The p62 Scaffold Regulates Nerve Growth Factor-induced NF-κB Activation by Influencing TRAF6 Polyubiquitination

Abstract

Sequestosome 1/p62 is a scaffolding protein with several interaction modules that include a PB1 dimerization domain, a TRAF6 (tumor necrosis factor receptor-associated factor 6) binding site, and a ubiquitin-associating (UBA) domain. Here, we report that p62 functions to facilitate K63-polyubiquitination of TRAF6 and thereby mediates nerve growth factor-induced activation of the NF-B pathway. In brain of p62 knock-out mice we did not recover polyubiquitinated TRAF6. The UBA domain binds polyubiquitin chains and deletion of p62-UBA domain or mutation of F406V within the ubiquitin binding pocket of the UBA domain abolished TRAF6 polyubiquitination. Likewise, deletion of p62 N-terminal dimerization domain or the TRAF6 binding site had similar effects on both polyubiquitination and oligomerization of TRAF6. Nerve growth factor treatment of PC12 cells induced TRAF6 polyubiquitination along with formation of a p62-TRAF6-IKK␤-PKC signal complex, while inhibition of the p62/TRAF6 interaction had an opposite effect. These results provide evidence for a mechanism whereby p62 serves to regulate the NF-B pathway. Nuclear Factor B (NF-B)2 plays an important role in the regulation of a number of genes involved in neuronal development and survival, as well as numerous other cellular functions, such as inflammation, immunity, and cellular differentiation (1, 2). Constitutive NF-B activity is necessary for neuronal survival (3) and neurite outgrowth (4). In its inactive state NF-B is bound by its inhibitor protein, IB, in the cytoplasm (1). Once a cell is activated, IB is phosphorylated by IB kinase (IKK) and tagged by ubiquitin for proteasomal degradation, freeing NF-B to enter the nucleus (2). Nerve growth factor (NGF) elicits its biological response by binding to two receptors, TrkA and p75. The p75 receptor activates NF-B by binding and interacting directly with TRAF6 (5) and a receptor-associated kinase, IRAK (interleukin receptor-associated kinase) (6). Through an adaptor protein, p62, the p75 receptor connects with the atypical protein kinase Cs (aPKCs), which serve as upstream kinases for phosphorylation and activation of IKK␤ (7) in NGF and IL-1 signaling (8), suggesting a common and conserved function for p62 in both the immune and nervous systems.Sequestosome 1/p62 was cloned as the interacting protein of the atypical PKCs (9, 10) and serves as a scaffold comprised of numerous protein interaction modules that allow effector proteins to converge and interact with their substrates, thus passing the signal downstream to activate 12). Both the aPKCs, and , and tumor necrosis factor receptor-associated factor (TRAF6) specifically interact with p62; aPKC interacts with the PB1 domain (amino acids 3-100), and TRAF6 interacts with the TRAF6 binding domain (amino acids 228 -254) (12). The interaction of p62 and TRAF family members is specific to p62 and TRAF6, as neither TRAF5 nor TRAF2 interact with p62 (8). The N terminus of p62 is also necessary for p62 dimerization (13), and p62 also possesses a UBA domain at its C terminu...

IκB kinase complexes: gateways to NF-κB activation and transcription

“…The relative contribution at these two levels upstream and downstream of IKK and the physiological regulation of b-arrestins in NF-kB pathways are not yet fully explored. Finally, sequestosome 1/p62, a scaffolding protein that also binds to TRAF6 and facilitates TRAF6 oligomerization and Ub Lys63 modification has been shown to be required for the nerve growth factor-induced IKK/NF-kB pathway (Wooten et al, 2005).…”

Identification of a consensus site for TRAF6/p62 polyubiquitination

“…Human embryonic kidney (HEK) 293 and nnr5 cells were grown as previously described [8]. HEK293 cells were transfected with the calcium phosphate method by using a Mammalian Cell Transfection Kit (Specialty Media), and nnr5 cells were transfected by using LipofectAMINE 2000 (Invitrogen Life Technologies).…”

“…Moreover, it appears that the E3 targets an accessible surface residue providing the selection process with a conformational recognition mechanism. The TRAF6-p62 signaling complex leads to autoactivation of TRAF6 [8]. The scaffold, p62, then recruits the substrate enabling the E3 to scan for the easily accessible lysine residues in the loops and helical structures on the surface of the substrate resulting in polyubiquitination at a specific lysine, if the flanking residues fit the consensus motif.…”

“…P62 serves as an adaptor bridge to recruit TRAF6 through its TRAF6 binding site [3,8,9]. Therefore, studies were undertaken to examine whether mutation at the primary ubiquitination site in the TrkB receptor impairs formation of a TRAF6/p62 signaling complex.…”

“…In this regard, p62 has been shown to act as an adaptor and interacts with the TRAF domain of TRAF6, resulting in its auto-activation [7,8]. Recent findings from our lab have revealed that both TrkA [9] and the neurotrophin receptor interacting factor (NRIF) [10] are K63-polyubiquitinated by the TRAF6/p62 complex.…”