The Cell Polarity Protein Par-3 To Regulate Dna Double-strand Break Repair Mechanism

Cell polarity is the fundamental characteristics of eukaryotic cells. Polarized localization of proteins is essential for asymmetric cell division during development, axon specification and tight junction assembly. The partitioning-defective 3 (Par-3), a key component in the conserved Par-3/Par-6/aPKC complex, plays critical roles in cell polarity. Recent studies have indicated that Par-3 is a multifunctional protein. For example, Par-3 is involved in liver cancer cell malignant growth andγ-irradiation-induced tumorigenesis. Moreover, Par-3 localizes on the membrane when Caco2 or MDCK cells are confluent, but translocates to the nucleus when cells are subconfluent. In addition, Par-3 may not complex with Par-6/aPKC but function as a"free"form in the cytosol. The data therefore indicate that Par-3 may be involved in other biological context in addition to establishing cell polarity. However, the possible operating mechanisms underlying these functions are elusive. Therefore, through screening novel Par-3 interaction proteins, we may identify new functions about Par-3.By means of in vitro binding assay and LC-MS/MS, we reported the identification of several proteins including nuclear protein complex Ku70/Ku80 that interacted with Par-3. Ku70/Ku80 proteins are two regulatory subunits of the DNA-dependent protein kinase (DNA-PK), which plays an essential role in repairing DNA double-strand breaks (DSBs). We determined that the association of Par-3 with Ku70/Ku80 in the nucleus was enhanced byγ-irradiation (IR), a potent DSB inducer. Furthermore, DNA-PKcs, the catalytic subunit of DNA-PK, interacted with the Par-3/Ku70/Ku80 complex in response to IR. Par-3 overexpression or knockdown was capable of up- or down-regulating DNA-PKcs activity, respectively. Moreover, the Par-3 knockdown cells were impaired in their ability to repair DSBs. Lastly, the Par-3 knockdown cells were radiosensitive. These findings may provide novel insight into cellular inherent connection between cell polarity and DSB repair.In addition, we also investigated the effect of pervanadate, PMA andγ-irradiation on tyrosine phosphorylation of Par-3 or Par-3 localization, and observed the localizations of three Par-3 isoforms in different cell cycle. Our results may uncover novel function of the evolutionarily conserved protein Par-3, which indicates that the cell operates as a whole to coordinate the function of Par-3 through regulating Par-3 localization, modification or interacting proteins with its different cell phase or state.