Universität zu Köln

Brenig, Julian (2015). Structural and mechanistic studies of the inhibitory effect of Liprin-alpha3 on mDia1 function. PhD thesis, Universität zu Köln.

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Abstract

Diaphanous-related formins (DRFs) are multi-domain proteins, that are involved in the nucleation and assembly of actin filaments. They are regulated by an autoinhibitory interaction of the C-terminal Diaphanous-autoregulatory domain (mDiaDAD) with the N-terminal Diaphanous-inhibitory domain (mDiaDID). Binding of active Rho proteins to the N-terminal domain leads to the release of the inhibitory state and plasma membrane localization of DRFs. Most recently, Liprin-alpha3 has been identified as a novel interaction partner of mDia1. It was shown, that overexpression of Liprin-alpha3 leads to a reduction in the amount of cellular F-actin and the translocation of mDia1 from the plasma membrane. The aim of this thesis was to structurally and mechanistically characterize the binding of mDia1 and Liprin-alpha3 and to investigate how the presence of Liprin-alpha3 influences the activity of mDia1. Comprehensive in vitro studies were performed to define a minimal Liprin-alpha3 fragment (Liprin-core region, LCR), that still binds to mDia1 and is able to induce a reduction in the amount of cellular F-actin. Using this fragment the interaction of Liprin-alpha3 and mDia1 was also characterized structurally. Presence of Liprin-alpha3 reduced the binding affinity of RhoA to mDiaN more efficiently than the binding of mDiaDAD to mDiaN, as determined by thermodynamic and kinetic analysis. Furthermore, the structural data revealed, that the dissociation of Liprin-alpha3 from mDia1 by RhoA is mediated allosterically, while mDiaDAD dissociates Liprin-alpha3 by competing for the a highly overlapping binding site on mDiaN. Additionally, overexpression of Liprin-alpha3 in HeLa and N2a cells displayed differences in Liprin-alpha3 localization and the reduction of the cellular amount of F-actin. This indicated the importance of cell-type dependent mechanisms that regulate the function of Liprin-alpha3. Based on the data obtained in this study the following mechanisms explaining the inhibition of mDia1 by Liprin-alpha3 were postulated. Firstly, the re-establishment of the mDia1 autoinhibitory state is supported by the altered binding affinities of RhoA and mDiaDAD for mDiaN. Secondly, RhoGAPs can compete more efficiently with mDiaN for RhoA binding. This leads to the downregulation of RhoA activity due to enhanced GTP hydrolysis and subsequently the inactivation of mDia1. Thirdly, Liprin-alpha3 inhibits the interactions of mDia1 with additional activating proteins, besides RhoA, and is able to recruit further regulatory proteins of mDia1 function. In conclusion, this thesis presents a model for the inhibition of mDia1 function by Liprin-alpha3, emphasizing the impact of the scaffold protein Liprin-alpha3 on the regulation of the actin cytoskeleton.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Brenig, Julian UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-65630
Date:	2015
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language Liprin-alpha; mDia; Formins; actin cytoskeleton English Liprin-alpha, mDia, Formine, Aktinzytoskelett German
Date of oral exam:	4 December 2015
Referee:	Name Academic Title Lammers, Michael Dr. Hofmann, Kay Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/6563