Targeting the INCENP IN-box-Aurora B interaction to inhibit CPC activity in vivo.

Florence H Gohard,Mike Tyers,William C Earnshaw,Daniel J St-Cyr

doi:10.1098/rsob.140163

Florence H Gohard, Mike Tyers + Show 2 more

Open Access

https://doi.org/10.1098/rsob.140163

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Abstract

The chromosome passenger complex (CPC) is an essential regulator of mitosis and cytokinesis. The CPC consists of Aurora B kinase, inner centromere protein (INCENP), and the targeting subunits survivin and borealin/Dasra B. INCENP is a scaffolding subunit for the CPC and activates Aurora B via its conserved IN-box domain. We show that overexpression of soluble IN-box in HeLa cells affects endogenous CPC localization and produces a significant increase in multinucleated and micronucleated cells consistent with CPC loss of function. The dominant-negative effect of soluble IN-box expression depends on residues corresponding to hINCENP W845 and/or F881, suggesting that these are essential for Aurora B binding in vivo. We then screened a targeted library of small (five to nine residues long) circular peptide (CP) IN-box fragments generated using split intein circular ligation of proteins and peptides (SICLOPPS) methodology. We identified a number of CPs that caused modest but reproducible increases in rates of multinucleated and micronucleated cells. Our results provide proof of concept that inhibition of the Aurora B–IN-box interaction is a viable strategy for interfering with CPC function in vivo.

Highlights

The chromosomal passenger complex (CPC), which consists of Aurora B kinase and the three non-enzymatic subunits inner centromere protein (INCENP) [1], survivin [2,3] and borealin/Dasra-B [4,5] orchestrates key events during mitosis [6]
As the interaction between INCENP and Aurora B is essential for activation and localization of the kinase, we have investigated the use of peptides and cyclic peptides to disrupt this interaction
We opted to the use the naturally split DnaE intein from the cyanobacterium Synechocystis species PCC6803 (Ssp) [31] because it has been used in previous SICLOPPS library applications in prokaryotes and lower eukaryotes (e.g. [32,33])

Summary

Introduction

The chromosomal passenger complex (CPC), which consists of Aurora B kinase and the three non-enzymatic subunits inner centromere protein (INCENP) [1], survivin [2,3] and borealin/Dasra-B [4,5] orchestrates key events during mitosis [6]. The C-terminus of INCENP contains the IN-box domain, which binds and activates Aurora B [9,10,11,12,13]. Upon INCENP binding, Aurora B phosphorylates a conserved threonine–serine–serine (TSS) motif located near the C-terminus of the IN-box [12,13]. This promotes CPC clustering, leading to trans phosphorylation of threonine 232 on the Aurora B activation loop and full kinase activation [14]

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