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.

Pauline Douglas,Ruiqiong Ye,Susan P. Lees-Miller,Suraj Radhamani,Jonathan Roveredo,Alexander Cobban,Edward Bartlett,Arminja N. Kettenbach,Nicole P. Jenkins

doi:10.1128/mcb.00191-19

Abstract

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.

Highlights

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have been reported
No DNA-PKcs protein was detected by Western blotting in the CRISPR–DNA-PKcs cells (Fig. 1A and B) and, in keeping with previous studies in the DNA-PKcs-null human cell line M059J [25, 26] and in mammalian cells treated with small interfering RNA (siRNA) to DNA-PKcs [6, 8, 27], cells with CRISPR/Cas9 deletion of DNA-PKcs had an approximately 80% reduction in expression of the related protein kinase, ataxia telangiectasia mutated (ATM)
We examined the expression of cyclin B1, anillin, and securin and phosphorylation of Aurora A, Pololike kinase 1 (PLK1), targeting protein for Xenopus Klp2 (TPX2), and MPM2-cross-reacting phosphoproteins in the panel of A549 cells

Summary

Introduction

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have been reported. 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 Cdc and Cdh to the APC/C. Small interfering RNA (siRNA) depletion of DNA-PKcs and/or inhibition of its catalytic activity results in chromosome misalignment and prolonged passage through mitosis in either nocodazole-treated cells, cells undergoing normal cellular progression [15, 16, 21], or cells exposed to ionizing radiation [22]. Reduced anillin, securin, and cyclin B1 levels in nocodazole-treated DNA-PKcs-deficient cells were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) with the cell-permeable small molecule proTAME (Pro-N-4-tosyl-L-arginine methyl ester), which disrupts loading of Cdc and Cdh onto the APC/C, but not with apcin, which disrupts interaction of Cdc and Cdh with APC/C target proteins [23, 24]. Our results suggest that loss of DNA-PKcs prevents activation of the spindle assembly checkpoint (SAC) and/or inactivation of the APC/C, causing inappropriate degradation of mitotic proteins, which in turn contributes to the multiple mitotic defects observed in DNA-PKcs-deficient cells

Methods

Results

Conclusion