Abstract 153: Phosphorylation Status Of Ser 58 Of 14–3–3 Proteins Is A Novel Molecular Switch Regulating Oxidant Stress-induced Death Via Ask-1
Background: SOK-1 (Ste20 oxidant stress kinase) is a member of the Mst family of kinases. It is Golgi-localized but exits the Golgi following oxidant stress, becoming a potent inducer of apoptosis. SOK-1 phosphorylates 14–3–3B6; on serine 58 (S58), but the consequences of this are not known. We hypothesized that since S58 is within the ligand binding groove of 14–3–3s, phosphorylation (P) of this residue might disrupt binding of pro-apoptotic factors to 14–3–3s, triggering cell death.
Methods: We expressed wild type (WT) 14–3–3B6;, or 14–3–3B6; with an S58 to Ala (SA) mutation, preventing P, or S58 to Asp (SD), mimicking P. Cells were exposed to 125 μM H2O2 for 3 hours to induce oxidant stress.
Results: 14–3–3/ASK-1 binding is dynamically regulated by oxidant stress since H2O2 disrupted binding of ASK-1 to WT. 14–3–3/ASK-1 binding is regulated specifically by S58 P status since ASK-1 readily co-immunoprecipitated with WT and SA, but not with SD. In addition, oxidant stress was unable to disrupt ASK-1 binding to SA. This suggests S58 P is both necessary and sufficient to disrupt ASK-1/14–3–3 interactions. Most strikingly, H2O2-induced cell death was significantly reduced by expression of SA, but was markedly increased by expression of SD (TUNEL+ cells in control (C), SA and SD were 35.5%*, 21.6%* and 60.8%* respectively). Thus P status of S58 regulates ASK-1 binding to 14–3–3s and oxidant stress-induced cell death. Confirming that ASK-1 was the mediator of cell death, expression of SD did not induce cell death in ASK-1−/ − mouse embryonic fibroblasts. Furthermore, in cells expressing SA, oxidant stress activation of the ASK-1 target, JNK, was markedly decreased, and a peptide antagonist of JNKs significantly decreased death in cells expressing SD, confirming a key role for the ASK1/JNK pathway. Finally, an S58 phospho-specific antibody demonstrated that P of S58 is dynamically regulated in situ, and that P is increased by oxidant stress.
Conclusion: We have identified a novel molecular switch that regulates oxidant stress-induced cell death: P status of S58, a residue conserved in most 14–3–3 proteins. P is catalyzed by SOK-1 when oxidant stress is extreme, and this leads to disruption of binding of ASK-1 to 14–3–3s, thereby leading to JNK activation and cell death.*, P<0.01 vs C