Proteomic identification of the TRAF6 regulation of vacuolar ATPase for osteoclast function.
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.
Ryu
Jiyoon
J
Department of Cell and Developmental Biology, Dental Research Institute, College of Dentistry, Seoul National University, Seoul, Korea.
Kim
Hyunsoo
H
Lee
Seung Ku
SK
Chang
Eun-Ju
EJ
Kim
Hyung Joon
HJ
Kim
Hong-Hee
HH
D000199:Actins / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000201:enzymology / Q000378:metabolism; D002352:Carrier Proteins / Q000502:physiology; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D006801:Humans; D047468:Immunoprecipitation; D008562:Membrane Glycoproteins / Q000502:physiology; D051379:Mice; D008813:Mice, Inbred ICR; D010010:Osteoclasts / Q000201:enzymology / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
D000199:Actins; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D048029:TNF Receptor-Associated Factor 6; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D025262:Vacuolar Proton-Translocating ATPases