Targets of JNK in acute hepatotoxicity.

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ABSTRACT Sustained activated c-Jun-N-terminal kinase (JNK) plays a pivotal role in mediating hepatotoxic cell death due to its phosphorylation of target proteins. This proposal focuses on two novel JNK targeted proteins, cytoplasmic - glutamyl cysteine ligase (GCL) and mitochondrial outer membrane SAB (SH3BP5) and their roles in mediating sustained JNK activation by promoting reactive oxygen (ROS) species dependent activation of the MAPK kinase cascade. Based on preliminary data we propose that (a) P-JNK targets GCLC subunit for rapid proteolytic degradation which enhances ROS exposure and consequent activation of MAPK cascade; (b) specific P-JNK docking and phosphorylation sites on SAB mediate SAB-dependent mitochondrial ROS production and together with GCL degradation sustain a P-JNK-mitoSAB-ROS activation loop; (c) a second isoform of SAB2 with a modified N-terminus is incapable of transducing an effect inside the mitochondria but retains ability of C-terminus to be a P-JNK substrate and therefore is a potential decoy. Our overarching hypothesis is that P-JNK targeting and phosphorylation of both novel targets, GCL and SAB, mediates sustained JNK activation leading to hepatotoxicity of acetaminophen (APAP) in a two-pronged mechanism and apoptosis in other models through SAB-dependent sustained P-JNK which then modulates apoptosis regulators. Thus, the aims of the proposal are: (1) Determine the role of JNK in the regulation of - glutamyl cysteine ligase (GCL) subunits and the impact on GSH recovery in acetaminophen (APAP) hepatotoxicity: Preliminary results show that GCLC is rapidly degraded after APAP treatment in conjunction with sustained JNK activation while expression of JNK resistant mutated GCLC dampens P-JNK before onset of necrosis in APAP induced liver injury. Both GCLC and GCLM contain a P-JNK docking site (KIM, kinase interaction motif) and possible phosphorylation sites but only GCLC contains a PEST cleavage site. (2) Characterize the role of specific JNK binding and phosphorylation sites of SAB in mediating JNK-dependent toxicity and relation to SAB oligomerization: SAB contains two possible P-JNK docking (KIM) sites and four serine (SP/L) sites for phosphorylation. We will perform site directed mutagenesis to elucidate which is/are indispensable in SAB-dependent signal transduction to inside of mitochondria. We assess the dependence of phosphorylation of SAB on the homo or hetero oligomerization of SAB and its role in promoting intramitochondrial signal transduction. (3) Define the expression and function of the two isoforms of mouse and human SAB (SAB1 and -2) in liver: We identified two isoforms of SAB. Mouse SAB2 has a unique N-terminal amino acid sequence whereas human SAB2 is shorter and truncated at N-terminus compared to SAB1. Preliminary data indicates that mouse SAB2 is in mitochondria, does not transduce a signal inside mitochondria, and, when overexpressed in wild type mice, protects against liver injury, suggesting it acts as a decoy substrate.