TRAF6 E3 LIGASE FUNCTION IN OSTEOCLASTOGENESIS

Summary

Principal Investigator: Bryant Darnay
Affiliation: The University of Texas
Country: USA
Abstract: RANK plays a central role in the maintenance of the bone homeostasis by regulating the differentiation and activation of osteoclasts. The binding of RANKL to its receptor results in the recruitment of TRAF6, which activates the NF-(B, JNK, p38, and MAPK pathways. TRAF6 is the critical RANK adaptor molecule since TRAF6-deficient mice develop severe osteopetrosis. Recent evidence indicates that the N-terminal RING domain of TRAF6 is required for its ability to signal, most likely by functioning as an E3 ubiquitin ligase, which together with the ubiquitin conjugating enzyme complex Ubc13/Uev1A catalyzes the synthesis of a unique polyubiquitin chain linked through lysine-63 (K63). However, this unique ubiquitin modification does not target TRAF6 for degradation, but rather the auto-ubiquitination of TRAF6 serves as a scaffold to recruit molecules required for the activation of kinase complexes including TAK1 and IKK. In this application, we provide preliminary data to address the functional role of TRAF6 auto-ubiquitination in RANK signaling. We show that upon RANKL stimulation endogenous TRAF6 is auto-ubiquitinated via K63 linkages, which is critical for NF-(B activation, induction of NFATc1 and subsequent osteoclast differentiation. Endogenous TAB2 and NEMO, which are potential TRAF6 downstream signaling molecules, are also ubiquitinated upon RANK activation. TRAF6 auto-ubiquitination is dependent on its RING domain and Ubc13 and mutations in either of these proteins disrupt RANK signaling. Strikingly, we identified a single critical Ub acceptor site residue (K124) in TRAF6 that is required for TRAF6 auto-ubiquitination. Mutation of this site abolished TRAF6-mediated TAB2 recruitment, TAB2 and NEMO ubiquitination, TAK1 and IKK activation, NF-(B activation, and osteoclast differentiation. Furthermore, we present evidence that the kinase activity of TAK1 is required for the efficient activation of IKK and JNK, induction of NFATc1, and osteoclast differentiation by RANKL. Through structural mapping studies, we have identified a unique domain in the C-terminus of TAK1 that is required to interact with TAB2 and its homologue TAB3. Expression of a GFP-fusion protein consisting of the last 100 residues of TAK1 acts as a dominant negative mutant that suppresses RANKL signaling and osteoclast differentiation. These results support a novel Ub-dependent TRAF6 signaling pathway that is required for efficient osteoclast differentiation. We hypothesize that upon RANKL stimulation of osteoclast progenitors, TRAF6 functions through its ubiquitin ligase activity to coordinate the recruitment (via site-specific ubiquitination of TAB2 and NEMO) and activation of the TAB2/TAK1 and IKK complexes to activate the NF-(B pathway, which is required for terminal osteoclast differentiation. To address this hypothesis, we propose three integrated specific aims. 1) Characterize non-proteasomal ubiquitination events in primary BMM after RANKL treatment. 2) Characterize the role TRAF6 auto-ubiquitination for its ability to recruit and ubiquitinate downstream targets. 3) Determine the physiological significance of TAK1 in osteoclastogenesis by generating and characterizing conditional knock-out mice deficient in TAK1 in the monocyte/macrophage linage. Taken together, these studies will reveal the molecular mechanism by which TRAF6 through ubiquitin modification regulates osteoclast differentiation. In particular, the data gathered from these experiments may provide insights to developing therapeutic strategies for a variety of bone related disease and possibly other diseases associated with immunity and inflammation
Funding Period: 2007-07-01 - 2011-06-30
more information: NIH RePORT

Top Publications

  1. pmc TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation
    Betty Lamothe
    Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Box 143, 1515 Holcombe Blvd, Houston, TX 77030, USA
    Biochem Biophys Res Commun 359:1044-9. 2007
  2. pmc Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20
    Su Chang Lin
    Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
    J Mol Biol 376:526-40. 2008
  3. pmc The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL
    Betty Lamothe
    Department of Experimental Therapeutics, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA
    J Biol Chem 283:24871-80. 2008
  4. pmc E2 interaction and dimerization in the crystal structure of TRAF6
    Qian Yin
    Weill Medical College of Cornell University, New York, New York, USA
    Nat Struct Mol Biol 16:658-66. 2009
  5. pmc Structural basis for the lack of E2 interaction in the RING domain of TRAF2
    Qian Yin
    Weill Medical College of Cornell University, New York, New York 10021, USA
    Biochemistry 48:10558-67. 2009

Detail Information

Publications5

  1. pmc TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation
    Betty Lamothe
    Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Box 143, 1515 Holcombe Blvd, Houston, TX 77030, USA
    Biochem Biophys Res Commun 359:1044-9. 2007
    ..These data establish a signaling cascade in which regulated Lys63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of osteoclast differentiation...
  2. pmc Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20
    Su Chang Lin
    Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
    J Mol Biol 376:526-40. 2008
    ....
  3. pmc The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL
    Betty Lamothe
    Department of Experimental Therapeutics, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA
    J Biol Chem 283:24871-80. 2008
    ..These data provide evidence for the critical role of the Ub ligase activity of TRAF6, which is coordinated via the RING domain and ZF1 to supply the necessary elements in signaling by cytokines dependent upon TRAF6...
  4. pmc E2 interaction and dimerization in the crystal structure of TRAF6
    Qian Yin
    Weill Medical College of Cornell University, New York, New York, USA
    Nat Struct Mol Biol 16:658-66. 2009
    ..The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors...
  5. pmc Structural basis for the lack of E2 interaction in the RING domain of TRAF2
    Qian Yin
    Weill Medical College of Cornell University, New York, New York 10021, USA
    Biochemistry 48:10558-67. 2009
    ..Our structural observation should prompt a re-evaluation of the role of TRAF2 in TNFalpha signaling and may indicate that TRAF2-associated proteins such as cIAPs may be the ubiquitin ligases for NF-kappaB signaling...