Dokument

Zusammenfassung:
Kleine GTPasen der Rho-Familie übernehmen die Funktion von molekularen Schaltern in komplexen Signal-Netzwerken, die eine Vielzahl unterschiedlicher Signalkaskaden miteinander verknüpfen und selektive Signalantworten erlauben. Gleichzeitig nehmen auch die Regulatoren der molekularen Schalter eine wichtige Rolle ein, da durch diese Proteine die spezifische Weiterleitung von Signalen sichergestellt wird. Weiterhin verhindern sie, dass es zu einer unspezifischen Aktivierung von multiplen Signalkaskaden kommt. In der vorliegenden Arbeit wurden mehrere Aspekte untersucht, wie in diesen komplexen Netzwerken sichergestellt wird, dass die richtige Signalantwort auf einen bestimmten Stimulus erfolgt. Während GTPasen in vielen unterschiedlichen Signalkaskaden aktiv sind, sind sowohl die regulatorischen Proteine - die GEFs und GAPs - als auch die Effektoren oftmals spezifisch für einzelne Signalwege. Es deutlich mehr GEFs und Effektoren, als das es kleine GTPasen gibt. Eine GTPase kann oftmals von mehr als einem GEF aktiviert werden und ihrerseits mehr als einen Effektor aktivieren. Diese Interaktion zwischen GTPase und Effektor ist nicht immer spezifisch. Viele Effektoren können mit mehr als einer GTPase interagieren. Es stellt sich also die Frage, wie sichergestellt wird, dass die richtige Signalantwort auf einen spezifischen Stimulus erfolgt. Während eine GTPase in einer Vielzahl unterschiedlicher Singalwegen aktiv ist, reguliert ein einzelner GEF oft nur eine spezifische Funktion der jeweiligen GTPase. Um die einzelnen Funktionen einer GTPase zu verstehen, ist es somit wichtig, die Komplexität zu verringern und auf einzelne Signalwege zu reduzieren. In der vorliegenden Arbeit wurde der RhoGEF Hot1 aus U. maydis näher charakterisiert. Hot1 ist ein untypischer RhoGEF, der anstelle des für RhoGEFs charakteristischen Domänen-Motivs eine DH-Domänen-assoziierte BAR-Domäne besitzt. Es konnte gezeigt werden, dass Hot1 als RhoGEF für Cdc42 wirken kann und an der Regulation der Zellmorphologie beteiligt ist. Hot1 hat damit eine komplementäre Funktion zum RhoGEF Don1, der die Cdc42-abhängige Zelltrennung reguliert. Die für einen RhoGEF ungewöhnliche BAR-Domäne von Hot1 erkennt spezifische Membrankrümmungen. Es konnte gezeigt werden, dass Hot1 an frühen endozytotischen Strukturen lokalisiert. Darüber hinaus bildet die BAR-Domäne von Hot1 Heterodimere mit dem BAR-Domänen Protein Hob3. Weiterhin wurde untersucht, wie Hot1 spezifisch sein Substrat, die GTPase Cdc42, erkennt. Die beiden GTPasen Cdc42 und Rac1 weisen einen hohen Grad an Sequenzähnlichkeit auf. Dennoch führt eine Deletion dieser Proteine in U. maydis zu unterschiedlichen Phänotypen. In früheren Arbeiten konnte gezeigt werden, dass einige RhoGEFs zwischen Cdc42 und Rac1 anhand einer einzigen konservierten Aminosäure an Position 56 unterscheiden. Ein Austausch dieser Aminosäure zwischen Cdc42 und Rac1 führt zu einer Änderung der Spezifität der Cdc42-spezifischen RhoGEFs Don1 und Its1 sowie des Rac1-spezifischen RhoGEFs Cdc24 in U. maydis. Auch aus anderen Organismen war bekannt, dass die Aminosäure an Position 56 eine wichtige Rolle für die spezifische Erkennung von Cdc42 und Rac1 durch RhoGEFs spielt. Überraschenderweise hat diese Aminosäure keinen Einfluss auf die Erkennung von Cdc42 durch Hot1. Vielmehr konnte im Rahmen der vorliegenden Arbeit gezeigt werden, dass Hot1, aber auch das humane Homolog TUBA, einen neuen Mechanismus benutzen, um zwischen Cdc42 und Rac1 zu unterscheiden. Anstatt der Aminosäure 56, erkennt Hot1 die GTPase Cdc42 anhand weniger unterschiedlicher Aminosäuren im N-Terminus von Cdc42. Als weiterer Punkt dieser Arbeit wurde untersucht, wie die spezifische Signalweiterleitung von kleinen GTPasen in komplexen Signalkaskaden funktioniert. Aufgrund der hohen Ähnlichkeit von Cdc42 und Rac1 und der teilweisen Redundanz ihrer Funktionen stellte sich die Frage, in wie weit diese GTPasen noch gemeinsame Funktionen übernehmen können und ob es zwangsläufig beide Proteine geben muss. Es konnte eine synthetische GTPase generiert werden, die zu großen Teilen die Funktionen von Cdc42 und Rac1 in einem Protein vereint. Diese GTPase kann von den meisten bekannten RhoGEFs von Cdc42 und Rac1 aktiviert werden und auch die meisten zellulären Funktionen übernehmen. Im Zuge dieser Untersuchungen konnte auch gezeigt werden, dass es einen spezifischen Effektor von Cdc42 geben muss, der auch anhand der Aminosäure an Position 56 zwischen Cdc42 und Rac1 unterscheidet. Schließlich konnte noch gezeigt werden, dass die C-terminale Region von Cdc42 und Rac1 einen wichtigen Einfluss auf die subzelluläre Lokalisation der GTPasen hat und dass diese Lokalisation kritisch für die korrekte Funktion dieser Proteine ist.

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