UBIMAPS · Ubiquitin-dependent regulation of the microtubule cytoskeleton
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2017-11-01 → 2020-03-02
- EU contribution
- €159,461
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
Ubiquitin-dependent regulation of the microtubule cytoskeleton
Background Ubiquitylation of proteins is catalyzed by ubiquitin E3 ligases and frequently leads to subsequent degradation of target proteins via the proteasome. Using comprehensive substrate screening on protein microarrays, we identified several microtubule-associated proteins as substrates of the multi-subunit ubiquitin E3 ligase SCFFbxw5 in vitro implicating its crucial role in the regulation of microtubules. After initial confirmation of these targets, our work focused on Kif2c/MCAK (mitotic centromere-associated kinesin), a microtubule depolymerase that plays important roles in mitosis. Medical relevance In agreement with its pivotal function in cell division, overexpression of Kif2c is frequently observed in different cancer types and is often associated with poor patient prognosis. It has been proposed that via its microtubule depolymerase activity, Kif2c overexpression may counteract the activity of the broadly applied anti-cancer drug paclitaxel (taxol) – a microtubule stabilizing agent. Hence, by elucidating a potential ubiquitylation and degradation of Kif2c by SCFFbxw5, our work may eventually contribute to the development of potential new therapy approaches for tumors overexpressing Kif2c. Objectives We first aimed at validating proteins identified in the screen as substrates of SCFFbxw5 (WP1) in order to focus on one specific target protein afterwards. Using different in vitro and in cellulo approaches we tried to identify binding sites on substrate for Fbxw5 and sought to elucidate the physiological role of the ubiquitylation reaction (WP2). In the last part, we wanted to address temporal and spatial characteristics of the SCFFbxw5-dependent regulation of the substrate and the molecular mechanism behind such a potential confinement (WP 3). Conclusions By combining different in vitro and in cellulo assays, we could establish Kif2c as a bona fide substrate of SCFFbxw5 in cells. In addition, we could demonstrate that the SCFFbxw5 targets Kif2c in the G2 stage of the cell cycle and that this process is required for subsequent ciliogenesis later in G0/G1. Importantly, knock down of Fbxw5 displayed also a severe synthetic lethality with Kif2c overexpression indicating a new therapeutic potential of Kif2c overexpressing cancer cells.
Data: CORDIS, © European Union
Project objective
The microtubule cytoskeleton is a dynamic array of cylindrical polymers that play important roles in various cellular processes including chromosome segregation and ciliogenesis. Hundreds of accessory factors – grouped under the term microtubule-associated proteins (MAPs) – orchestrate this network by governing the interaction profile and growth behaviour of microtubules. Due to this critical function, the precise abundance and distribution of MAPs within cells is of high importance and their deregulation is associated with various human diseases, such as ciliopathies and cancer. Surprisingly little is known about mechanisms involved in the regulation of MAPs and so far only a few of them have been identified as targets of the major proteolytic control machinery in cells, i.e. ubiquitin-proteasome system. In this work, I will further explore how MAPs are regulated by ubiquitin-dependent degradation by focussing on a multi-subunit ubiquitin E3 ligase called SCFFbxw5 that seems to play an important role in microtubule control according to preceding experiments of the host lab. Hence, I will investigate in detail two MAPs that were identified as substrates of SCFFbxw5 by the host lab in order to unravel the functional, temporal, and spatial characteristics of their ubiquitylation by SCFFbxw5. Since these MAPs are known to fulfil crucial functions in ciliogenesis and chromosome segregation, this work will promote our knowledge about how ubiquitin-dependent degradation contributes to these processes and may open up novel therapeutic strategies with the associated diseases.
Original text from CORDIS.
Participants
- RUPRECHT-KARLS-UNIVERSITAET HEIDELBERG · HeidelbergCoordinatorGermany
Links
Data: CORDIS, © European Union