Src activation by Chk1 promotes actin patch formation and prevents chromatin bridge breakage in cytokinesis

In cytokinesis with chromatin bridges, cells delay abscission and retain actin patches at the intercellular canal to prevent chromosome breakage. In this study, we show that inhibition of Src, a protein-tyrosine kinase that regulates actin dynamics, or Chk1 kinase correlates with chromatin breakage and impaired formation of actin patches but not with abscission in the presence of chromatin bridges. Chk1 is required for optimal localization and complete activation of Src. Furthermore, Chk1 phosphorylates human Src at serine 51, and phosphorylated Src localizes to actin patches, the cell membrane, or the nucleus. Nonphosphorylatable mutation of S51 to alanine reduces Src catalytic activity and impairs formation of actin patches, whereas expression of a phosphomimicking Src-S51D protein rescues actin patches and prevents chromatin breakage in Chk1-deficient cells. We propose that Chk1 phosphorylates Src-S51 to fully induce Src kinase activity and that phosphorylated Src promotes formation of actin patches and stabilizes chromatin bridges. These results identify proteins that regulate formation of actin patches in cytokinesis

Phosphorylation at serine 331 is required for Aurora B activation

Chk1 phosphorylates Aurora B to promote its full activation during mitotic prometaphase

Chk1 is required for correction of merotelic attachments during mitotic cell division

Merotelic kinetochore attachments can induce mis-segregated chromosomes in anaphase. In the present study, we show that Chk1 kinase, a component of the DNA damage and DNA replication responses, protects vertebrate cells against merotelic attachments and lagging chromosomes and regulates correction of merotelic attachments during a prolonged metaphase. Decreased Chk1 activity leads to hyperstable kinetochore-microtubules, unstable binding of MCAK, Kif2b and Mps1 to centromeres or kinetochores and reduced phosphorylation of Hec1 by Aurora-B kinase. Chk1 phosphorylates Aurora-B Serine 331 at kinetochores and this phosphorylation is high in prometaphase and decreases significantly when chromosomes bi-orient. We propose that phosphorylation of Serine 331 by Chk1 is required for localization of MCAK, Kif2b and Mps1 to centromeres or kinetochores and for Hec1 phosphorylation. Furthermore, inhibition of Mps1 activity diminishes initial recruitment of MCAK and Kif2b to centromeres or kinetochores, impairs mitotic Hec1 phosphorylation and exacerbates merotelic attachments in the absence of Chk1 function. We propose that Chk1 and Mps1 jointly regulate Aurora-B, MCAK, Kif2b and Hec1 functions to correct merotelic attachments. These results suggest a previously unreported role for Chk1 and Mps1 in error correction.Αν δεν επιδιορθωθούν, οι μεροτελικές προσδέσεις μπορούν να οδηγήσουν στο λανθασμένο διαχωρισμό των χρωμοσωμάτων στην ανάφαση. Στην παρούσα διατριβή, δείχνουμε ότι η κινάση Chk1 προστατεύει τα κύτταρα των σπονδυλωτών από τις μεροτελικές προσδέσεις και τα χρωμοσώματα που καθυστερούν και απαιτείται για επιδιόρθωση των μεροτελικών προσδέσεων κατά τη διάρκεια παρατεταμένης μετάφασης. Μειωμένη ενεργότητα της Chk1 οδηγεί σε σταθερούς μικροσωληνίσκους των κινητοχώρων, ασταθή πρόσδεση των MCAK, Kif2b και Mps1 στα κεντρομερή ή στους κινητοχώρους και μειωμένη φωσφορυλίωση της Hec1 από την Aurora-B. Η φωσφορυλίωση της Aurora-B στη σερίνη 331 από την Chk1 είναι υψηλή στην προμετάφαση και μειώνεται σημαντικά στη μετάφαση. Προτείνουμε ότι η φωσφορυλίωση στη σερίνη 331 είναι απαραίτητη για τον εντοπισμό των MCAK, Kif2b και Mps1 στα κεντρομερή ή στους κινητοχώρους και για τη φωσφορυλίωση της Hec1. Επιπροσθέτως, αναστολή της ενεργότητας της Mps1 μειώνει την αρχική πρόσδεση των MCAK και Kif2b στα κεντρομερή ή στους κινητοχώρους, ελαττώνει τη φωσφορυλίωση της Hec1 και προκαλεί μεροτελικές προσδέσεις στα κύτταρα με μειωμένη Chk1. Προτείνουμε ότι η Chk1 και η Mps1 ρυθμίζουν από κοινού τις Aurora-B, MCAK, Kif2b και Hec1 προκειμένου να επιδιορθωθούν οι μεροτελικές προσδέσεις. Αυτά τα αποτελέσματα υποδεικνύουν ένα ρόλο της Chk1 και της Mps1 στην επιδιόρθωση των λανθασμένων προσδέσεων

An ATM-CHK2-INCENP pathway prevents chromatin breakage by regulating the abscission checkpoint

In response to chromatin bridges, the chromosomal passenger complex (CPC) delays completion of cytokinesis (abscission) to prevent chromosome breakage. Here, we discuss recent findings from our lab showing that an ATM-CHK2-INCENP pathway imposes the abscission checkpoint in human cells by regulating CPC midbody-localization

CHMP4C: A novel regulator of the mitotic spindle checkpoint

The mitotic spindle checkpoint delays anaphase onset until all chromosomes have achieved stable kinetochore-microtubule attachments. Here, we discuss recent findings showing that CHMP4C, a component of the endosomal sorting complex required for transport (ESCRT) machinery, protects human cells against chromosome missegregation by promoting localisation of the ROD–ZW10–ZWILCH (RZZ) spindle checkpoint complex to unattached kinetochores

Chk1 protects against chromatin bridges by constitutively phosphorylating BLM serine 502 to inhibit BLM degradation

Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Bloom's syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM