Service Communautaire d'Information sur la Recherche et le Développement - CORDIS

FP7

APCACTIVATION Résumé de rapport

Project ID: 328641
Financé au titre de: FP7-PEOPLE
Pays: United Kingdom

Final Report Summary - APCACTIVATION (Deciphering the dynamics and molecular mechanisms of APC/C activity in vitro and in live cells)

The anaphase promoting complex or cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase, ensures unidirectionality of mitotic progression by targeting several mitotic proteins for degradation. Despite its central role in mitotic progression, the molecular mechanisms activating the APC/C towards different substrates are poorly defined. The aim of this project was to decipher the dynamics and molecular mechanisms of APC/C activity in live cells and in vitro.
I Measuring APC/C-dependent ubiquitylation dynamics in live cells

In order to characterise APC/C-dependent ubiquitylation activity in live cells, we proposed to establish new tools and protocols to quantify Foerster Resonance Energy Transfer (FRET) between Cyclin B1 tagged with a FRET donor fluorophore, and ubiquitin tagged with an acceptor fluorophore.
We created human RPE1 cell lines expressing endogenously tagged Cyclin B1-Venus (1) and mKate2-Ubiquitin ectopically expressed from an inducible promoter. In parallel, we expressed and purified recombinant mKate2-Ubiquitin. Ubiquitylation assays using APC/C pulled down from mitotic HeLa cell extracts verified that mKate2-Ubiquitin was incorporated into ubiquitylation products in vitro. In collaboration with Clemens Kaminski at the Department of Chemical Engineering and Biotechnology, University of Cambridge, we set up protocols to quantify FRET by sensitized emission (2).
Regrettably, after setting up the system, none of the different mKate2-linker-Ubiquitin constructs allowed for the detection of an increase in FRET efficiency between Cyclin B1-Venus and mKate2-Ubiquitin during mitotic progression. In order to exclude the possibility that the rapid degradation of ubiquitylated products impeded the detection of FRET, we quantified FRET between Cyclin B1-Venus and mKate2-Ubiquitin in mitotic cells treated with the proteasome inhibitor MG132, after release of the spindle assembly checkpoint (SAC) by Reversine, a chemical inhibitor of the SAC kinase Mps1. Despite these efforts to increase the sensitivity of the system, we could not measure a significant increase in FRET efficiency after inhibition of the SAC.
Therefore, the system we developed does not seem to be suitable to quantify substrate ubiquitylation in live cells. One potential problem of our approach is the hardly characterised activity of deubiquitylating proteins (DUBs), which shortens the half-life of ubiquitylation products even in the absence of proteasome activity. Another possible limitation are geometrical constraints in the structure of ubiquitylation products that may not allow for an orientation of donor and acceptor fluorophores that favours FRET. Due to these reasons, we had to divert from the workflow outlined in our research proposal. As we got very promising results for the second part of the project (see below), I concentrated on these experiments during the last months of my fellowship.

II Reconstituting APC/C activity in vitro

Next to setting up a system to measure APC/C-dependent ubiquitylation in live cells, the other aim of this project was to dissect how individual subunits, cofactors, E2 enzymes and post-translational modifications (PTMs) contribute to APC/C activity by reconstituting the APC/C in vitro from recombinant proteins. As outlined in the original research proposal, we are collaborating with David Barford at the MRC LMB, Cambridge, whose lab recently succeeded in expressing and purifying recombinant human APC/C (3-4).
At the start of this project, we obtained purified recombinant human APC/C from David Barford’s laboratory and created all other components required to reconstitute APC/C activity in vitro. Specifically, we generated different fluorescently labelled substrates to compare the ubiquitylation activity of the recombinant APC/C towards checkpoint-independent (Cyclin A2), checkpoint-dependent (Cyclin B1 and Securin) and late mitotic substrates (Aurora A). We further generated different recombinant co-factors and E2 enzymes, and established protocols to quantify and compare APC/C kinetics and processivity.
In accordance with the milestones set in our proposal, we tested the requirement of different co-factors for substrate ubiquitylation by the APC/C. These experiments revealed that recombinant Cdc20 does not activate recombinant APC/C, in contrast to recombinant Cdh1. Mutating individual or several Cdk1-dependent phosphorylation sites on Cdc20 (5-7) did not alter its activity towards recombinant APC/C.
In collaboration with Barbara Di Fiore, a postdoc in our lab, we started to address to decipher how the mitotic checkpoint complex (MCC) composed of BubR1, Cdc20, Bub3 and Mad2, inhibits the APC/C. We created recombinant MCC and mutated recently identified binding motifs for Cdc20 (8 and unpublished data) to test the impact of these mutations on the inhibition of the APC/C by recombinant MCC. Interestingly, we found that mutation of two but not all of these binding sites abrogates the potential to inhibit the APC/C. The results from these experiments will be included in a publication characterising the binding motifs in more detail.

References

1. Mansfeld, J., Collin, P., Collins, M. O., Choudhary, J. S. & Pines, J. APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nat. Cell Biol. 13, 1234–1243 (2011).
2. Elder, A. D. & Domin, A. A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission. … Interface (2009).
3. Chang, L., Zhang, Z., Yang, J., McLaughlin, S. H. & Barford, D. Molecular architecture and mechanism of the anaphase-promoting complex. Nature 513, 388–+ (2014).
4. Zhang, Z. et al. Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C). Biochem. J. 449, 365–371 (2013).
5. Labit, H., Fujimitsu, K., Bayin, N. S. & Takaki, T. Dephosphorylation of Cdc20 is required for its C‐box‐dependent activation of the APC/C. The EMBO … (2012).
6. Kramer, E. R., Scheuringer, N., Podtelejnikov, V., Mann, M. & Peters, J. M. Mitotic regulation of the APC activator proteins CDC20 and CDH1. Mol. Biol. Cell 11, 1555–1569 (2000).
7. Yudkovsky, Y., Shteinberg, M., Listovsky, T., Brandeis, M. & Hershko, A. Phosphorylation of Cdc20/Fizzy negatively regulates the mammalian cyclosome/APC in the mitotic checkpoint. Biochem Biophys Res Commun 271, 299–304 (2000).
8. Di Fiore, B., Davey, N.E., Hagting, A., Izawa, D., Mansfeld, J., Gibson, T.j., Pines, J. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators. Dev Cell 32(3), 358-372 (2015).

Informations connexes

Contact

Renata Schaeffer, (EC Policy Manager)
Tél.: +44 1223 333543
Fax: +44 1223 332988
E-mail
Numéro d'enregistrement: 182624 / Dernière mise à jour le: 2016-05-11
Source d'information: SESAM