Glutathione S-transferase P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus

c-Jun N-terminal kinase (JNK)-mediated cell signaling pathways are regulate d endogenously in part by protein-protein interactions with glutathione S-t ransferase P1-1 (GSTP1-1) (1). Using purified recombinant proteins, combine d with fluorescence resonance energy transfer technology, we have found tha t the C terminus of JNK is critical to the interaction with GSTP1-1. The ap parent K-d for full-length JNK was 188 nM and for a C-terminal fragment (re sidues 200-424) 217 nM. An N-terminal fragment (residues 1-206) did not bin d to GSTP1-1. Increased expression of the C-terminal JNK fragment in a tetr acycline-inducible transfected NIH3T3 cell line produced a concentration-de pendent increase in the kinase activity of JNK under normal, unstressed gro wth conditions indicating a dominant-negative effect. This suggests that th e fragment can compete with endogenous full-length functional JNK resulting in dissociation of the GSTP1-1-JNK interaction and concomitant JNK enzyme activation. By using an antibody to hemagglutinin-tagged C-JNK, a concentra tion-dependent co-immunoprecipitation of GSTP1-1 was achieved. These data p rovide evidence for direct interactions between the C-terminal of JNK and G STP1-1 and a rationale for considering GSTP1-1 as a critical ligand-binding protein with a role in regulating kinase pathways.