XB-ART-56885Mol Cell Biol January 1, 2020; 40 (13):
Nocodazole-Induced Expression and Phosphorylation of Anillin and Other Mitotic Proteins Are Decreased in DNA-Dependent Protein Kinase Catalytic Subunit-Deficient Cells and Rescued by Inhibition of the Anaphase-Promoting Complex/Cyclosome with proTAME but Not Apcin.
The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121. Moreover, reduced nocodazole-induced expression of anillin, securin, and cyclin B1 and phosphorylation of PLK1, Aurora A, and TPX2 were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) by proTAME, which prevents binding of the APC/C-activating proteins Cdc20 and Cdh1 to the APC/C. Altogether, our studies suggest that loss of DNA-PKcs prevents inactivation of the APC/C in nocodazole-treated cells.
PubMed ID: 32284347
PMC ID: PMC7296215
Article link: Mol Cell Biol
Genes referenced: anln atm atr aurka ccdc25 ccnb1 cdc20 cdh1 cdk1 kif15 mcc mtor myt1 plk1 ppp6c prss1 tpx2 wee1
Article Images: [+] show captions
|FIG 3. Nocodazole-induced expression of anillin and securin is reduced in HeLa–CRISPR–DNA-PKcs cells. (A) HeLa-CRISPR-control and HeLa–CRISPR–DNA-PKcs cells were incubated in the absence (0) or presence of nocodazole (40 ng/ml) and harvested after 6 or 16 h, as indicated. Whole-cell extracts were generated, run on SDS-PAGE, and immunoblotted as for Fig. 2. (B and D) Quantitation of anillin and securin expression from three separate experiments as for Fig. 2. Statistical analysis was carried out as for Fig. 2. (C) Whole-cell extracts (lanes E) from nocodazole-treated (40 ng/ml; 16 h) HeLa-CRISPR-control (lanes 1 to 3) or HeLa–CRISPR–DNA-PKcs (lanes 4 to 6) cells were run on SDS-PAGE either directly (lanes E) or after incubation with 0.5 μl lambda phosphatase at 30°C for 10 min (lanes P) or incubated without phosphatase (mock treated; lanes M). Extracts were run on SDS-PAGE and immunoblotted for anillin and Ku80, as shown. The error bars indicate standard deviations.|
|FIG 5. Depletion of PP6 does not rescue nocodazole-induced downregulation of mitotic proteins in DNA-PKcs-deficient HeLa cells. (A) HeLa–CRISPR–DNA-PKcs cells were transfected with either scrambled control siRNA (siRNA-sc) or siRNA to PP6c (siRNA-PP6c) (100 nM), and after 56 h, the cells were treated with nocodazole (40 ng/ml) and harvested immediately (0) or after 3, 6, or 16 h, as indicated. Extracts were probed for mitotic proteins as described above. The Western blots are representative of 3 separate experiments. (B to K) Quantitation shown as in Fig. 2 and 4. The asterisk on the securin Western blot indicates a nonspecific band. siRNA-sc, scrambled siRNA control. The error bars indicate standard deviations.|
|FIG 6. Depletion of PP6 does not rescue nocodazole-induced downregulation of mitotic proteins in DNA-PKcs-deficient A549 cells. The experiment was carried out exactly as for Fig. 5, but A549–CRISPR–DNA-PKcs cells were used. The asterisk on the securin blot indicates a nonspecific band. The error bars indicate standard deviations.|
|FIG 7. Inhibition of APC/C with proTAME rescues expression and phosphorylation of mitotic proteins in nocodazole-treated DNA-PKcs-deficient HeLa cells. HeLa-CRISPR-control and HeLa–CRISPR–DNA-PKcs cells were preincubated with apcin (48 μM), proTAME (12 μM), or DMSO control for 1 h, and then nocodazole was added to 40 ng/ml and cells were harvested after a further 16 h. Representative Western blots (A) and quantitation from 3 separate experiments (B to M) are shown as in Fig. 5 and 6. (A, B, C, D, E, G, H, J, K, and M) Proteins were normalized to Ku80. (F, I, and L) Phosphoproteins were normalized to their respective total protein controls. The error bars indicate standard deviations. *, P < 0.05; ns, not significant.|
|FIG 8. Inhibition of APC/C with proTAME rescues expression and phosphorylation of mitotic proteins in nocodazole-treated DNA-PKcs-deficient A549 cells. Experiments were carried out exactly as for Fig. 7, except that A549-CRISPR-control and CRISPR–DNA-PKcs cells were used. The error bars indicate standard deviations. *, P < 0.05; ns, not significant.|
|FIG 9. Model for the role of DNA-PK in regulation of APC/C activity in nocodazole-treated cells. (A) Nocodazole causes microtubule depolymerization, preventing attachment of kinetochores to the mitotic spindle. In controls cells, this leads to activation of the SAC; Cdc20 is sequestered by the MCC; APC/C is not activated; and levels of anillin, securin, cyclin B1, and other proteins (not shown) rise. (B) In DNA-PKcs-deficient cells, Cdc20 and Cdh1 interact with and activate the APC/C, causing degradation of mitotic proteins, such as anillin, securin, and cyclin B1, even in the presence of unattached kinetochores. (C) In DNA-PKcs-deficient cells treated with ProTAME, APC/C-mediated degradation of anillin, securin, cyclin B, and other proteins (not shown) is inhibited, causing levels of proteins to rise.|
References [+] :
Blackford, ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. 2017, Pubmed