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Forum on Immunopathological Diseases and Therapeutics
SJR: 0.309 SNIP: 0.041 CiteScore™: 0.18

ISSN 印刷: 2151-8017
ISSN オンライン: 2151-8025

Archives: Volume 1, 2010 to Volume 7, 2016

Forum on Immunopathological Diseases and Therapeutics

DOI: 10.1615/ForumImmunDisTher.v2.i1.60
pages 47-58

Locostatin Disrupts Association of Raf Kinase Inhibitor Protein With Binding Proteins by Modifying a Conserved Histidine Residue in the Ligand-Binding Pocket

Anwar B. Beshir
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA
Christian E. Argueta
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA
Lochana C. Menikarachchi
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA
Jose A. Gascon
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA
Gabriel Fenteany
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA

要約

Raf kinase inhibitor protein (RKIP) interacts with a number of different proteins and regulates multiple signaling pathways. Here, we show that locostatin, a small molecule that covalently binds RKIP, not only disrupts interactions of RKIP with Raf-1 kinase, but also with G protein-coupled receptor kinase 2. In contrast, we found that locostatin does not disrupt binding of RKIP to two other proteins: inhibitor of κB kinase α and transforming growth factor β-activated kinase 1. These results thus imply that different proteins interact with different regions of RKIP. Locostatin’s mechanism of action involves modification of a nucleophilic residue on RKIP. We observed that after binding RKIP, part of locostatin is slowly hydrolyzed, leaving a smaller RKIP-butyrate adduct. We identified the residue alkylated by locostatin as His86, a highly conserved residue in RKIP’s ligand-binding pocket. Computational modeling of the binding of locostatin to RKIP suggested that the recognition interaction between small molecule and protein ensures that locostatin’s electrophilic site is poised to react with His86. Furthermore, binding of locostatin would sterically hinder binding of other ligands in the pocket. These data provide a basis for understanding how locostatin disrupts particular interactions of RKIP with RKIP-binding proteins and demonstrate its utility as a probe of specific RKIP interactions and functions.