Identification of a consensus site for TRAF6/p62 polyubiquitination

Abstract

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an ubiquitin ligase that regulates a diverse array of physiological processes via forming Lys-63 linked polyubiquitin chains. In this study, the lysine selection process for TRAF6/p62 ubiquitination was examined. The protein sequence of two characterized TRAF6/p62 substrates, NRIF and TrkA, revealed a conserved consensus pattern for the ubiquitination site of these two TRAF6 substrates. The consensus pattern established in the verified substrates was common to the other Trk receptor family members, TrkB and TrkC. Interestingly, Lysine 811 in TrkB was selected for ubiquination, and mutation of Lysine 811 diminished the formation of TRAF6/p62 complex that is necessary for effective ubiquination. Moreover, downstream signaling was affected upon binding of BDNF to the mutant TrkB receptor. These findings reveal a possible selection process for targeting a specific lysine residue by a single E3 ligase and underscore the role of the scaffold, p62, in this process.

Fig. 1

Conserved sequences flanking the TRAF6/p62 ubiquitin acceptor site. An alignment of TRAF6/p62 ubiquitination acceptor site in NRIF and TrkA shown here with maximum number of matches. Amino acids of the same typed are marked as (*) hydrophobic; (!) polar, (X) any amino acid residue and (K) the acceptor Lysine residue.

Fig. 2

TrkB is ubiquitinated at Lysine 811. HEK cells were transfected with either WT-TrkA, K485R-TrkA, WT-TrkB or K118R-TrkB along with His/Myc-tagged ubiquitin constructs. The cells were treated with or without, NGF for TrkA; BDNF for TrkB for 15 min. The cells were then lysed with SDS lysis buffer and the extent of ubiquitination was determined by immunoprecipitating the lysate with Trk antibody and Western blotting with anti-ubiquitin (upper panel). As a control, a fraction of lysate (50 µg) was blotted with anti-Trk (lower panel). This experiment was replicated three independent times with similar results.

Fig. 3

Mutation of Trk receptors impairs interaction with TRAF6/p62. HEK cells were transfected with WT-TrkA, WT-TrkB or their point mutants K485R-TrkA and K811R-TrkB. The cells were stimulated with or without NGF and BDNF for 15 min. The cells were then lysed in Triton lysis buffer and the cell lysate was immunoprecipitated with Trk antibody and Western blotted with Trk, TRAF6 or p62 antibody. As a control, a fraction of lysate (50 µg) was blotted with anti-Trk and anti-TRAF6 antibody. This experiment was replicated three independent times with similar results.

Fig. 4

Receptor ubiquitination regulates downstream signaling. HA-tagged WT-TrkA, WT-TrkB, or their mutants were transfected in NNR5 cells and treated with either NGF or BDNF for 15 min. The lysates from transfected cells were blotted with phospho-MAPK and stripped, and reblotted with nonphospho-MAPK antibody as shown. Alternatively, the lysates were also blotted with phospho-AKT and stripped, and reblotted with nonphospho-AKT antibody. The expression of Trk receptors in the lysate was also examined. This experiment was replicated three independent times with similar results.