5-Lipoxygenase interacts with coactosin-like protein

We have recently identified coactosin-like protein (CLP) in a yeast two-hyb rid screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonst rate a direct interaction between 5LO and CLP, 5LO associated with CLP, whi ch was expressed as a glutathione S-transferase fusion protein, in a dose-d ependent manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates, In reciprocal expe riments, 5LO was detected in CLP immunoprecipitates. Non-denaturing polyacr ylamide gel electrophoresis and cross-linking experiments showed that 5LO b inds CLP in a 1:1 molar stoichiometry in a Ca2+-independent manner. Site-di rected mutagenesis suggested an important role for lysine 131 of CLP in med iating 5LO binding. In view of the ability of CLP to bind 5LO and filamento us actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments. However, no F-actin-CLP.5LO ternary complex was observed. In contrast, 5LO appeared to compete with F-actin for the binding of CLP. M oreover, 5LO was found to interfere with actin polymerization. Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusi ve and suggest a modulatory role for 5LO in actin dynamics.