Department of Cell and Developmental Biology, Dental Research Institute, College of Dentistry, Seoul National University, Seoul, Korea.
Osteoclasts are cells specialized for bone resorption. For osteoclast activation, tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role. To find new molecules that bind TRAF6 and have a function in osteoclast activation, we employed a proteomic approach. TRAF6-binding proteins were purified from osteoclast cell lysates by affinity chromatography and their identity was disclosed by MS. The identified proteins included several heat shock proteins, actin and actin-binding proteins, and vacuolar ATPase (V-ATPase). V-ATPase, documented for a great increase in expression during osteoclast differentiation, is an important enzyme for osteoclast function; it transports proton to resorption lacunae for hydroxyapatite dissolution. The binding of V-ATPase with TRAF6 was confirmed both in vitro by GST pull-down assays and in osteoclasts by co-immunoprecipitation and confocal microscopy experiments. In addition, the V-ATPase activity associated with TRAF6 increased in osteoclasts stimulated with receptor activator of nuclear factor kappaB ligand (RANKL). Furthermore, a dominant-negative form of TRAF6 abrogated the RANKL stimulation of V-ATPase activity. Our study identified V-ATPase as a TRAF6-binding protein using a proteomics strategy and proved a direct link between these two important molecules for osteoclast function.