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TRAF6 binds YOD1. 42 / 51
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"Therefore, we investigated whether YOD1 and p62 are binding simultaneously to TRAF6 and potentially forming a tripartite complex or if binding of the two MATH domain interactors is mutually exclusive and possibly even competitive."
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"To increase the physiological relevance, we show endogenous interaction of TRAF6 and YOD1 in different cell lines and also primary human umbilical vein endothelial cells (HUVEC) ( xref )."
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"Co-IP revealed binding of reconstituted YOD1 to TRAF6 in unstimulated cells and the interaction was reduced after IL-1β stimulation ( xref and xref )."
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"The authors show that YOD1 interacts with the C-terminal MATH domain of TRAF6, and inhibits TRAF6 binding with p62/Sequestosome-1."
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"Indeed, co-IP experiments using MYC-TRAF6 together with Crimson-p62 and GFP-YOD1 revealed that YOD1 was able to inhibit the association of TRAF6 and p62, while p62 did not alter the binding of YOD1 to TRAF6 ( xref )."
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"We show that YOD1 binds to TRAF6 in unstimulated cells and that binding is decreased after IL-1 stimulation."
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"To assess the selectivity of YOD1 and TRAF6 interaction, we compared association of YOD1 to TRAF2 and TRAF6 in HEK293 cells (XREF_FIG)."
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"Co-immunoprecipitations (IP) using anti-HA or anti-FLAG antibodies confirmed the interaction of TRAF6 and YOD1 (XREF_FIG)."
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"YOD1 and TRAF6 binding is apparently independent of p97, suggesting that YOD1 affects NF-kappaB signaling in response to IL-1 independent of this multifunctional AAA-ATPase."
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"However, whereas YOD1 binds to TRAF6 in resting cells affecting C-terminal substrate binding, A20 is associating with TRAF6 only upon prolonged IL-1 stimulation to counteract binding of the E2 enzyme UBC13 to the RING-Z1 of TRAF6 ( xref )."
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"The current study further shows that in two cell lines – HeLa and HEK293, YOD1 associates constitutively with TRAF6 and dissociates from it in response to IL-1."
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"We find that YOD1 directly associates with TRAF6 and competes with p62 for TRAF6 binding and activation."
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"Further, specificity of TRAF6 and YOD1 interaction was confirmed by the decreased co-precipitation of YOD1 in TRAF6 knock-down HeLa cells (XREF_FIG)."
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"YOD1 associates with the C-terminal MATH domain of TRAF6."
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"However, whereas YOD1 binds to TRAF6 in resting cells affecting C-terminal substrate binding, A20 is associating with TRAF6 only upon prolonged IL-1 stimulation to counteract binding of the E2 enzyme UBC13 to the RING-Z1 of TRAF6."
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"We discovered a novel interaction of YOD1 with full length TRAF6, but not with a fragment comprising the catalytic RING-Zinc Finger1 (Z1) domain ( xref ; xref )."
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"We find that YOD1 directly associates with TRAF6 and competes with p62 for TRAF6 binding and activation."
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"To obtain data on the selectivity of YOD1 and TRAF6 interaction, we checked binding of YOD1 to a panel of E3 ligases as well as other proteins involved in regulatory ubiquitination by Y2H (XREF_FIG)."
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"YOD1 associates with TRAF6 in unstimulated cells but is released upon IL-1β stimulation, thereby facilitating TRAF6 auto-ubiquitination as well as NEMO/IKKγ substrate ubiquitination."
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"However, it appears that IL-1β stimulation actually increases association of WT YOD1 with TRAF6 while reducing that with YOD1[C160S]."
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"Since YOD1 has been described as a cellular co-factor of p97, we checked for YOD1/TRAF6 interaction in p97 knock-down HEK293 cells and found that binding of YOD1 to TRAF6 was not significantly altered upon p97 depletion ( xref )."
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"Nevertheless, YOD1 specifically binds to TRAF6 and not to TRAF2, and consequently YOD1 counteracts TRAF6-dependent NF-κB signaling from the IL-1R and not the TNFR."
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"To confirm that TRAF6 and YOD1 are directly associating and to narrow down the YOD1 binding site on TRAF6, we expressed and purified recombinant YOD1 and TRAF6 proteins and performed pull-down (PD) experiments ( xref and xref )."
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"To gain insights into the role of YOD1 and TRAF6 interaction, we determined the cellular localization of both proteins upon overexpression in U2OS and HeLa cells by confocal fluorescence microscopy."
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"Further, we also show binding data in primary cells: YOD1 and TRAF6 interact in primary human umbilical vein endothelial cells (HUVEC) ( xref )."
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"TRAF6 and YOD1 binding was visible in all cells and there was a tendency that more TRAF6 and YOD1 association was observed in cells that expressed more TRAF6 (U2OS and HEK293)."
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"Indeed, co-IP experiments using MYC-TRAF6 together with Crimson-p62 and GFP-YOD1 revealed that YOD1 was able to inhibit the association of TRAF6 and p62, while p62 did not alter the binding of YOD1 to TRAF6 (XREF_FIG)."
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"Interaction of TRAF6 and YOD1 did not rely on YOD1 catalytic activity, because like YOD1 WT, the DUB mutant YOD1 C160S was co-localizing ( xref and xref ) and co-precipitating with TRAF6 ( xref )."
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"1) To test the specificity of YOD1 interaction with TRAF6, a panel of E3 ligases and other Ub regulatory proteins was tested for interaction with YOD1 by yeast two-hybrid assay ( xref )."
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"Co-immunoprecipitations (IP) using anti-HA or anti-FLAG antibodies confirmed the interaction of TRAF6 and YOD1 ( xref )."
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"2) It is surprising that mutation of the putative TRAF6 interaction motif in YOD1 does not abolish the interactions between YOD1 and TRAF6."
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"Since YOD1 has been described as a cellular co-factor of p97, we checked for YOD1 and TRAF6 interaction in p97 knock-down HEK293 cells and found that binding of YOD1 to TRAF6 was not significantly altered upon p97 depletion (XREF_FIG)."
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"Since YOD1 and TRAF6 interaction takes place in unstimulated cells, TRAF6 oligomerization is apparently not required, indicating that monomeric TRAF6 has a preference for binding to YOD1 over p62."
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"To investigate whether YOD1 and TRAF6 also interact in cells, we co-expressed full length FLAG-YOD1 and HA-TRAF6 in HEK293 cells."
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"Thus, it is not strictly clear whether there is still residual endogenous YOD1 bound or an influence of the endogenous in the case of YOD1 C160S. We show decreased binding of YOD1 to TRAF6 after IL-1 stimulation in two reconstituted HeLa KO cell clones ( xref and xref ) and of endogenous TRAF6/YOD1 in HeLa cells and HUVEC ( xref ), providing evidence that interaction is reduced after IL-1 stimulation."
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"To confirm that TRAF6 and YOD1 are directly associating and to narrow down the YOD1 binding site on TRAF6, we expressed and purified recombinant YOD1 and TRAF6 proteins and performed pull-down (PD) experiments (XREF_FIG and XREF_FIG)."
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"In fact, IL-1-induced TRAF6 ubiquitination correlates with a decreased binding of YOD1 to TRAF6 after IL-1 stimulation (see XREF_FIG and XREF_FIG), and we asked, whether reduced binding is also visible at the level of endogenous proteins in HeLa cells and primary HUVEC cells (XREF_FIG)."
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