Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydrophobic patch MGF as well as the conserved leucine in helix 2 are necessary for binding polyubiquitinated proteins and for sequestration-aggregate formation. We report that p62 interacts with the proteasome by pull-down assay, coimmunoprecipitation, and colocalization. Depletion of p62 levels results in an inhibition of ubiquitin proteasome-mediated degradation and an accumulation of ubiquitinated proteins. Altogether, our results support the hypothesis that p62 may act as a critical ubiquitin chain-targeting factor that shuttles substrates for proteasomal degradation.Sequestosome 1/p62 is a cellular protein which was initially identified as a phosphotyrosine-independent ligand of the Src homology 2 (SH2) domain of p56 lck (27). The protein has been cloned by two independent groups as a cointeracting protein of the atypical protein kinase C (aPKC) and is also named ZIP for PKC-interacting protein (47, 50). p62 has been shown to bind ubiquitin noncovalently (56), and its overexpression results in large cytoplasmic aggregates (47). We have recently determined that p62 possesses sequence homology with other proteins possessing a ubiquitin-associated (UBA) domain at their C terminus, amino acids 386 to 434 (16), in p62. Interestingly, p62 contains several structural motifs which suggest that it might participate in the formation of multimeric signaling complexes. These domains include an acidic interaction domain (AID/ORCA/PC/PB1) that binds the aPKC, a ZZ finger, a binding site for the RING finger protein TRAF6, two PEST sequences, and the UBA domain (16).Ubiquitin is a small polypeptide of 76 amino acids that can be covalently attached to other proteins. Monoubiquitination serves as a novel endocytosis signal (19), whereas polyubiquitin chains target substrates for degradation by the proteasome (45). p62 has been shown to interact in a noncovalent fashion with polyubiquitin chains (5, 53), which is consistent with reports demonstrating that proteins possessing UBA domains are more likely to bind polyubiquitin chains over monoubiquitin (59). Conjugation of ubiquitin to substrate proteins requires three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Initially, E1 activates ubiquitin by forming a high-energy thioester intermediate with the C-terminal glycine using ATP. The activated ubiquitin is sequentially transferred to E2 and then to E3 which catalyzes isopeptide bond formation between the activated C-terminal glycine of ubiquitin and an ε-amino group of a lysine residue in the substrate (45). However, only HECT-type E3s can form thioester bon...

Nerve growth factor (NGF) binding to p75(NTR) influences TrkA signaling, yet the molecular mechanism is unknown. We observe that NGF stimulates TrkA polyubiquitination, which was attenuated in p75(-/-) mouse brain. TrkA is a substrate of tumor necrosis factor receptor-associated factor 6 (TRAF6), and expression of K63R mutant ubiquitin or an absence of TRAF6 abrogated TrkA polyubiquitination and internalization. NGF stimulated formation of a TrkA/p75(NTR) complex through the p62 scaffold, recruiting the E3/TRAF6 and E2/UbcH7. Peptide targeted to the TRAF6 binding site present in p62 blocked interaction with TRAF6 and inhibited ubiquitination of TrkA, signaling, internalization, and NGF-dependent neurite outgrowth. Mutation of K485 to R blocked TRAF6 and NGF-dependent polyubiquitination of TrkA, resulting in retention of the receptor on the membrane and an absence in activation of specific signaling pathways. These findings reveal that polyubiquitination serves as a common platform for the control of receptor internalization and signaling.

Inclusions isolated from several neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by ubiquitin-positive proteinaceous aggregates. Employing confocal and immunoelectron microscopy, we find that the ubiquitin-associating protein sequestosome1/p62, co-localizes to aggregates isolated from AD but not control brain, along with the E3 ubiquitin ligase, TRAF6. This interaction could be recapitulated by co-transfection in HEK293 cells. Employing both in vitro and in vivo approaches, tau was found to be a substrate of the TRAF6, possessing lysine 63 polyubiquitin chains. Moreover, tau recovered from brain of TRAF6 knockout mice, compared with wild type, was not ubiquitinated. Tau degradation took place through the ubiquitinproteasome pathway and was dependent upon either the K63-polyubiquitin chains or upon p62. In brain lysates of p62 knockout mice, tau fails to co-interact with Rpt1, a proteasomal subunit, thereby indicating a requirement for p62 shuttling of tau to the proteasome. Our results demonstrate that p62 interacts with K63-polyubiquitinated tau through its UBA domain and serves a novel role in regulating tau proteasomal degradation. We propose a model whereby either a decline in p62 expression or a decrease in proteasome activity may contribute to accumulation of insoluble/aggregated K63-polyubiquitinated tau.

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...

High fat diet-induced obesity is associated with insulin resistance (IR) and other chronic, diet related illnesses, including dementia. Alzheimer disease is the most common form of dementia, and is characterized by the presence of amyloid plaques and neurofibrillary tangles in brain. This study was designed to determine whether diet-induced changes in peripheral insulin sensitivity could contribute to alterations in brain insulin signaling and cognitive functions. Six week old, male C57BL/6NHsd mice were randomly assigned a high fat diet (40% energy from fat) with 42g/L liquid sugar (HFS) added to the drinking water or a normal chow diet (12% energy from fat) for 14weeks. Metabolic phenotypes were characterized for energy expenditure, physical activity, and food intake, and glucose and insulin tolerance tests. In addition, we examined the changes in protein expression related to brain insulin signaling and cognitive function. Mice fed HFS exhibited a statistically significant increase in obesity, and lower glucose and insulin tolerance as compared to animals fed the normal chow diet. In brain, HFS elicited IR as evidenced by a significant decrease in tyrosine phosphorylation of insulin receptor and an increase serine phosphorylation of IRS-1. These changes were accompanied by inflammatory (NFκB, JNK) and stress responses (p38 MAPK, CHOP) in whole brain lysate. In addition, HFS mouse brain exhibited biochemical changes related to increased amyloid beta deposition and neurofibrillary tangle formation, and decreased synaptic plasticity. These results suggested changes in insulin sensitivity might contribute to cognitive impairment associated with the HFS diet in mice.