TRAF6 and A20 Regulate Lysine 63–Linked Ubiquitination of Beclin-1 to Control TLR4-Induced Autophagy

Abstract

Autophagy delivers cytoplasmic constituents to autophagolysosomes and is linked to both innate and adaptive immunity. Toll-like receptor 4 (TLR4) signaling induces autophagy and recruits Beclin-1, the mammalian homolog of yeast Atg6, to the receptor complex. We found that tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-mediated, Lys(63) (K63)-linked ubiquitination of Beclin-1 is critical for TLR4-triggered autophagy in macrophages. Two TRAF6-binding motifs in Beclin-1 facilitated the binding of TRAF6 and the ubiquitination of Beclin-1. Lys(117), which is strategically located in the Bcl-2 homology 3 (BH3) domain of Beclin-1, was a major site for K63-linked ubiquitination. The deubiquitinating enzyme A20 reduced the extent of K63-linked ubiquitination of Beclin-1 and limited the induction of autophagy in response to TLR signaling. Treatment of macrophages with either interferon-gamma or interleukin-1 also triggered the K63-linked ubiquitination of Beclin-1 and the formation of autophagosomes. These results indicate that the status of K63-linked ubiquitination of Beclin-1 plays a key role in regulating autophagy during inflammatory responses.

The Beclin 1 network regulates autophagy and apoptosis

“…27 The K63-linked ubiquitination of Beclin 1 facilitates oligomerization of Beclin 1 and the activation of PI3KC3. This helps trigger the formation of autophagosomes.…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…136,137 For instance, ubiquitination of BECN1 through Lys-11-linked 138 or Lys-48-linked 139 ubiquitin chains promotes the degradation of BECN1, which possibly decreases autophagy, whereas Lys-63-linked ubiquitination enhances autophagy through dissociating BECN1 from BCL2. 140 More importantly, BECN1 serves as a platform to coordinate various regulators of autophagy, which has been termed the "BECN1 interactome." 85,141 These secondary interacting proteins of class III PtdIns3K complexes, some of which are viewed as the peripheral components of class III PtdIns3K complexes, include AMBRA1, HMGB1 (high mobility group box 1), BIRC5/survivin, PINK1 (PTEN induced putative kinase 1), CISD2/NAF-1 (CDGSH iron sulfur domain 2), and the abovementioned regulators including BCL2; these proteins can either promote (e.g., AMBRA1, HMGB1, BIRC5, PINK1) or inhibit (e.g., CISD2, BCL2) autophagy.…”

Autophagy: for better or for worse

“…Following activation of TLR-1, -3, -4, -5, -6 or -7, myeloid differentiation primary response gene 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) recruit Beclin-1 into the TLR signaling complex and thereby reduce the autophagy-inhibiting interaction between Beclin-1 and Bcl-2 [102]. In addition, TNF receptor-associated factor 6 (TRAF6)-mediated ubiquitination of Beclin-1 was shown to be required for TLR4-triggered autophagy in macrophages [103]. The NLRs (NOD1 (nucleotide-binding oligomerization domaincontaining protein 1) and NOD2) direct Atg16L1 to the site of bacterial entry at the plasma membrane, a process critical for elimination of bacteria by autophagy [104].…”

“…For example, several BH3-only proteins, such as Bcl-2-associated death promoter protein (Bad), tBid (truncated BH3 interacting domain death agonist) and BNIP3, abrogate the Bcl-2-Beclin-1 interaction by competing with Beclin-1 for Bcl-2 binding. Furthermore, other apoptotic modulators (death-associated protein kinase, c-jun N-terminal kinase, TRAF6 and A20) mediate Beclin-1 or Bcl-2 post-translational modifications that enable dissociation of Beclin-1 from Bcl-2 [103,142,143]. Surprisingly, caspases were also implicated in autophagy-mediated survival.…”