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. 2011 May;23(5):772-7.
doi: 10.1016/j.cellsig.2010.12.004. Epub 2010 Dec 23.

Mutational Analysis of TRAF6 Reveals a Conserved Functional Role of the RING Dimerization Interface and a Potentially Necessary but Insufficient Role of RING-dependent TRAF6 Polyubiquitination Towards NF-κB Activation

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Mutational Analysis of TRAF6 Reveals a Conserved Functional Role of the RING Dimerization Interface and a Potentially Necessary but Insufficient Role of RING-dependent TRAF6 Polyubiquitination Towards NF-κB Activation

Charilaos Megas et al. Cell Signal. .
Free PMC article

Abstract

TRAF6 is an E3 ubiquitin ligase that plays a pivotal role in the activation of NF-κB by innate and adaptive immunity stimuli. TRAF6 consists of a highly conserved carboxyl terminal TRAF-C domain which is preceded by a coiled coil domain and an amino terminal region that contains a RING domain and a series of putative zinc-finger motifs. The TRAF-C domain contributes to TRAF6 oligomerization and mediates the interaction of TRAF6 with upstream signaling molecules whereas the RING domain comprises the core of the ubiquitin ligase catalytic domain. In order to identify structural elements that are important for TRAF6-induced NF-κB activation, mutational analysis of the TRAF-C and RING domains was performed. Alterations of highly conserved residues of the TRAF-C domain of TRAF6 did not affect significantly the ability of the protein to activate NF-κB. On the other hand a number of functionally important residues (L77, Q82, R88, F118, N121 and E126) for the activation of NF-κB were identified within the RING domain of TRAF6. Interestingly, several homologues of these residues in TRAF2 were shown to have a conserved functional role in TRAF2-induced NF-κB activation and lie at the dimerization interface of the RING domain. Finally, whereas alteration of Q82, R88 and F118 compromised both the K63-linked polyubiquitination of TRAF6 and its ability to activate NF-κB, alteration of L77, N121 and E126 diminished the NF-κB activating function of TRAF6 without affecting TRAF6 K63-linked polyubiquitination. Our results support a conserved functional role of the TRAF RING domain dimerization interface and a potentially necessary but insufficient role for RING-dependent TRAF6 K63-linked polyubiquitination towards NF-κB activation in cells.

Figures

Fig. 1
Fig. 1
Schematic representation of the proteins TRAF6 and TRAF2. Previously defined domains are shown as darker rectangles. Vertical lines show the relative positions of the amino acid alterations that were introduced in both proteins. RF: RING finger, ZnF: Zinc fingers.
Fig. 2
Fig. 2
NF-κB activation potential of TRAF-C mutants of TRAF6. (A) Relative activity of a NF-κB-responsive luciferase reporter in response to expression of FLAG-tagged wild type or the indicated TRAF6 mutants in HEK293T cells. The results are the means ± SE of relative luciferase activity from at least three independent experiments. The asterisks designate the TRAF6 mutants that show a statistically significant difference in their ability to activate NF – κB relative to wild type TRAF6. (B) Representative Western blots showing the expression of FLAG-tagged wild type or mutated TRAF6 isoforms and β-actin in cell lysates analyzed in A.
Fig. 3
Fig. 3
NF-κB activation and dimerization potential of RING domain mutants of TRAF6. (A) Relative activity of a NF-κB-responsive luciferase reporter in response to expression of FLAG-tagged wild type or the indicated TRAF6 mutants in HEK293T cells (upper panel). The results are the means ± SE of relative luciferase activity from at least three independent experiments. The asterisks designate the TRAF6 mutants that show a statistically significant difference in their ability to activate NF-κB relative to wild type TRAF6. Representative Western blots showing the expression of FLAG-tagged wild type or mutated TRAF6 isoforms and β-actin in cell lysates analyzed in A (lower panels). (B) Molecular modeling of the RING domain of TRAF6. A dimer of the RING domain is shown. Residues that were mutated are shown as sticks. Grey: residues on RING dimer interface. (C) Superimposed gel filtration profiles of wild-type (WT) and RING domain mutants of TRAF6 (50-211).
Fig. 4
Fig. 4
NF-κB activation potential of RING domain mutants of TRAF2. (A) Relative activity of a NF-κB-responsive luciferase reporter in response to expression of FLAG-tagged wild type or the indicated TRAF2 mutants in HEK293T cells (upper panel). The results are the means ± SE of relative luciferase activity from at least three independent experiments. The asterisks designate the TRAF2 mutants that show a statistically significant difference in their ability to activate NF – κB relative to wild type TRAF2. Representative Western blot showing the expression of wild type or mutated TRAF2 isoforms and β-actin in cell lysates analyzed in A (lower panel). (B) Molecular modeling of the RING domain of TRAF2. A dimer of the RING domain is shown. Residues that were mutated are shown as sticks. Grey: residues on RING dimer interface.
Fig. 5
Fig. 5
Analysis of TRAF6 polyubiquitination. HEK293T cells were mock transfected (control) or transfected with FLAG-tagged wild type (WT) or the indicated mutated forms of TRAF6 along with a vector expressing ubiquitin. The TRAF6 proteins were immunoprecipitated 16 hours post transfection and analyzed by immunoblotting using the anti-FLAG M5 monoclonal antibody or antibodies that recognize ubiquitin or K63-linked polyubiquitin chains. (A) Representative Western blot showing poyubiquitinated (poly-Ub) and non-ubiquitinated (TRAF6) TRAF6 proteins. (B) Representative Western blot showing poyubiquitinated by K63-linked chains (K63 poly-Ub) and non-ubiquitinated (TRAF6) TRAF6 proteins.

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