In addition to DNA damage response, DNA-PKcs are elicited spontaneously upon mitosis entrance and plays a critical role in maintaining chromosomal stability and cell cycle progression through mitosis.
DNA-PKcs is spontaneously activated and phosphorylated by itself in mitosis and is critical for cell cycle progression through mitosis. DNA-PKcs phosphorylation occurs at both centrosomes and kinetochores, suggesting that it plays an important role in regulating microtubule dynamics and chromosomal segregation. Indeed, in cells lacking DNA-PKcs or treated with DNA-PKcs inhibitor, there is a delay in mitotic transition due to dysregulation of mitotic spindle formation and defects in chromosomal alignment/segregation.
Mitotic activation of DNA-PKcs is required for phosphorylation of downstream target proteins, including Chk2 kinase and heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1). DNA-PKcs-dependent Chk2 phosphorylation promotes the Chk2-Brca1 signaling pathway, which is known to impact microtubule dynamics and chromosomal segregation during mitosis, and influences tumor response to microtubule inhibitor (e.g. Taxane) therapy. Whereas DNA-PKcs-dependent hnRNP-A1 phosphorylation stimulates its binding to single-stranded telomeric DNA, this displaces RPA from newly replicated telomeres to allow telomere-cap formation. Disruption of DNA-PKcs dependent hnRNP-A1 regulation results in significant elevation of fragile telomeres, including telomere-associated γH2AX foci and sister telomere fusions.
Our further investigations reveal that deficiency in DNA-PKcs activity causes a delay in mitotic entry due to dysregulation of key mitotic kinases, including Cdk1 and Plk1 kinases. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs-deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases, but also required for cellular resistance to mitotic stress.