Mechanismen der Erkennung der kleinen GTPasen Cdc42 und Rac1durch Guanin-Nukleotid-Austausch-Faktoren in U. maydis

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 wi...

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Bibliographic Details
Main Author: Tillmann, Britta Anna Maria
Contributors: Bölker, Michael (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2013
Online Access:PDF Full Text
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All cells contain complex molecular signaling networks. Small GTPases of the Rho family act as molecular switches in these networks. They link different signaling cascades and enable the cell to react selectively to different stimuli. The regulators of these molecular switches play an important part, since they guarantee the selective transmission of signals. Moreover, they also restrict the unselective activation of multiple signaling cascades. The work presented here investigates how it can be ensured that in complex signaling networks only specific signal responses are elicited by a certain stimulus. While RhoGTPases can act in multiple signaling pathways, both their regulatory proteins - GEFs and GAPs - and their effectors are often specific for single signaling pathways. This is highlighted by the fact that there are more GEFs and effectors than RhoGTPases, and that a single GTPase can be activated by multiple GEFs and signal through multiple effectors. Moreover, the interaction between GTPase and effector is not always highly specific, since many effectors can be activated by more than one GTPase. This poses the question how it can be guaranteed that a certain stimulus results in a specific response. An important part of this specificity is mediated by the activators of small GTPases, the GEFs. A GTPase can act in multiple different signaling pathways; however, many GEFs regulate a GTPase only in the context of a specific pathway or a specific function. Therefore, in order to understand the role of a GTPase in complex signaling networks, it is often necessary to reduce this complexity and analyze their role in single signal transduction pathways, for example by analyzing rather the function of the pathway-specific GEFs than the more universal GTPases. This work characterizes the function of the RhoGEF Hot1 in the phytopathogenic fungus Ustilago maydis. Hot1 is an atypical RhoGEF which contains a catalytic DH domain in combination with a BAR domain instead of the canonical combination of catalytic DH domain and membrane-associated PH domain. In the work presented here it could be shown that Hot1 acts as a RhoGEF for Cdc42 and is involved in the regulation of cellular morphology. Hot1 acts as a complimentary factor to the GEF Don1, which regulates the Cdc42-dependent cell separation. The BAR domain of Hot1, an unusual feature for Rho-GEFs, is able to recognize membranes with a specific curvature, and localizes Hot1 to early endocytic structures. This is probably archived by the ability of the Hot1 BAR domain to form heterodimers with the BAR-domain protein Hob3, a regulator of early endocytosis and cell division. A main goal of this study was to investigate how Hot1 is able to specifically recognize its substrate, the GTPase Cdc42. Cdc42 and the closely related GTPase Rac1 show a high degree of sequence similarity, yet they appear to have functions in distinct signaling cascades since their deletion causes completely different phenotypes. In earlier works, it was shown that a number of RhoGEFs distinguishes between Cdc42 and Rac1 by a single amino acid at position 56 of these GTPases. The exchange of this residue between Cdc42 and Rac1 results in a changed recognition by the Cdc42-specific GEFs Don1 and Its1 and the Rac1-specific GEF Cdc24 in U. maydis. Also from other organisms it is known that the amino acid 56 is crucial for the specific recognition of Cdc42 and Rac1 by their respective RhoGEFs. Notably, this amino acid plays no role for the specific recognition of Cdc42 by Hot1. As the here presented work demonstrates, Hot1 , but also its mammalian homolog TUBA, recognizes Cdc42 by virtue of several specific amino acids at the N-terminus of Cdc42. Another aspect addressed by the work presented here is the question how specific signal transduction by small GTPases can be achieved in complex signaling networks. Cdc42 and Rac1 show a high degree of sequence similarity and have partially redundant functions. This poses the question which common functions these two proteins have and if there is a requirement that both proteins need to exist. In the course of this work, it was possible to generate a synthetic GTPase that embodies the functions of Cdc42 and Rac1 in a single protein. This GTPase can be activated by nearly all known GEFs for Cdc42 and Rac1 and can also replace Cdc42 and Rac1 in the majority of their cellular functions. Finally, it could be shown that the C-terminal region of Cdc42 and Rac1 is critical for the subcellular localization of these GTPases and is also required for the correct function of these proteins. This specific subcellular localization is critical for the correct signal transduction since an area of the C-terminus, the polybasic region, concentrates Cdc42 and Rac1 in certain subdomains of the membrane and thereby restricts and focusses the activity of these GTPases.