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Research Interest

 

A Drosophila melanogaster cell at anaphase.

 

 

 

 

 

How does proteolysis of different substrates contribute to the success of cytokinesis?

The pathways and machinery of proteolysis in all eukaryotes centre on the small protein ubiquitin, that is used to tag other proteins for immediate destruction by the proteasome. A series of enzymes known as the ubiquitin-proteasome system (UPS) 'primes' ubiquitin then conjugates it onto target proteins, with the downstream E3 activity (or ubiquitin ligase) acting to mediate recognition of the target protein. Multiple rounds of tagging with ubiquitin gives rise to poly-ubiquitin chains that are recognized and bound by the proteasome. The target protein is then fed into the proteasome core and cleaved by the proteolytic enzymes that reside there (Fig 1).

Poly-ubiquitin conjugates are difficult to purify, not least because they are inherently unstable but also because the poly-ubiquitinated conjugates of a given protein usually represent only a small fraction of the total pool of that protein in the cell (by our calculation less than 1%, see Fig 3).We have developed novel tools for purification of ubiquitinated proteins from dividing and synchronized human cells, in collaboration with Dr Ugo Mayor who pioneered this approach in Drosophila. The approach uses in vivo biotinylation of ubiquitin to label ubiquitin conjugates that can then be purified on streptavidin under highly stringent conditions (Fig 2). Analysis of purified proteins by mass spectrometry has allowed us to identify many proteins targeted for poly-ubiquitination during exit from mitosis, and to identify sites of ubiquitination on target proteins.

We are currently studying newly identified targets of poly-ubiquitination, using time-lapse microscopy in living cells to understand exactly when these target proteins are destroyed, and how their proteolytic destruction contributes to the sequence of steps that comprises cytokinesis. We are finding that the majority of poly-ubiquitination that occurs at this time is mediated by one particular E3, called the anaphase-promoting complex (APC/C). We are also carrying out bioinformatic studies of sites of ubiquitination on target proteins of the APC/C to extract principles of substrate recognition by the proteolytic machinery.

 



 

How is substrate proteolysis regulated in space and time?

We are pursuing this question through detailed studies of Aurora A and Aurora B kinases. Both are essential regulators of mitosis, closely related kinases that show distinct patterns of localization and function during cell division (Fig 1). They both

 

 

 

 

 

 

 

 

 

 

 

undergo proteolysis during mitotic exit (Fig 2). The relationships between their different binding partners, functions and proteolysis of these kinases are not well understood. However, forced expression of either Aurora kinase can lead to failure of cytokinesis and generation of aneuploid cells (characteristic of aggressive cancers) showing that these relationships are important for the stability of the genome.

In one project we are studying how APC/C-mediated proteolysis of Aurora B contributes to its dynamic localization and functions during mitotic exit. A further comparative study of ubiquitination and proteolysis of the Aurora kinases has enabled us to identify critical molecular determinants of the stability of these kinases. Aurora A is strongly linked with several types of cancers and is regarded as a particularly promising target for therapeutic intervention. Therefore we aim to describe molecular events in the turnover of Aurora A that can have value in cancer diagnostics and treatment. Finally, in a collaborative project with the group of Professor David Glover we are carrying out a systematic identification of Aurora A binding partners at different times during the cell cycle, in both human cells and Drosophila embryos. A more complete understanding of how specific Aurora A functions are regulated can lead to better targeted interventions in cancer.

 

 

References

1. Franco M, Seyfried NT, Brand AH, Peng JM, Mayor U. A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development. Molecular & Cellular Proteomics 2011; 10: M110.002188.
2. Pines J. Mitosis: a matter of getting rid of the right protein at the right time.Trends in Cell Biology 2006; 16: 55-63.
3. Floyd S, Pines J, Lindon C. APC/C-Cdh1 Targets Aurora Kinase to Control Reorganization of the Mitotic Spindle at Anaphase. Current Biology 2008; 18: 1649-1658.
4. Carmena M, Earnshaw WC. The cellular geography of aurora kinases. Nature Reviews Molecular Cell Biology 2003; 4: 842-854.