Scientists of the University of Crete found the way to prevent cancer?

Clks 1, 2 and 4 prevent chromatin breakage by regulating the Aurora B-dependent abscission checkpoint

by Eleni Petsalaki & George Zachos

When chromatin is trapped at the intercellular bridge, cells delay completion of cytokinesis (abscission) to prevent chromosome breakage. Here we show that inhibition of Cdc-like kinases (Clks) 1, 2 or 4 accelerates midbody resolution in normally segregating cells and correlates with premature abscission, chromatin breakage and generation of DNA damage in cytokinesis with trapped chromatin. Clk1, Clk2 and Clk4 localize to the midbody in an interdependent manner, associate with Aurora B kinase and are required for Aurora B–serine 331 (S331) phosphorylation and complete Aurora B activation in late cytokinesis.

Phosphorylated Aurora B–S331 localizes to the midbody centre and is required for phosphorylation and optimal localization of the abscission protein Chmp4c. In addition, expression of phosphomimetic mutants Aurora B–S331E or Chmp4c-S210D delays midbody disassembly and prevents chromatin breakage in Clk-deficient cells. We propose that Clks 1, 2 and 4 impose the abscission checkpoint by phosphorylating Aurora B–S331 at the midbody.

INTRODUCTION

Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles or daughter nuclei and have been linked to chromosomal instability in human tumours and tumourigenesis in mice. In response to chromatin bridges or to lagging chromosomes that are trapped in the intercellular bridge in late cytokinesis, eukaryotic cells delay abscission, the final cut of the narrow cytoplasmic canal that connects the daughter cells, to prevent chromosome breakage or tetraploidization by regression of the cleavage furrow. In mammals, this abscission delay is called ‘the abscission checkpoint’ and is dependent on Aurora B kinase. Aurora B localizes to the midbody and imposes the abscission checkpoint by phosphorylating the endosomal sorting complex required for transport-III (ESCRT-III) subunit charged multivesicular body protein 4C (Chmp4c) on serines 210, 214 and 215 in human cells. This phosphorylation has been proposed to target Chmp4c to the midbody centre, to prevent downstream endosomal sorting complex required for transport components including the ATPase Vps4 from relocalizing to the abscission site and deliver the final cut. In addition, in normally segregating cells, that is, in the absence of trapped chromatin at the intercellular bridge, inhibition of Aurora B accelerates abscission, suggesting that the abscission checkpoint may function more generally as an abscission timer. However, the mechanism of Aurora B activation in the abscission checkpoint is a matter of active investigation.

Complete Aurora B kinase activity requires phosphorylation at S331. The DNA damage kinases Chk1 and Chk2 phosphorylate Aurora B–S331 in mitosis: Chk2 phosphorylates Aurora B–S331 in early prometaphase, while Chk1 phosphorylates S331 in late prometaphase and metaphase. However, the kinase that activates Aurora B in the late stages of cytokinesis has not been previously reported.

The Cdc-like kinases (Clk1–4 in human cells) are an evolutionary conserved family of dual specificity protein kinases, which can autophosphorylate at tyrosine residues and phosphorylate their substrates on serine/threonine residues. Clks localize in the cytoplasm and in the nucleus where they regulate alternative splicing through phosphorylation of serine/arginine-rich domains on splicing factors. Clks recognize the minimum consensus sequence R-x-x-S/T also shared by Chk1 and Chk2; however, our current understanding of Clk biological targets and function is relatively limited.

In the present study, we show that depletion of Clk1, Clk2 or Clk4 by small interfering RNA (siRNA) or pharmacological inhibition of Clk catalytic activity accelerates midbody resolution in normally segregating human cells. Furthermore, Clk-deficient cells exhibit premature abscission, chromatin breakage and generation of DNA damage in cytokinesis with chromatin bridges. Clks 1, 2 and 4 phosphorylate Aurora B–S331 in vitro and are required for optimal Aurora B–phosphorylation and complete Aurora B activation in late cytokinesis. In addition, Clk1, Clk2 and Clk4 localize to the midbody in an interdependent manner and associate with Aurora B in cell extracts after enrichment of cells in cytokinesis. Using cells transiently expressing siRNA-resistant forms of wild-type (WT) or phosphomimetic S331E Aurora B after depletion of the endogenous protein, we propose that Clk-dependent Aurora B–S331 phosphorylation is required for phosphorylation and optimal localization of Chmp4c to the midbody centre in late cytokinesis, in the absence or the presence of DNA bridges. In addition, expression of S331E Aurora B or overexpression of the phosphomimetic mutant S210D Chmp4c delays midbody disassembly and prevents chromatin breakage in Clk-deficient cells. On the basis of these findings, we propose that Clk1, Clk2 and Clk4 impose the abscission checkpoint by phosphorylating Aurora B–S331 at the midbody.

RESULTS

Clk inhibition accelerates midbody disassembly

To investigate a role for Cdc-like kinases in midbody resolution, human colon carcinoma BE cells transiently expressing α-tubulin fused to mCherry (mCherry:tubulin) were monitored by time-lapse microscopy and the kinetics of tubulin disassembly at the midbody determined. In control cells, the midbody remained visible for a median time of 35±5min after formation. In contrast, treatment of cells with 1μM TG003, an inhibitor of Clk1, Clk2 and Clk4 catalytic activity at this concentration, accelerated midbody disassembly (t=18±3min, n=8) compared with controls. This correlated with reduced frequency of cells at midbody stage after treatment with TG003 or depletion of Clk1, Clk2 or Clk4 by two independent siRNAs, but not with an increase in binucleate or multinucleate cells compared with controls. Furthermore, Clk-deficient and control cells exhibited similar frequency of cells in prometaphase, suggesting that Clk inhibition does not prevent mitotic entry and that Clk-deficient cells can progress through abscission and disassemble their midbodies more rapidly than controls. We propose that Clks 1, 2 and 4 regulate proper timing of midbody resolution in normally segregating cells.

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