Jun activation domain-binding protein 1 is required for mitotic checkpoint activation via its involvement in hyperphosphorylation of 53BP1

Purpose: p53-binding protein 1 (53BP1), a participant in the DNA damage response pathway, has also been implicated in the cellular response to mitotic stress conditions. Here, we sought to broaden our understanding of the protein network surrounding 53BP1 by identifying and characterizing a 53BP1-interacting protein. Method: Yeast two-hybrid screening was performed to identify possible binding partners of 53BP1. To investigate the functional meaning of the interaction, knock-down cells were established by introduction of antisense construct or siRNA into HeLa cells. The hyperphosphorylation of 53BP1 after treatment with nocodazole, a microtubule-interfering agent, was monitored by immunoblotting. And the cell cycle arrest at mitotic phase was measured by flow cytometry after staining with phospho-(Ser10)-histone H3 antibody. Results: Jun activation domain-binding protein 1 (Jab1) was identified as a 53BP1-binding protein, and the interaction between them was confirmed to occur in mammalian cells. We found that nocodazole-induced 53BP1 hyperphosphorylation was abolished in Jab1 knock-down cells. In addition, ectopic overexpression of Jab1 augmented 53BP1 hyperphosphorylation. On the cellular level, Jab1 knock-down cells exhibited reduced mitotic arrest upon exposure to nocodazole, resulting in cellular resistance to the drug. Conclusion: Taken together, these results suggest that Jab1 is required for the hyperphosphorylation of 53BP1 upon mitotic stress conditions and is involved in proper activation of mitotic checkpoint mechanism. Our study also suggests the possibility that modulation of Jab1 activity may be an intriguing approach for enhancing the efficacy of microtubule-interfering anticancer drugs.