Proteomic identification of the TRAF6 regulation of vacuolar ATPase for osteoclast function

Jiyoon Ryu, Hyunsoo Kim, Seung Ku Lee, Eun Ju Chang, Hyung Joon Kim, Hong Hee Kim

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

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 ιB 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.

Original languageEnglish (US)
Pages (from-to)4152-4160
Number of pages9
JournalProteomics
Volume5
Issue number16
DOIs
StatePublished - Nov 2005
Externally publishedYes

Keywords

  • MALDI-TOF
  • Osteoclast
  • Tumor necrosis factor receptor-associated factor 6
  • Vacuolar ATPase
  • Western blot analysis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Proteomic identification of the TRAF6 regulation of vacuolar ATPase for osteoclast function'. Together they form a unique fingerprint.

Cite this