Abstract
The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is known to have a critical role in DNA double-strand break repair. We have previously reported that DNA-PKcs is activated when cells enter mitosis and functions in mitotic spindle assembly and chromosome segregation. Here we report that DNA-PKcs is the upstream regulator of the Chk2–Brca1 pathway, which impacts microtubule dynamics, kinetochore attachment and chromosomal segregation in mitosis. Downstream from Chk2, Brca1 promotes monoubiquitination of γ-tubulin to inhibit microtubule nucleation and growth. We found that DNA-PKcs is essential for mitotic Chk2 phosphorylation at Thr68. As in Chk2- and Brca1-deficient cells, loss of DNA-PKcs resulted in chromosome misalignment and lagging during anaphase owing to elevation in microtubule dynamics. Importantly, these mitotic aberrations in DNA-PKcs-defective cells were alleviated by the overexpression of phosphomimetic Chk2 or Brca1 mutant proteins but not their wild-type counterparts. Taken together, these results demonstrate that DNA-PKcs regulates mitotic spindle organization and chromosomal instability via the Chk2–Brca1 signaling pathway.
Highlights
Chromosome instability (CIN), characterized by increased frequency of gain or loss of the whole chromosomes, has long been implicated in tumorigenesis, poor patient prognosis and drug resistance.[1]
We report that DNA-PKcs is the upstream regulator of the Chk2–Brca[1] pathway, which impacts microtubule dynamics, kinetochore attachment and chromosomal segregation in mitosis
These results demonstrate that DNA-PKcs mediates Chk[2] phosphorylation to regulate microtubule nucleation and the spindle damage response
Summary
Chromosome instability (CIN), characterized by increased frequency of gain or loss of the whole chromosomes, has long been implicated in tumorigenesis, poor patient prognosis and drug resistance.[1] The most common cause of CIN in cancer cells is a defect in the dynamics of kinetochore-microtubule (k-MT) attachment during mitosis.[2] An evolutionarily conserved spindle assembly checkpoint mechanism has evolved to ensure proper completion of all k-MT attachments before chromosomal segregation.[3] Certain improper k-MT attachments (for example, merotelic attachment with microtubules orientated from both spindle poles) can escape from spindle assembly checkpoint surveillance and persist into the lagging chromosome; these are observed at the spindle midzone during anaphase.[2] Abnormal k-MT attachments occur frequently in early mitosis but are efficiently corrected in normal cells.[4] In cancer cells with CIN, stability of k-MT attachments increases, hindering the ability to correct the abnormal k-MT attachment.[5] In addition, abnormal spindle assembly with extra centrosomes significantly increases the frequency of lagging chromosomes during anaphase, indicating that centrosome instability is a major source of abnormal k-MT attachment.[6] Centrosome hypertrophy is an accepted cause of CIN and is frequently correlated with tumorigenesis.[7] centrosome instability is highly linked to certain tumor suppressor protein mutations (for example, those in Brca1).[8,9]
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