cellular processes, structural modeling and biochemical studies were integrated in this work. Computational modeling of the mKSR1(KD) protein structure revealed strong hydrogen bonding between phospho-Tyr728 and the residues surrounding Arg649. Remarkably, this pattern was altered when Tyr728 was non-phosphorylated or substituted. As confirmed by biochemical analysis, Arg649 may serve as a major anchor point for phospho-Tyr728 in order to stabilize internal structures of KSR1. In line with the protein modeling results, mutational studies revealed that substitution of Tyr728 by phenylalanine leads to a less compact interaction between KSR1 and MEK, a facilitated KSR1/B-RAF binding and an increased phosphorylation of MEK in complex with KSR1. From these findings it can be concluded that phospho-Tyr728 is involved in tightening the KSR1/MEK interaction interface and in regulating the phosphorylation of KSR1-bound MEK by either RAF or KSR1 kinases. Beside the Tyr728, Ser722 was identified as a novel regulatory phosphorylation site. Amino acid exchanges at the relevant position demonstrated that Ser722 regulates KSR1-bound MEK phosphorylation without affecting KSR1/MEK binding per se. Due to its localization, Ser722 might consequently control the catalytic activity of KSR1 by interfering with the access of substrate (possibly MEK) to the active site of KSR1 kinase. Together with Ser722, phosphorylated Tyr728 may further positively affect the kinase activity of KSR1 as a consequence of its vicinity to the activation and catalytic loop in the KSR1(KD). As revealed by structural modeling, phospho-Tyr728 builds a hydrogen bond with the highly conserved Lys685. Consequently, phospho-Tyr728 has a stabilizing effect on internal structures involved in the catalytic reaction and possibly enhances the phosphate transfer within the catalytic cleft in KSR1. Considering these facts, it seems very likely that the LCK-dependent phosphorylation of Tyr728 plays a crucial role in the regulation of KSR1 catalytic activity. Results of fractionation and morphology analyses revealed that KSR1 recruits LCK to cytoskeleton for its phosphorylation at Tyr728 suggesting that this residue may regulate cytoskeleton dynamics and, consequently, cell motility. Beside that, phosphorylation of Tyr728 is involved in the regulation of cell proliferation, as shown by a significantly reduced population doubling time of KSR1-Y728F cells compared to cells expressing wild type KSR1. Taken together, tyrosine phosphorylation in KSR1 uncovers a new link between Src family kinases and MAPK signaling. Tyr728, the novel regulatory phosphorylation site in murine KSR1, may coordinate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses. N2 - KSR1 fungiert bei Säugetieren als zentrales Gerüstprotein, welches die Anordnung von RAF/MEK/ERK-Komplexen koordiniert und die intrazelluläre Signalweiterleitung nach extrazellulärer Stimulation reguliert. Eine abweichende Aktivierung des entsprechenden MAPK-Signalwegs wurde mit vielen humanen Krebsformen und einigen Entwicklungsstörungen in Verbindung gebracht. Der Mechanismus der KSR1-Regulierung ist hochgradig komplex und involviert mehrfach Schritte der Phosphorylierung/Dephosphorylierung. In der vorliegenden Studie wurden etliche neue in-vivo-Phosphorylierungsstellen in mKSR1 mittels massenspektrometrischer Analyse entdeckt. Neben anderen wurde Tyr728 als besonderer regulatorischer Rest identifiziert, welcher durch LCK, einem Mitglied der Src-Kinase-Familie, phosphoryliert wird. Um zu verstehen wie die Phosphorylierung von Tyr728 die Funktion von KSR1 innerhalb der Signalweiterleitung und zellulärer Prozesse regulieren könnte, wurden strukturelle Modellierungen und biochemische Untersuchungen in diese Arbeit integriert. Die Computermodellierung der mKSR1(KD)-Proteinstruktur zeigte starke Wasserstoff- brückenbindungen zwischen Phospho-Tyr728 und den Resten in der Umgebung von Arg649 auf. Dieses Muster war auffällig verändert, wenn Tyr728 nicht phosphoryliert oder substituiert war. Wie anhand biochemischer Analyse untermauert wurde, könnte Arg649 für phospho-Tyr728 als Hauptankerpunkt dienen, um interne Strukturen in KSR1 zu stabilisieren. In Übereinstimmung mit den Ergebnissen der Proteinmodellierung enthüllten die Mutationsstudien, dass die Substitution von Tyr728 mit Phenylalanin zu einer weniger kompakten Interaktion zwischen KSR1 und MEK, einer erleichterten KSR1/B-RAF-Bindung und einer ansteigenden Phosphorylierung von MEK im Komplex mit KSR1 führt. Anhand dieser Erkenntnisse kann man rückschließen, dass Phospho-Tyr728 in die Verstärkung der Interaktionen innerhalb der KSR1/MEK-Grenzfläche und in die Regulierung der Phosphorylierung von KSR1-gebundenem MEK durch entweder RAF- oder KSR1-Kinasen involviert ist. Neben Tyr728 wurde Ser722 als eine neuartige regulatorische Phosphorylierungsstelle identifiziert. Aminosäureaustausche an der betreffenden Position demonstrierten, dass Ser722 die Phosphorylierung von KSR1-gebundenem MEK reguliert ohne die KSR1/MEK-Bindung selbst zu beeinträchtigen. Bedingt durch seine Lokalisierung könnte Ser722 folglich die katalytische Aktivität von KSR1 kontrollieren, indem es den Zugang des Substrates (möglicherweise MEK) zur aktiven Seite der KSR1-Kinase behindert. Zusammen mit Ser722 könnte phosphoryliertes Tyr728 ferner die Kinaseaktivität von KSR1 positiv beeinflussen, infolge von dessen Nähe zur Aktivierungs- und katalytischen Schleife in der KSR1(KD). Wie mittels Strukturmodellierung offengelegt wurde, bildet Phospho-Tyr728 eine Wasserstoffbrücke mit dem hochgradig konservierten Lys685 aus. Folglich hat Phospho-Tyr728 einen stabilisierenden Effekt auf interne Strukturen, welche in die katalytische Reaktion involviert sind, und erleichtert möglicherweise den Phosphattransfer innerhalb der katalytischen Spalte in KSR1. In Anbetracht dieser Fakten scheint es sehr wahrscheinlich, dass die LCK-abhängige Phosphorylierung von Tyr728 eine äußerst wichtige Rolle in der Regulierung der katalytischen Aktivität von KSR1 spielt. Die Ergebnisse der Fraktionierungs- und Morphologieanalysen enthüllten, dass KSR1 für die Phosphorylierung an Tyr728 LCK zum Zytoskelett rekrutiert, was darauf hindeutet, dass dieser Rest die Dynamik des Zytoskeletts und folglich Zellmotilität regulieren könnte. Darüber hinaus ist die Phosphorylierung von Tyr728 in die Regulierung der Zellproliferation involviert, wie anhand einer bedeutend reduzierten Populationsverdopplungszeit von KSR1-Y728F-Zellen im Vergleich zu Zellen, welche wildtypisches KSR1 exprimieren, gezeigt wurde. Zusammenfassend lässt sich sagen, dass die Tyrosin-Phosphorylierung in KSR1 eine neue Verknüpfung zwischen Kinasen der Src-Familie und der MAPK-Signalwirkung enthüllt. Tyr728, die neuartige regulatorische Phosphorylierungsstelle in Maus-KSR1, könnte den Übergang zwischen der Gerüst- und der katalytischen Funktion von KSR1 koordinieren und damit als Kontrollpunkt dienen, um zelluläre Reaktionen fein abzustimmen. KW - MAP-Kinase KW - Signaltransduktion KW - Regulation KW - tyrosine phosphorylation KW - KSR1 KW - LCK KW - MAPK KW - phosphorylation KW - signaling Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114672 ER - TY - JOUR A1 - Röder, Pia V. A1 - Geillinger, Kerstin E. A1 - Zietek, Tamara S. A1 - Thorens, Bernard A1 - Koepsell, Hermann A1 - Daniel, Hannelore T1 - The Role of SGLT1 and GLUT2 in Intestinal Glucose Transport and Sensing JF - PLOS ONE N2 - Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion. KW - rat small-intestine KW - brush border membrane KW - apical GLUT2 KW - incretin secretion KW - diffusive component KW - sugar absorption KW - mice KW - calcium absorption KW - phosphorylation KW - cotransporter Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117262 VL - 9 IS - 2 ER - TY - JOUR A1 - Rudel, Thomas A1 - Faulstich, Michaela A1 - Böttcher, Jan-Peter A1 - Meyer, Thomas F. A1 - Fraunholz, Martin T1 - Pilus Phase Variation Switches Gonococcal Adherence to Invasion by Caveolin-1-Dependent Host Cell Signaling JF - PLoS Pathogens N2 - Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorBIA, in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorBIA-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection. KW - antibodies KW - bacterial pathogens KW - cell membranes KW - intracellular pathogens KW - neisseria gonorrhoeae KW - phosphates KW - phosphorylation KW - pili and fimbriae Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96679 ER - TY - JOUR A1 - Rauert-Wunderlich, Hilka A1 - Siegmund, Daniela A1 - Maier, Eduard A1 - Giner, Tina A1 - Bargou, Ralf C. A1 - Wajant, Harald A1 - Stühmer, Thorsten T1 - The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NF kappa B Transcription Factors JF - PLoS ONE N2 - Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells. KW - signal inhibition KW - necrotic cell death KW - cell viability testing KW - cell death KW - small interfering RNAs KW - HT29 cells KW - phosphorylation KW - multiple myeloma Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130140 VL - 8 IS - 3 ER - TY - JOUR A1 - Navdaev, Alexey A1 - Subramanian, Hariharan A1 - Petunin, Alexey A1 - Clemetson, Kenneth J. A1 - Gambaryan, Stepan A1 - Walter, Ulrich T1 - Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation JF - PLoS ONE N2 - von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways. KW - tyrosine KW - ERK signaling cascade KW - integrins KW - phosphorylation KW - polystyrene KW - platelet activation KW - platelet aggregation KW - platelets Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119815 SN - 1932-6203 VL - 9 IS - 4 ER -