The c-Jun N-terminal Kinases (JNKs) play important roles in cell responses to stress or growth factor stimulation. The JNK1 alpha 1 isoform shares >90% identity with a predominantly neuronal JNK3 alpha 1 isoform, but JNK3 alpha 1 also includes a distinctive 38 amino acid N-terminal sequence. To address the outstanding question of the potential for these JNK isoforms to have different binding partners that mediate different biological actions, the work presented here refined the yeast two-hybrid approach to identify and categorize binding partners for JNK1 alpha 1 and JNK3 alpha 1. Specifically, site-directed mutagenesis of the JNK1 alpha 1 common docking (CD) domain that mediates typical JNK-binding domain (JBD)-dependent interactions, truncation of the distinctive JNK3 N-terminal domain (i.e. Delta N JNK3 alpha 1), and interaction evaluation in the yeast two-hybrid system defined the interacting partners as either JNK1-specific interactors (ATF7, FUS, KCNE4, PIAS1, SHANK1, TKT), typical JBD-dependent interactors shared by JNK1 alpha 1 and JNK3 alpha 1 (AKAP6, BMPR2, EEF1A1, GFAP, GRIP2, GTF2F1, HDAC2, MAP1B, MYO9B, PTPN2, RABGAP1, RUSC2, SUMO1, SYPL1, TOPBP1, ZNF668), or JNK3-specific partners (ATXN1, NNAT, PTGDS) dependent on interaction with the JNK3 N-terminal extension. The interacting partners ATF7, AKAP6, and ATXN1 were explored further as representatives of these different classes. Two potential JBDs were identified in ATF7 as important for its interaction with JNK1 alpha 1, but additionally an interaction between ATF7 and Delta N JNK3 alpha 1 was shown to be JBD-dependent, suggesting that the JNK3 alpha 1 N-terminus prevents interaction with some proteins. For the shared partner AKAP6, one of the multiple potential JBDs predicted by sequence analysis was important for the AKAP6-JNK interaction in the yeast screening system as well as in mammalian cells. Finally, the ATXN1-JNK3 alpha 1 interaction was dependent on the JNK3 alpha 1 N-terminus in a mammalian cell context. These studies therefore highlight a diversity of potential JNK-interacting partners with both JBD-dependent as well as JBD-independent modes of interaction. (C) 2014 Elsevier Inc. All rights reserved.