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Arrestin-3 binds the MAP kinase JNK3?2 via multiple sites on both domains.

ABSTRACT: 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?2. Using purified JNK3?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?2 and identified one element on the N-domain and two on the C-domain that directly interact with JNK3?2. Using in vitro competition we confirmed that JNK3?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?2, suggesting that it is the key site for JNK3?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?2 are suitable for mapping of arrestin-3 sites involved in interactions with other partners.

SUBMITTER: Zhan X

PROVIDER: S-EPMC3936466 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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

Zhan Xuanzhi X Perez Alejandro A Gimenez Luis E LE Vishnivetskiy Sergey A SA Gurevich Vsevolod V VV

Cellular signalling 20140108 4

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 JNK3α2. Using purified JNK3α2 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 JNK3α2 and identified one element on the N-domain and two on the C-domain that directly interact wi ...[more]

PMID: 24412749

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Project description:The Helicobacter pylori chemoreceptor TlpA plays a role in dampening host inflammation during chronic stomach colonization. TlpA has a periplasmic dCache_1 domain, a structure that is capable of sensing many ligands; however, the only characterized TlpA signals are arginine, bicarbonate, and acid. To increase our understanding of TlpA's sensing profile, we screened for diverse TlpA ligands using ligand binding arrays. TlpA bound seven ligands with affinities in the low- to middle-micromolar ranges. Three of these ligands, arginine, fumarate, and cysteine, were TlpA-dependent chemoattractants, while the others elicited no response. Molecular docking experiments, site-directed point mutants, and competition surface plasmon resonance binding assays suggested that TlpA binds ligands via both the membrane-distal and -proximal dCache_1 binding pockets. Surprisingly, one of the nonactive ligands, glucosamine, acted as a chemotaxis antagonist, preventing the chemotaxis response to chemoattractant ligands, and acted to block the binding of ligands irrespective of whether they bound the membrane-distal or -proximal dCache_1 subdomains. In total, these results suggest that TlpA senses multiple attractant ligands as well as antagonist ones, an emerging theme in chemotaxis systems. IMPORTANCE Numerous chemotactic bacterial pathogens depend on the ability to sense a diverse array of signals through chemoreceptors to achieve successful colonization and virulence within their host. The signals sensed by chemoreceptors, however, are not always fully understood. This is the case for TlpA, a dCache_1 chemoreceptor of H. pylori that enables the bacterium to induce less inflammation during chronic infections. H. pylori causes a significant global disease burden, which is driven by the development of gastric inflammation. Accordingly, it is essential to understand the processes by which H. pylori modulates host inflammation. This work uncovers the signals that TlpA can sense and highlights the underappreciated ability to regulate chemotactic responses by antagonistic chemoreceptor ligands, which is an emerging theme among other chemotactic systems.

Dataset Information

Arrestin-3 binds the MAP kinase JNK3?2 via multiple sites on both domains.

Publications

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

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