Abstract
The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.
Highlights
OCs are multinucleated giant cells that are formed by the fusion of monocyte/macrophage lineage precursors through the process of OC differentiation or osteoclastogenesis (2)
The downstream consequence of RANK-RANKL signaling is the activation of osteoclastogenic transcription factors, including NF-B, activator protein 1 (AP-1), cAMP response element-binding protein (CREB), and nuclear factor of activated T cell c1 (NFATc1), which induce osteoclastogenic genes, such as Trap, Dcstamp, Atp6v0d2, and Oscar; these genes code for the proteins tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), V-ATPase subunit d2 (Atp6v0d2), and OC-associated receptor (OSCAR), respectively (1–3)
TRAF6 Interacts with Vav3—To identify novel protein partners that interact with TRAF6, lysates of FLAG-TRAF6-expressing 293T cells were immunoprecipitated with FLAG beads, and the TRAF6-bound protein band at ϳ90 kDa was identified by nanoscale liquid chromatography coupled with tandem mass spectrometry (Table 1)
Summary
TRAF6 Interacts with Vav3—To identify novel protein partners that interact with TRAF6, lysates of FLAG-TRAF6-expressing 293T cells were immunoprecipitated with FLAG beads, and the TRAF6-bound protein band at ϳ90 kDa was identified by nanoscale liquid chromatography coupled with tandem mass spectrometry (Table 1). To further confirm the TRAF6-Vav interaction at the endogenous protein level, we conducted an endogenous TRAF6 IP assay with an anti-TRAF6 antibody in bone marrow-derived macrophages (BMMs) and observed the interaction between endogenous TRAF6 and Vav (Fig. 1B). Consistent with these results, TRAF6 co-localized with Vav in subcellular localization analysis (Fig. 1C). The DH Domain of Vav Physically Interacts with the CC Domain of TRAF6 —To identify the specific domain of Vav that interacts with TRAF6, we constructed expression plasmids for Vav deletion mutants lacking the N- and C-terminal regions (Fig. 2A). Among the N-terminal deletion mutants of Vav, TRAF6 bound to the
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