15 research outputs found

    Charakterisierung des RanGAP1-RanBP2 Komplexes in Mitose

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    Der RanGAP1-RanBP2 Komplex stellt ein faszinierendes makromolekulares Gebilde dar, welches mindestens zwei enzymatische AktivitĂ€ten umfasst. Zum einen beinhaltet der Komplex die GTP-Hydrolyse aktivierende Funktion, die RanGAP1 zusammen mit RanBP2 ausĂŒbt, zum anderen besitzt RanBP2 zusammen mit Ubc9 Sumo-konjugierende AktivitĂ€t. Gemeinsam sind diese Proteine essentielle Regulatoren des nukleo-zytoplasmatischen Transportes in Interphasezellen, und spielen des Weiteren eine wichtige, bislang jedoch kaum verstandene Rolle fĂŒr die Funktion der Kinetochore in Mitose.Um den RanGAP1-RanBP2 Komplex spezifisch in mitotischen Zellen nĂ€her zu untersuchen, habe ich spezifisch in Mitose nach Interaktionspartnern gesucht. Dies fĂŒhrte zur Identifikation des Kernexportrezeptors Crm1 und der GTPase Ran als stabile Komponenten in einem Komplex mit RanGAP1, RanBP2 und Ubc9 in mitotischen Zellen. ZusĂ€tzlich schien dieser Komplex zahlreiche weitere Proteine in substöchiometrischen Mengen zu enthalten. Diese könnten beispielsweise NES-enthaltende Interaktionspartner von Crm1 und/oder Substrate fĂŒr RanBP2-abhĂ€ngige Sumoylierung sein. Da bisher RanBP2-abhĂ€ngige Sumo-Substrate weitgehend unbekannt sind, habe ich eine Strategie entwickelt, um sumoylierte Proteine aus immungereinigtem RanGAP1-RanBP2 Komplex anzureichern. Dies ermöglichte die massenspektrometrische Identifizierung von ungefĂ€hr 90 potentiellen Sumo-Substraten, die spezifisch in mitotischen RanGAP1 Komplexen angereichert waren; 6 dieser Substrate wurden zur weiteren Charakterisierung ausgewĂ€! hlt. Alle Kandidaten assoziierten mit mitotischen RanGAP1 Komplexen (Topo II alpha, TACC2, CKAP-5, Plk1, USP7, PIAS1). Die meisten davon konnten entweder in vitro mit rekombinanten Faktoren (TACC2, Plk1) oder als endogene Proteine, die mit mitotischem RanGAP1-RanBP2 Komplex als Quelle der Sumo E3 Ligase-AktivitĂ€t assoziiert waren (TopoII alpha, Plk1, USP7), sumoyliert werden. AuffĂ€llig war die Co-Reinigung der Sumo E3 Ligase PIAS1 mit RanGAP1 aus mitotischen Zellen; diese wurde ebenfalls effizient sumoyliert. Weitere Analysen deuteten darauf hin, dass RanGAP1 in einem Komplex mit PIAS1 enthalten ist, der sich vom RanGAP1-RanBP2 Komplex unterscheidet.In einem Nebenprojekt konnte ich zeigen, dass das Sumo-konjugierende Enzym Ubc9 in Zellen an Lysin 14 sumoyliert werden kann. Dieser Befund war wichtig, um eine biochemische Studie von Knipscheer et al. zu ergĂ€nzen, die einen neuen Mechanismus der Selektion von Sumo-Substraten identifizierte; diese Ergebnisse flossen in die Veröffentlichung Knipscheer, Flotho, Klug et al. (2008) Mol Cell ein

    The Ran GTPase-activating protein (RanGAP1) is critically involved in smooth muscle cell differentiation, proliferation and migration following vascular injury: implications for neointima formation and restenosis.

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    Differentiation and dedifferentiation, accompanied by proliferation play a pivotal role for the phenotypic development of vascular proliferative diseases (VPD), such as restenosis. Increasing evidence points to an essential role of regulated nucleoporin expression in the choice between differentiation and proliferation. However, whether components of the Ran GTPase cycle, which is of pivotal importance for both nucleocytoplasmic transport and for mitotic progression, are subject to similar regulation in VPD is currently unknown. Here, we show that differentiation of human coronary artery smooth muscle cell (CASMC) to a contractile phenotype by stepwise serum depletion leads to significant reduction of RanGAP1 protein levels. The inverse event, dedifferentiation of cells, was assessed in the rat carotid artery balloon injury model, a well-accepted model for neointima formation and restenosis. As revealed by temporospatial analysis of RanGAP1 expression, neointima formation in rat carotid arteries was associated with a significant upregulation of RanGAP1 expression at 3 and 7 days after balloon injury. Of note, neointimal cells located at the luminal surface revealed persistent RanGAP1 expression, as opposed to cells in deeper layers of the neointima where RanGAP1 expression was less or not detectable at all. To gain first evidence for a direct influence of RanGAP1 levels on differentiation, we reduced RanGAP1 in human coronary artery smooth muscle cells by siRNA. Indeed, downregulation of the essential RanGAP1 protein by 50% induced a differentiated, spindle-like smooth muscle cell phenotype, accompanied by an upregulation of the differentiation marker desmin. Reduction of RanGAP1 levels also resulted in a reduction of mitogen induced cellular migration and proliferation as well as a significant upregulation of the cyclin-dependent kinase inhibitor p27KIP1, without evidence for cellular necrosis. These findings suggest that RanGAP1 plays a critical role in smooth muscle cell differentiation, migration and proliferation in vitro and in vivo. Appropriate modulation of RanGAP1 expression may thus be a strategy to modulate VPD development such as restenosis

    RanGAP1 expression in the rat carotid artery injury model.

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    <p>To determine the spatiotemporal expression pattern of RanGAP1 during the course of neointima formation, the rat carotid injury model was applied. Immunohistochemical staining revealed upregulation of RanGAP1 at day 3 (b, f, j, and day 7 (c, g, k) whereas RanGAP1 expression ceased when SMC proliferation decreases at day 14 (d, h, l) subsequent to balloon injury.</p

    siRNA Sequences.

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    <p>All molecules consist additionally of dTdT overhangs. n/a indicates not avaible (commercially available scrambled siRNA obtained from Ambion).</p

    Downregulation of RanGAP1 in arrested, differentiated smooth muscle cells (A).

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    <p>To induce differentiation, CASMCs were depleted from serum. RanGAP1 expression in CASMC was assessed by Western Blotting 12(∌70 kDa) form as well as the SUMO-1 conjugated (∌90 kDa) form of the RanGAP-1 protein. Both bands revealed reduced RanGAP1 expression over time in cells entering quiescence. α-SM Actin protein, a marker of cell differentiation, is increased over time. Actin levels are displayed as loading control. <b>Effect of posttranscriptional gene silencing of RanGAP1 by small interfering RNA (siRNA) on cell cycle and differentiation markers (B).</b> siRNA mediated gene silencing of RanGAP1 was able to reduce the 90 kD band by 31.8±21.2% (90 kD band) and 75%±14.7% (70 kD band) 48 h post transfection, respectively (average of three different experiments). RanGAP1 depletion was associated with a strong increase of p27<sup>Kip1</sup> expression by 60±34%. RanGAP1 deficiency was also associated with a sharp increase in desmin expression with levels even higher than in quiescent cells. CASMC denotes coronary artery smooth muscle cells; (+) denotes serum stimulated CASMC; (−) denotes quiescent CASMC (≄72 h serum depletion); “control” denotes oligofectamine transfected cells without siRNAs; siRNA-SCR denotes scrambled (control) siRNA.</p

    Quantitative morphometric and immunohistochemical analysis of neointima formation and RanGAP1 expression in the rat carotid artery injury model.

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    <p>No significant difference was detectable between the injured group compared to non-injured control arteries with respect to medial area at 3 days, 7 days and 14 days post injury (A) (p = 0.58). Of note, we observed in the media a trend towards a cellular upregulation of RanGAP1 (p = 0.08) 3 days after injury concomitantly with the beginning of cellular proliferation as a response to vascular injury. The increase of neointimal area was detectable at day 7 and peaked at 14 days following vascular injury (B) (p<0.0001). RanGAP1 expression in the media was the highest at the initiation of cellular proliferation and decreased to barely detectable levels at the completion of neointima formation (C). 3 days post injury, almost all cells in the neointima stained positive for RanGAP1 and subsequently, levels decrementally decreased at later time points, e.g. at day 7 and day 14 (D). In non-injured control sections, RanGAP1 expression was virtually undetectable.</p
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