Thangiah Geetha

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.

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.

Anti-inflammatory activity of lupeol and lupeol linoleate in rats.

Two pentacyclic triterpenes, namely lupeol and lupeol linoleate, were investigated for their anti-inflammatory, antinociceptive, anti-pyretic and ulcerogenic properties in comparison with the commonly used non-steroidal anti-inflammatory drug, indomethacin in rats. Lupeol, lupeol linoleate and indomethacin showed a reduction in paw swelling by 39, 58 and 35%, respectively, in adjuvant arthritis. Triterpenes were devoid of any antinociceptive, anti-pyretic and ulcerogenic actions. However, indomethacin exhibited a positive response to these properties. These results suggest that the mechanism of action of triterpenes is different from the non-steroidal anti-inflammatory drug.

Signaling, Polyubiquitination, Trafficking, and Inclusions: Sequestosome 1/p62's Role in Neurodegenerative Disease

Aggregated misfolded proteins are hallmarks of most neurodegenerative diseases. In a chronic disease state, including pathologic situations of oxidative stress, these proteins are sequestered into inclusions. Accumulation of aggregated proteins can be prevented by chaperones, or by targeting their degradation to the UPS. If the accumulation of these proteins exceeds their degradation, they may impair the function of the proteasome. Alternatively, the function of the proteasome may be preserved by directing aggregated proteins to the autophagy-lysosome pathway for degradation. Sequestosome 1/p62 has recently been shown to interact with polyubiquitinated proteins through its UBA domain and may direct proteins to either the UPS or autophagosome. P62 is present in neuronal inclusions of individuals with Alzheimers disease and other neurodegenerative diseases. Herein, we review p62s role in signaling, aggregation, and inclusion formation, and specifically as a possible contributor to Alzheimers disease. The use of p62 as a potential target for the development of therapeutics and as a disease biomarker is also discussed.

Nerve growth factor stimulates multisite tyrosine phosphorylation and activation of the atypical protein kinase C's via a src kinase pathway

ABSTRACT Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-ι becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-ι were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-ι in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-ι were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-ι. Recruitment of PKC-ι into the complex was dependent on the tyrosine phosphorylation state of PKC-ι. The association of src and PKC-ι was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-ι (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-ι. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-ι, whereas the Y325F mutation significantly reduced src-induced activation of PKC-ι. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-κB, with significant impairment by the Y325F PKC-ι mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGFs ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.

TRAF6‐mediated ubiquitination regulates nuclear translocation of NRIF, the p75 receptor interactor

TRAF6 is an E3 ubiquitin ligase that mediates signaling from members of the tumor necrosis factor and Toll‐like receptor superfamilies, including the p75 neurotrophin receptor. Recently, TRAF6 was shown to bind to another p75 cytoplasmic interactor, NRIF, and promote its nuclear localization. Here, we demonstrate that NRIF is a substrate for TRAF6‐mediated K63 polyubiquitination and that this modification is necessary for its nuclear translocation. Activation of p75 resulted in NRIF polyubiquitination, association with TRAF6 and nuclear localization. NRIF was polyubiquitinated by TRAF6 in vitro and in cultured cells, and this was abrogated by mutation of K19 in the amino‐terminus of NRIF. The K19R mutant NRIF displayed reduced TRAF6 association and neurotrophin‐dependent nuclear localization. In neurons from traf6−/− mice, NRIF failed to enter the nucleus in response to p75 activation, and polyubiquitination and nuclear localization were attenuated in traf6−/− brain. Finally, unlike wild‐type NRIF, the K19R NRIF failed to reconstitute p75‐mediated apoptosis in nrif−/− neurons. These results reveal a unique mechanism of p75 signaling and a novel role for K63‐linked ubiquitin chains.

Association of the Atypical Protein Kinase C-interacting Protein p62/ZIP with Nerve Growth Factor Receptor TrkA Regulates Receptor Trafficking and Erk5 Signaling

Previous work demonstrated an essential role for the atypical protein kinase C interacting protein, p62, in neurotrophin survival and differentiation signaling. Here we show that p62 interacts not only with TrkA but also with TrkB and TrkC, which are the primary receptors for brain-derived neurotrophic factor and neurotrophin-3. The interaction of p62 with TrkA requires the kinase activity of TrkA. Mapping analysis indicates that p62 does not compete with Shc for binding to TrkA, and p62 association was confined to the juxtamembrane region of TrkA, amino acids 472–493. By immunofluorescence the colocalization of p62 and TrkA was observed 30 min post-nerve growth factor treatment within overlapping vesicular structures. Upon subcellular fractionation, activated TrkA colocalized to an endosomal compartment and p62 was coassociated with the receptor post-nerve growth factor stimulation. Moreover, an absence of p62 blocked internalization of TrkA without an effect on phosphorylation of either TrkA or MAPK; however, Erk5 signaling was selectively abrogated. We propose that p62 plays a novel role in connecting receptor signals with the endosomal signaling network required for mediating TrkA-induced differentiation.