The Aurora B specificity switch is required to protect from non-disjunction at the metaphase/anaphase transition

Abstract

The Aurora B abscission checkpoint delays cytokinesis until resolution of DNA trapped in the cleavage furrow. This process involves PKCε phosphorylation of Aurora B S227. Assessing if this PKCε-Aurora B module provides a more widely exploited genome-protective control for the cell cycle, we show Aurora B phosphorylation at S227 by PKCε also occurs during mitosis. Expression of Aurora B S227A phenocopies inhibition of PKCε in by-passing the delay and resolution at anaphase entry that is associated with non-disjunction and catenation of sister chromatids. Implementation of this anaphase delay is reflected in PKCε activation following cell cycle dependent cleavage by caspase 7; knock-down of caspase 7 phenocopies PKCε loss, in a manner rescued by ectopically expressing/generating a free PKCε catalytic domain. Molecular dynamics indicates that Aurora B S227 phosphorylation induces conformational changes and this manifests in a profound switch in specificity towards S29 TopoIIα phosphorylation, a response necessary for catenation resolution during mitosis.

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics

“…Although CDK complexes such as Cyclin B-CDK1 phosphorylate a plethora of crucial M-phase substrates, containing minimal [S/T]P and full consensus [S/T]PXX[L/R] motifs [94][95][96][97], it is now clear that at least one CDK enzyme complex can also phosphorylate non-[S/T]P consensus motifs [98], operating through a combined 'multisite' code [99], differential substrate co-localisation and ordered phosphorylation that is dependent on substrate affinity [100,101]. Similar findings have also been made with the abscission-controlling PKCε-Aurora B complex, whose substrate specificity can switch at different phases of the cell cycle [102]. In the context of PLK4, further studies with full-length proteins are justified to examine whether PLK4 substrate specificity might be altered depending upon the subcellular repertoire of regulatory proteins/substrates formed.…”

A cancer-associated, genome protective programme engaging PKCε

“…1 ). It has been shown that physiologically, caspase activation can also drive V3 domain cleavage ( Basu et al, 2002 ) and this turns out to be central to an M-Phase engagement of PKCε (see further below ( Kelly et al, 2020 )). A second non-canonical activation pathway for PKCε involves the assembly of a PKCε-14-3-3 complex ( Saurin et al, 2008 ).…”

“…More recently the mechanism of PKCε engagement in this process has been addressed and this is of particular interest given the chromatin-associated changes involved in the SAC silencing delay and the conventional activation of PKCε in membrane compartments (see above). Notably in the context of this compartmental issue, it has been reported that there is a constitutive cell cycle dependent activation of PKCε in a chromatin sub-compartment that is effected by V3 domain cleavage ( Kelly et al, 2020 ). Proteolysis occurs principally through Caspase 7 acting at Asp383 (the critical, activating V3 domain site) and Asp451 (kinase domain site), in line with earlier caspase site mapping work ( Basu et al, 2002 ).…”

“…Proteolysis occurs principally through Caspase 7 acting at Asp383 (the critical, activating V3 domain site) and Asp451 (kinase domain site), in line with earlier caspase site mapping work ( Basu et al, 2002 ). This non-apoptotic caspase action is absolutely required for the delay to anaphase entry and mutation of the PKCε Asp383 caspase cleavage site blocks any delay to anaphase onset in a manner that is artificially reversible through regulated cleavage of an engineered TEV site or via expression of the kinase domain of PKCε but not the full length non-cleavable protein ( Kelly et al, 2020 ).…”

“…The kinase domain generated in this membrane-independent M-phase process triggers downstream events that act to promote resolution of chromatin catenation, based on the extent of retained sister chromatid catenation, and also to impose the delay to checkpoint silencing, as determined by the timing of anaphase entry ( Kelly et al, 2020 ). Interestingly, an essential element in the actions of PKCε is again the phosphorylation of Aurora B at Ser277 (as at cytokinesis; see above) and in this context it was shown that this is critical to the activation of Topo2A through a switch in Aurora B phosphorylation of Topo2A ( Kelly et al, 2020 ). There are however other targets that are necessary for this arrest/resolution function based upon a genetic code expansion-based substrate screen and knock-down (Davis, Martini and Parker unpublished).…”