Marie W. Wooten

Sequestosome 1/p62 Is a Polyubiquitin Chain Binding Protein Involved in Ubiquitin Proteasome Degradation

ABSTRACT 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.

Structure and functional properties of the ubiquitin binding protein p62

Several highly conserved p62 homologs have recently been isolated, e.g. the rat atypical protein kinase C‐interacting protein (ZIP), the murine A170/signal transduction and adapter protein, and the human p62, a protein that binds the Src homology 2 domain of p56lck. These proteins share striking similarity in amino acid sequence and structural motifs, thereby suggesting conserved functional properties. ZIP/p62 has been shown to play an important role as a scaffold leading to the activation of the transcription factor nuclear factor κB. In addition, a nuclear form of p62 has been characterized that can serve as a transcriptional co‐activator. Moreover, p62 is capable of binding ubiquitin (Ub) non‐covalently through its Ub‐associated domain. In this review, we will focus on the structure and function of ZIP/p62.

Sequestosome 1/p62 shuttles polyubiquitinated tau for proteasomal degradation

Inclusions isolated from several neurodegenerative diseases, including Alzheimers 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 ubiquitin–proteasome 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.

Essential Role of Sequestosome 1/p62 in Regulating Accumulation of Lys63-ubiquitinated Proteins

Sequestosome 1 (SQSTM1)/p62 is an interacting partner of the atypical protein kinase C ζ/ι and serves as a scaffold for cell signaling and ubiquitin binding, which is critical for several cell functions in vivo such as osteoclastogenesis, adipogenesis, and T cell activation. Here we report that in neurons of p62–/– mouse brain there is a detectable increase in ubiquitin staining paralleled by accumulation of insoluble ubiquitinated proteins. The absolute amount of each ubiquitin chain linkage measured by quantitative mass spectrometry demonstrated hyperaccumulation of Lys63 chains in the insoluble fraction recovered from the brain of p62–/– mice, which correlated with increased levels of Lys63-ubiquitinated TrkA receptor. The increase in Lys63 chains was attributed in part to diminished activity of the TRAF6-interacting the Lys63-deubiquitinating enzyme (DUB), cylindromatosis tumor suppressor (CYLD). The interaction of CYLD with TRAF6 was dependent upon p62, thus defining a mechanism that accounts for decreased activity of CYLD in the absence of p62. These findings reveal that p62 serves as an adapter for the formation of this complex, thereby regulating the DUB activity of CYLD by TRAF6 interaction. Thus, p62 has a bifunctional role in regulation of an E3 ubiquitin-protein ligase, TRAF6, and a DUB, CYLD, to balance the turnover of Lys63-polyubiquitinated proteins such as TrkA.

Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration.

The signaling adapter p62 plays a coordinating role in mediating phosphorylation and ubiquitin‐dependent trafficking of interacting proteins. However, there is little known about the physiologic role of this protein in brain. Here, we report age‐dependent constitutive activation of glycogen synthase kinase 3β, protein kinase B, mitogen‐activated protein kinase, and c‐Jun‐N‐terminal kinase in adult p62−/− mice resulting in hyperphosphorylated tau, neurofibrillary tangles, and neurodegeneration. Biochemical fractionation of p62−/− brain led to recovery of aggregated K63‐ubiquitinated tau. Loss of p62 was manifested by increased anxiety, depression, loss of working memory, and reduced serum brain‐derived neurotrophic factor levels. Our findings reveal a novel role for p62 as a chaperone that regulates tau solubility thereby preventing tau aggregation. This study provides a clear demonstration of an Alzheimer‐like phenotype in a mouse model in the absence of expression of human genes carrying mutations in amyloid‐beta protein precursor, presenilin, or tau. Thus, these findings provide new insight into manifestation of sporadic Alzheimer disease and the impact of obesity.

Sequestosome 1/p62 – More than just a scaffold

The interaction of proteins with ubiquitin receptors is key to solving the mystery that surrounds the functional role ubiquitin chains play in directing traffic. The specificity of these interactions is largely mediated by UbL/UBA domains. Sequestosome 1/p62 is a protein that is gaining attention as it is intimately involved in cell signaling, receptor internalization, and protein turnover. Herein we review recent advances in the field.

Sequestosome 1/p62 links familial ALS mutant SOD1 to LC3 via an ubiquitin-independent mechanism

The p62/sequestosome 1 protein has been identified as a component of pathological protein inclusions in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). P62 has also been implicated in autophagy, a process of mass degradation of intracellular proteins and organelles. Autophagy is a critical pathway for degrading misfolded and/or damaged proteins, including the copper‐zinc superoxide dismutase (SOD1) mutants linked to familial ALS. We previously reported that p62 interacted with ALS mutants of SOD1 and that the ubiquitin‐association domain of p62 was dispensable for the interaction. In this study, we identified two distinct regions of p62 that were essential to its binding to mutant SOD1: the N‐terminal Phox and Bem1 (PB1) domain (residues 1–104) and a separate internal region (residues 178–224) termed here as SOD1 mutant interaction region (SMIR). The PB1 domain is required for appropriate oligomeric status of p62 and the SMIR is the actual region interacting with mutant SOD1. Within the SMIR, the conserved W184, H190 and positively charged R183, R186, K187, and K189 residues are critical to the p62–mutant SOD1 interaction as substitution of these residues with alanine resulted in significantly abolished binding. In addition, SMIR and the p62 sequence responsible for the interaction with LC3, a protein essential for autophagy activation, are independent of each other. In cells lacking p62, the existence of mutant SOD1 in acidic autolysosomes decreased, suggesting that p62 can function as an adaptor between mutant SOD1 and the autophagy machinery. This study provides a novel molecular mechanism by which mutant SOD1 can be recognized by p62 in an ubiquitin‐independent fashion and targeted for the autophagy–lysosome degradation pathway.

Nucleolin is a protein kinase C-zeta substrate. Connection between cell surface signaling and nucleus in PC12 cells.

We have previously shown that protein kinase C (PKC)-ζ is activated and required for nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma PC12 cells (Wooten, M. W., Zhou, G., Seibenhener, M. L., and Coleman, E. S. (1994) Cell Growth & Diff. 5, 395–403; Coleman, E. S., and Wooten, M. W. (1994) J. Mol. Neurosci.5, 39–57). Here we report the characterization and identification of a 106-kDa nuclear protein as a specific substrate of PKC-ζ. NGF treatment of PC12 cells resulted in translocation of PKC-ζ and coincident phosphorylation of a protein that was localized within the nucleoplasm of nuclei isolated from PC12 cells. Addition of PKC-ζ pseudosubstrate peptide in vitro or myristoylated peptidein vivo diminished phosphorylation of pp106 in a dose-dependent fashion. Likewise, addition of purified PKC-ζ, but neither PKC-α nor δ, to nuclear extracts resulted in an incremental increase in the phosphorylation of pp106. Expression of dominant-negative PKC-ζ inhibited NGF-induced phosphorylation of pp106, by comparison overexpression of PKC-ζ enhanced basal phosphorylation without a noticeable effect upon NGF-induced effects. Amino acid sequence analysis of four peptides derived from purified pp106 revealed that this protein was homologous to nucleolin. Using anin vitro reconstitution system, purified nucleolin was likewise shown to be phosphorylated by purified PKC-ζ. The staining intensity of both enzyme and substrate in the nucleus increased upon treatment with NGF. In vivo labeling with32Pi and stimulation of PC12 cells with NGF followed by immunoprecipitation with anti-nucleolin antibody corroborated the in vitro approach documenting enhanced phosphorylation of nucleolin by NGF treatment. Taken together, the findings presented herein document that nucleolin is a target of PKC-ζ that serves to relay NGF signals from cell surface to nucleus in PC12 cells.

Atypical PKC ζ is activated by ceramide, resulting in coactivation of NF-κb/JNK kinase and cell survival

Both protein kinase C (PKC) and ceramide play a critical role in cell signaling, but the relationship between PKC and ceramide is unclear. Low concentrations of ceramide were observed to transiently stimulate PKC ζ activity in vitro and in vivo, whereas high doses of ceramide lead to inhibition of PKC ζ. Inhibition of activity was accompanied by enhanced binding of the negative regulator, Par4 to PKC ζ. Treatment of PC12 cells with low doses of ceramide promoted survival in serum‐free media and activation of nuclear factor‐κB, whereas higher doses (>2.5 μM) resulted in cell death. Overexpression of either aPKC isoform, PKC ζ or ι, resulted in enhanced survival of PC12 cells at high doses of ceramide and in ceramide‐stimulated Jun N‐terminal kinase (JNK), without any apparent effect on mitogen‐activated kinase. These findings support a role for ceramide‐induced PKC ζ activity in the control of cell survival signaling via a pathway that also activates JNK kinase. J. Neurosci. 55:293–302, 1999. 

Unc-51-like kinase 1/2-mediated endocytic processes regulate filopodia extension and branching of sensory axons

The molecular mechanism and significance of endocytic processes involved in directional axon elongation are not well understood. The Unc-51 family of serine/threonine kinases was shown to be important for axon growth and was also linked to endocytosis, providing an entry point to study this problem. We found that mouse Unc-51-like kinase 1/2 (Ulk1/2) proteins are localized to vesicular structures in growth cones of mouse spinal sensory neurons. RNAi-mediated knockdown of Ulk1 and/or Ulk2 resulted in impaired endocytosis of nerve growth factor (NGF), excessive axon arborization, and severely stunted axon elongation. The evidence also indicates that Ulk1/2 mediates a non-clathrin-coated endocytosis in sensory growth cones. Interestingly, NGF can induce the interaction of Ulk1 with TrkA receptor complexes through promoting K63-polyubiquitination of Ulk1 and binding of Ulk1 to the scaffolding protein p62. These results and additional studies suggest that Ulk1/2 proteins regulate filopodia extension and neurite branching during sensory axon outgrowth, probably through regulating TrkA receptor trafficking and signaling.