Arrestin-3 binds the MAP kinase JNK3α2 via multiple sites on both domains

Although arrestins bind dozens of non-receptor partners, the interaction sites for most signaling proteins remain unknown. Here we report the identification of arrestin-3 elements involved in binding MAP kinase JNK3 alpha 2. Using purified JNK3 alpha 2 and MBP fusions containing separated arrestin-3 domains and peptides exposed on the non-receptor-binding surface of arrestin-3 we showed that both domains bind JNK3 alpha 2 and identified one element on the N-domain and two on the C-domain that directly interact with JNK3 alpha 2. Using in vitro competition we confirmed that JNK3 alpha 2 engages identified N-domain element and one of the C-domain peptides in the full-length arrestin-3. The 25-amino acid N-domain element has the highest affinity for JNK3 alpha 2, suggesting that it is the key site for JNK3 alpha 2 docking. The identification of elements involved in protein-protein interactions paves the way to targeted redesign of signaling proteins to modulate cell signaling in desired ways. The tools and methods developed here to elucidate the molecular mechanism of arrestin-3 interactions with JNK3 alpha 2 are suitable for mapping of arrestin-3 sites involved in interactions with other partners. (C) 2014 Elsevier Inc. All rights reserved.