Co-ordinated control of the Aurora B abscission checkpoint by PKCε complex assembly, midbody recruitment and retention.

Lisa Watson,Ulrike Eggert,Joanna Kelly,Victoria Crossland,Khalil Davis,Nicola Lockwood,John D Scott,Neil Q Mcdonald,Xiaoping Yang,Steven Lynham,Peter J Parker,Alan Armstrong,Tanya N Soliman,David J Mann

doi:10.1042/bcj20210283

Abstract

A requirement for PKCε in exiting from the Aurora B dependent abscission checkpoint is associated with events at the midbody, however, the recruitment, retention and action of PKCε in this compartment are poorly understood. Here, the prerequisite for 14-3-3 complex assembly in this pathway is directly linked to the phosphorylation of Aurora B S227 at the midbody. However, while essential for PKCε control of Aurora B, 14-3-3 association is shown to be unnecessary for the activity-dependent enrichment of PKCε at the midbody. This localisation is demonstrated to be an autonomous property of the inactive PKCε D532N mutant, consistent with activity-dependent dissociation. The C1A and C1B domains are necessary for this localisation, while the C2 domain and inter-C1 domain (IC1D) are necessary for retention at the midbody. Furthermore, it is shown that while the IC1D mutant retains 14-3-3 complex proficiency, it does not support Aurora B phosphorylation, nor rescues division failure observed with knockdown of endogenous PKCε. It is concluded that the concerted action of multiple independent events facilitates PKCε phosphorylation of Aurora B at the midbody to control exit from the abscission checkpoint.

Highlights

Checkpoints acting through the cell cycle serve as critical safeguards of genome integrity and determine the timing of progression
We previously reported that knockdown of endogenous PKCε or expression of mutants which could not form a PKCε/14-3-3 complex induced a telophase delay, failing cytokinesis resulting in an accumulation of binucleated cells [27]
The investigation here indicates that there are at least three independent elements to this, each of which appears to be necessary to drive the PKCε dependent Aurora B S227 phosphorylation at the midbody required for checkpoint exit, protecting from division failure and consequent binucleation

Summary

Introduction

Checkpoints acting through the cell cycle serve as critical safeguards of genome integrity and determine the timing of progression Amongst these is the Aurora B (S/T protein kinase) dependent abscission checkpoint that acts at the ultimate stage of cell division to control the timing of the final act of division and the separation of presumptive daughter cells (recently reviewed [1,2]). This checkpoint was originally identified in budding yeast and termed the NoCut Pathway [3], requiring Ipl (Aurora) to delay cytokinesis, so protecting from chromosome breakage and likely associated with chromatin in the midzone. The relationship to the abscission machinery is afforded by the Aurora B phosphorylation of CHMP4C at the midbody [5] and the ANCHR-dependent sequestration of Vps4 [7]

Methods

Results

Conclusion