Iain Hagan - Cell Division

Protein Phosphatase 2A in cell division and signalling

Protein phosphorylation is used widely to change the flux through the signalling pathways that determine cell fate. Consequently, the protein kinases that execute these phosphorylation events are frequently hyper-activated in cancer through mutation or overexpression and kinase inhibitors are widely deployed in cancer therapy. Although the phosphatases that remove the phosphate put on targets by kinases are mutated as frequently as kinases, far less is known about their biology. 95% of the serine/threonine phosphatase activity of a human cell is executed by protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A). PP1, acts as a monomer that is recruited to defined docking motifs from where it dephosphorylates substrates. PP2A is a hetero-trimeric enzyme composed of single catalytic, structural and regulatory subunits. PP2A harbouring the B55 and B56 regulatory subunits execute many functions during cell division.

Commitment to mitosis is driven by Cdk1-Cyclin B kinase activation of a cohort of downstream kinases that drive chromosome condensation and formation of the mitotic spindle. Cyclin B is degraded once the chromosomes are correctly aligned in order to generate a state of low Cdk1-Cyclin B kinase activity that supports chromosome segregation into two daughter cells and exit from mitosis. These changes in protein kinase activity are coordinated with the equally important control of the counteracting protein phosphatase activities. Thus, while Cdk1-Cyclin B cannot drive cells into mitosis if the antagonistic PP2A-B55 phosphatase activity is not simultaneously repressed, PP2A-B55 activity is able to inappropriately drive cells out of division if Cdk1-Cyclin B activity declines below a critical threshold. Such inappropriate exit is known as mitotic slippage. The microtubule perturbing drug Taxol kills cells because it induces a sustained block to mitotic progression that triggers apoptosis to eliminate the cancerous cells. Some tumours resist killing by taxol because they slip out of mitosis before death can be triggered. Blocking PP2A-B55 activity in these cells would trap them in mitosis long enough to trigger death and so confer sensitivity to Taxol onto this cancer.

We have used fission yeast as a model system in which to address the complex question of protein phosphatase function in cell division. We identified an unanticipated control of PP2A-B55 and PP2A-B56 by PP1 that appears to be conserved in humans (Grallert, Boke et al. 2015 Nature 517:94-98). The activity of all three phosphatases is repressed upon mitotic commitment. PP1 is inhibited through direct phosphorylation by Cdk1-CyclinB. Inhibition of PP2A-B55 and PP2A-B56 holo-enzymes is relieved by PP1 to promote exit from mitosis. PP1 recruitment to each PP2A complex is regulated. For PP2A-B56, PP1 recruitment is controlled via phosphorylation within the PP1 docking site, however it is unclear how PP1 recruitment to PP2A-B55 is modulated. PP1 only binds PP2A-B55 from mitotic commitment until the chromosomes are segregated in anaphase.

This studentship will address the hypothesis that phosphorylation of the B55 subunit modulates PP1 affinity. The native locus encoding fission yeast B55 will be mutated to change sites at which we have found phosphorylation to alanine/valine in order to block phosphorylation. The impact of each mutation on PP1 affinity and mitotic progression will be assessed. Sites that change affinity will be mutated to glutamic and aspartic acid, to mimic constitutive phosphorylation, and the functional analyses repeated. Antibodies that only recognise B55 when phosphorylated will monitor phosphorylation on key residues as cultures progress synchronously through division and determine the impact of mutating the 106 kinases of fission yeast upon phosphorylation at this site. The impact of phosphorylation upon phosphatase activity will be assessed with established assays, while a combination of in vitro biochemistry and in vivo cell biology will determine how the identified kinase controls phosphorylation at this site in different signalling contexts.

Of the 8 sites of phosphorylation that we have identified on fission yeast B55, 7 are conserved in humans. Once the work in fission yeast has identified which sites play defining roles in controlling PP2A-B55 function, genome editing with CRISPR-cas9 technology will mutate the loci encoding B55 in h-TERT immortalised human RPE-1 diploid cells. The impact of phospho-mimetic and phospho-blocking mutations upon PP2A-B55 function will be monitored with a range of biochemical and cell biological assays. Antibodies that recognise the target site when phosphorylated will be generated to chart the timing of modification and identify the kinase responsible for phosphorylating the residue and interrogate the relevant signalling network in normal and transformed cells.