Abstract 1354 Understanding Kinase Substrate Interactions with Phospho-PCA and Deep Mutational Scanning

Normal cells express functional tumor suppressor WW domain-containing oxidoreductase (WWOX), designated WWOXf.UV irradiation induces WWOXf cells to undergo bubbling cell death (BCD) -an event due to overproduction of nuclear nitric oxide (NO) gas that pushes the nuclear and cell membranes to form one or two bubbles at room temperature (22 C) and below.When WWOX-deficient or -dysfunctional (WWOXd) cells are exposed to UV and/or cold shock, the cells undergo nuclear pop-out explosion death (POD) without NO.We aimed to determine whether BCD can be converted to POD.WWOXf and WWOXd cells were exposed to UV followed by measuring BCD or POD by time-lapse microscopy at 4, 22, or 37 C to visualize morphological changes.UV induces internucleosomal DNA fragmentation during apoptosis at 37oC, but no DNA fragmentation in BCD and POD at lower temperatures.UV induced co-translocation of the WWOX/ TRAF2 complex to the nuclei, in which the prosurvival TRAF2 blocked the proapoptotic WWOX via its zinc finger domain.Without WWOX, TRAF2 did not relocate to the nuclei.Cold shock caused the dissociation of the WWOX/TRAF2 complex in the nucleus needed for BCD.In contrast, the formation of the WWOX/TRAF2 complex, plus p53, was strengthened at 37 C required for apoptosis.Clathrin inhibitor pitstop 2 blocks endocytosis of serum MIF and complement proteins, and exogenous EGFP and WWOX-EGFP cDNA constructs in UV-treated cells, which results in conversion of UV-mediated BCD to POD in WWOXf cells at 22oC.In conclusion, functional WWOX is needed for UV-mediated BCD, which requires endocytosis of serum MIF and certain complement proteins to support NO generation in the nucleus.Without endocytosis, BCD turns into POD due to failure in nuclear NO production.