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1.
J Cell Sci ; 127(Pt 18): 4052-63, 2014 Sep 15.
Article in English | MEDLINE | ID: mdl-25015291

ABSTRACT

The dynein motor protein complex is required for retrograde transport of vesicular cargo and for transport of aggregated proteins along microtubules for processing and degradation at perinuclear aggresomes. Disruption of this process leads to dysfunctional endosome accumulation and increased protein aggregation in the cell cytoplasm, both pathological features of neurodegenerative diseases. However, the exact mechanism of dynein functionality in these pathways is still being elucidated. Here, we show that the scaffolding protein SQSTM1 directly interacts with dynein through a previously unidentified dynein-binding site. This interaction is independent of HDAC6, a known interacting protein of both SQSTM1 and dynein. However, knockdown of HDAC6 increases the interaction of SQSTM1 with dynein, indicating a possible competitive interaction. Using different dynein cargoes, we show that SQSTM1 is required for proper dynein motility and trafficking along microtubules. Based on our results, we propose a new model of competitive interaction between SQSTM1 and HDAC6 with dynein. In this model, SQSTM1 would not only affect the association of polyubiquitylated protein aggregates and endosomes with dynein, but would also be required for normal dynein function.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cytoplasmic Dyneins/metabolism , Heat-Shock Proteins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Animals , Cytoplasmic Dyneins/genetics , Endosomes/metabolism , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Histone Deacetylase 6 , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Mice , Mice, Knockout , Microtubules/metabolism , Protein Binding , Protein Structure, Tertiary , Protein Transport , Sequestosome-1 Protein
2.
Behav Brain Res ; 248: 94-103, 2013 Jul 01.
Article in English | MEDLINE | ID: mdl-23591541

ABSTRACT

Affective spectrum and anxiety disorders have come to be recognized as the most prevalently diagnosed psychiatric disorders. Among a suite of potential causes, changes in mitochondrial energy metabolism and function have been associated with such disorders. Thus, proteins that specifically change mitochondrial functionality could be identified as molecular targets for drugs related to treatment for affective spectrum disorders. Here, we report generation of transgenic mice overexpressing the scaffolding and mitophagy related protein Sequestosome1 (SQSTM1/p62) or a single point mutant (P392L) in the UBA domain of SQSTM1/p62. We show that overexpression of SQSTM1/p62 increases mitochondrial energy output and improves transcription factor import into the mitochondrial matrix. These elevated levels of mitochondrial functionality correlate directly with discernible improvements in mouse behaviors related to affective spectrum and anxiety disorders. We also describe how overexpression of SQSTM1/p62 improves spatial learning and long term memory formation in these transgenic mice. These results suggest that SQSTM1/p62 provides an attractive target for therapeutic agents potentially suitable for the treatment of anxiety and affective spectrum disorders.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Anxiety/genetics , Depression/genetics , Mitochondria/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Behavior, Animal/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/genetics , Mutation/genetics , Transcription Factor TFIIH
3.
Biochim Biophys Acta ; 1833(3): 452-9, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23147249

ABSTRACT

As a signaling scaffold, p62/sequestosome (p62/SQSTM1) plays important roles in cell signaling and degradation of misfolded proteins. While localization of p62 to mitochondria has been reported, a description of its function once there, remains unclear. Here, we report that p62 is localized to mitochondria in non-stressed situations and demonstrate that deficiency in p62 exacerbates defects in mitochondrial membrane potential and energetics leading to mitochondrial dysfunction. We report on the relationship between mitochondrial protein import and p62. In a p62 null background, mitochondrial import of the mitochondrial transcription factor TFAM is disrupted. When p62 is returned, mitochondrial function is restored to more normal levels. We identify for the first time that p62 localization plays a role in regulating mitochondrial morphology, genome integrity and mitochondrial import of a key transcription factor. We present evidence that these responses to the presence of p62 extend beyond the protein's immediate influence on membrane potential.


Subject(s)
Adaptor Proteins, Signal Transducing/physiology , DNA, Mitochondrial/genetics , DNA-Binding Proteins/metabolism , Genome, Mitochondrial , Heat-Shock Proteins/physiology , High Mobility Group Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Turnover , Adenosine Triphosphate/metabolism , Animals , Blotting, Western , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Genomic Instability , Immunoenzyme Techniques , Membrane Potential, Mitochondrial , Mice , Mice, Knockout , Reactive Oxygen Species/metabolism , Sequestosome-1 Protein
4.
Hippocampus ; 19(4): 392-406, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19004011

ABSTRACT

SQSTM1/p62 is a multidomain/scaffold for the atypical protein kinase Cs (aPKC). Phosphorylation of AMPA receptors by PKC has been shown to regulate their insertion in the postsynaptic membrane. Here, we directly tested whether p62 could interact with AMPA receptor subunits and influence their trafficking and phosphorylation. GluR1 receptor intracellular loop L2-3 and the ZZ-type zinc finger domain of p62 are essential for the interaction between these two proteins. In this context, both p62 and aPKC-mediated phosphorylation were necessary for surface delivery of the receptor. Our findings reveal that p62 is the first protein identified that interacts with a region of the GluR receptor other than the C-terminal tail. Furthermore, mice deficient in p62 displayed impaired hippocampal CA1 long-term potentiation (LTP), along with diminished surface expression of GluR1 and phosphorylation of S818. Lastly, we identify a conserved sequence (ISExSL) shared by all p62 interacting-aPKC substrates. These findings support a model where p62 interaction and aPKC phosphorylation act together to mediate AMPA receptor trafficking and long-term synaptic plasticity in the hippocampus.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Heat-Shock Proteins/metabolism , Neuronal Plasticity/physiology , Receptors, AMPA/metabolism , Synaptic Transmission , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Animals , Binding Sites/physiology , Cell Line , Cell Membrane/metabolism , Conserved Sequence , Heat-Shock Proteins/genetics , Hippocampus/physiology , Humans , Membrane Potentials , Mice , Mice, Knockout , Molecular Sequence Data , Phosphorylation , Protein Conformation , Protein Kinase C/metabolism , Sequestosome-1 Protein
5.
Biochem Biophys Res Commun ; 374(1): 33-7, 2008 Sep 12.
Article in English | MEDLINE | ID: mdl-18598672

ABSTRACT

The scaffold protein p62 is involved in internalization and trafficking of TrkA. The receptor is deubiquitinated by the proteasomes prior to degradation by lysosomes. Here we demonstrate that p62 serves as a shuttling protein for interaction of ubiquitinated TrkA with Rpt1, one of the six ATPases of 19S regulatory particle of the 26S proteasome. In p62(-/-) mouse brain TrkA failed to interact with the Rpt1. The interaction of TrkA with Rpt1 was reduced in proteasomes isolated from p62(-/-) brain, but was restored by addition of p62. The UBA domain of p62 interacts with TrkA and its PB1/UbL domain with AAA-ATPase cassette in the C-terminal region of Rpt1. Last, neurotrophin-dependent turnover of TrkA was impaired by reduction in the level of p62. These findings reveal that p62 serves as a shuttling factor for interaction of ubiquitinated substrates with the proteasome and could promote localized protein turnover in neurons.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Brain/enzymology , Heat-Shock Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Receptor, trkA/metabolism , Animals , Brain/cytology , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Knockout , Neurons/enzymology , Protein Structure, Tertiary , Sequestosome-1 Protein , Ubiquitination
6.
J Biol Chem ; 283(11): 6783-9, 2008 Mar 14.
Article in English | MEDLINE | ID: mdl-18174161

ABSTRACT

Sequestosome 1 (SQSTM1)/p62 is an interacting partner of the atypical protein kinase C zeta/iota 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.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Gene Expression Regulation , Heat-Shock Proteins/chemistry , Lysine/chemistry , Ubiquitin/chemistry , Adaptor Proteins, Signal Transducing/physiology , Animals , Cysteine Endopeptidases/metabolism , Deubiquitinating Enzyme CYLD , Heat-Shock Proteins/physiology , Humans , Mass Spectrometry/methods , Mice , Mice, Knockout , Mice, Transgenic , Polyubiquitin/chemistry , Proteasome Endopeptidase Complex/metabolism , Rats , Receptor, trkA/chemistry , Receptor, trkA/metabolism , Sequestosome-1 Protein , TNF Receptor-Associated Factor 6/metabolism
7.
FEBS Lett ; 581(2): 175-9, 2007 Jan 23.
Article in English | MEDLINE | ID: mdl-17188686

ABSTRACT

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.


Subject(s)
Proteins/chemistry , Proteins/metabolism , Adaptor Proteins, Signal Transducing , Humans , Protein Structure, Tertiary , Proteins/genetics , Sequestosome-1 Protein , Ubiquitin/metabolism
8.
J Biomed Biotechnol ; 2006(3): 62079, 2006.
Article in English | MEDLINE | ID: mdl-17047309

ABSTRACT

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 Alzheimer's disease and other neurodegenerative diseases. Herein, we review p62's role in signaling, aggregation, and inclusion formation, and specifically as a possible contributor to Alzheimer's disease. The use of p62 as a potential target for the development of therapeutics and as a disease biomarker is also discussed.

9.
FEBS Lett ; 579(22): 5029-34, 2005 Sep 12.
Article in English | MEDLINE | ID: mdl-16129434

ABSTRACT

The polyubiquitin-binding protein p62 has been shown to localize in aggregates common to several types of diseases. Here, we report that p62 forms independent fibrillar aggregates in vitro in a time- and concentration-dependent manner. FTIR spectra and ThT fluorescence assay of p62 reveals increased beta-sheet content as aggregates form compared to the native protein. The fibrillar nature of the aggregates was observed by transmission electron microscopy. Overexpression of p62 in HEK cells results in aggregate formation that may protect cells from apoptosis. Altogether, these results suggest that p62 fibrils may influence cell viability and indicates an important role for p62 in aggresome formation.


Subject(s)
Cell Survival , Proteins/chemistry , Proteins/metabolism , Adaptor Proteins, Signal Transducing , Cell Line , Humans , Multiprotein Complexes/ultrastructure , Protein Conformation , Proteins/genetics , Proteins/ultrastructure , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequestosome-1 Protein , Spectroscopy, Fourier Transform Infrared
10.
J Biol Chem ; 280(42): 35625-9, 2005 Oct 21.
Article in English | MEDLINE | ID: mdl-16079148

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-kappaB 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-IKKbeta-PKC iota 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-kappaB pathway.


Subject(s)
Heat-Shock Proteins/physiology , NF-kappa B/metabolism , Proteins/physiology , TNF Receptor-Associated Factor 6/metabolism , Ubiquitin/metabolism , Adaptor Proteins, Signal Transducing , Animals , Binding Sites , Blotting, Western , Brain/metabolism , Cell Line , Dimerization , Dose-Response Relationship, Drug , Gene Deletion , Glycerol/chemistry , Heat-Shock Proteins/chemistry , Humans , Immunoprecipitation , Mice , Mice, Knockout , Models, Genetic , Nerve Growth Factor/metabolism , PC12 Cells , Protein Binding , Protein Structure, Tertiary , Proteins/chemistry , Rats , Sequestosome-1 Protein , Signal Transduction , Time Factors , Ubiquitin/chemistry , Ultracentrifugation
11.
Mol Cell Biol ; 24(18): 8055-68, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15340068

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.


Subject(s)
Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Polyubiquitin/metabolism , Proteins/metabolism , Ubiquitin/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Binding Sites/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Cell Survival , Humans , Molecular Sequence Data , Proteasome Endopeptidase Complex , Protein Structure, Tertiary , Proteins/chemistry , Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Sequestosome-1 Protein , Signal Transduction , TNF Receptor-Associated Factor 6
12.
J Biol Chem ; 279(6): 4161-5, 2004 Feb 06.
Article in English | MEDLINE | ID: mdl-14684752

ABSTRACT

We have previously shown that the activity of the interleukin-1 (IL-1) receptor-associated kinase (IRAK) is required for nerve growth factor (NGF)-induced activation of NF-kappaB and cell survival ((2002) J. Biol. Chem. 277, 28010-28018). Herein we demonstrate that NGF induces co-association of IRAK with atypical protein kinase C iota (PKC) and that the iota PKC.IRAK complex is recruited to the p75 neurotrophin receptor. Recruitment of IRAK to the receptor was dependent upon the activity of the iota PKC. Moreover, transfection of kinase-dead iota PKC blocked both NGF- and IL-1-induced IRAK activation and the activity of NF-kappaB. Hence, iota PKC lies upstream of IRAK in the kappaB pathway. Examining the primary structure of IRAK, we identified three putative PKC phosphorylation sites; iota PKC selectively phosphorylated peptide 1 (RTAS) within the death domain domain at Thr66, which is highly conserved among all IRAK family members. Mutation of Thr66 to Ala impaired the autokinase activity of IRAK and reduced its association with iota PKC but not TRAF6, resulting in impaired NGF- as well as IL-1-induced NF-kappaB activation. These findings provide insight into the underlying mechanism whereby IRAK regulates the kappaB pathway and reveal that IRAK is a substrate of iota PKC.


Subject(s)
Isoenzymes/metabolism , Protein Kinase C/metabolism , Protein Kinases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line , DNA, Complementary/genetics , Humans , In Vitro Techniques , Interleukin-1 Receptor-Associated Kinases , Isoenzymes/genetics , Mutagenesis, Site-Directed , Nerve Growth Factor/pharmacology , PC12 Cells , Phosphorylation , Protein Kinase C/genetics , Protein Kinases/deficiency , Protein Kinases/genetics , Rats , Receptor, Nerve Growth Factor , Receptors, Nerve Growth Factor/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Transfection
13.
J Cell Biochem ; 85(1): 42-53, 2002.
Article in English | MEDLINE | ID: mdl-11891849

ABSTRACT

Herein, we employed a combined approach of molecular modeling and site-directed mutagenesis to address the role of tyrosine phosphorylation in transport of atypical protein kinase C (aPKC) into the nucleus. Computer modeling of the three-dimensional structure of the aPKC catalytic core, reveals that tyrosine 256 (Tyr256) is located at the lip of the activation loop and is conserved among members of the aPKC family, iota/lambda and zeta. Based on these findings, we examined whether tyrosine phosphorylation of aPKC on the activation lip may facilitate nuclear import. An antiserum was generated that selectively recognizes the phosphorylated Tyr256 residue in aPKC. By isolating nuclei of PC12 cells and immunoprecipitating aPKC with Ab-PY256, we observed that Tyr256 is rapidly phosphorylated upon NGF treatment prior to entry of aPKC into the nucleus. aPKC was observed to exclusively bind to importin-beta. The interaction between importin-beta and aPKC was enhanced upon tyrosine phosphorylation of aPKC and binding was abrogated when Tyr256 was mutated to phenylalanine. We propose that phosphorylation of aPKC at Tyr256 induces a conformation, whereby, the arginine-rich NLS is exposed, which then binds importin-beta leading to import of aPKC into the nucleus. Altogether, these findings document a novel role for the tyrosine phosphorylation in regulating import of atypical PKC into the nucleus.


Subject(s)
Cell Nucleus/metabolism , Protein Kinase C/metabolism , Tyrosine/metabolism , Active Transport, Cell Nucleus/physiology , Animals , Antibodies/analysis , Antibodies/immunology , Binding Sites , Catalytic Domain/physiology , Cell Line , Humans , Models, Molecular , Moths , Mutagenesis, Site-Directed , Nerve Growth Factor/pharmacology , Nuclear Localization Signals/metabolism , PC12 Cells/drug effects , Phosphorylation , Protein Conformation , Protein Kinase C/immunology , Rats , beta Karyopherins/metabolism , src-Family Kinases/metabolism
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