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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.
PLoS One ; 8(9): e76016, 2013.
Article in English | MEDLINE | ID: mdl-24086678

ABSTRACT

Protein aggregates can form in the cytoplasm of the cell and are accumulated at aggresomes localized to the microtubule organizing center (MTOC) where they are subsequently degraded by autophagy. In this process, aggregates are engulfed into autophagosomes which subsequently fuse with lysosomes for protein degradation. A member of the class II histone deacetylase family, histone deacetylase 6(HDAC6) has been shown to be involved in both aggresome formation and the fusion of autophagosomes with lysosomes making it an attractive target to regulate protein aggregation. The scaffolding protein sequestosome 1(SQSTM1)/p62 has also been shown to regulate accumulation and autophagic clearance of protein aggregates. Recent studies have revealed colocalization of HDAC6 and p62 to ubiquitinated mitochondria, as well as, ubiquitinated protein aggregates associated with the E3 ubiquitin ligase TRIM50. HDAC6 deacetylase activity is required for aggresome formation and can be regulated by protein interaction with HDAC6. Due to their colocalization at ubiquitinated protein aggregates, we sought to examine if p62 specifically interacted with HDAC6 and if so, if this interaction had any effect on HDAC6 activity and/or the physiological function of cortactin-F-actin assembly. We succeeded in identifying and mapping the direct interaction between HDAC6 and p62. We further show that this interaction regulates HDAC6 deacetylase activity. Data are presented demonstrating that the absence of p62 results in hyperactivation of HDAC6 and deacetylation of α-tubulin and cortactin. Further, upon induction of protein misfolding we show that p62 is required for perinuclear co-localization of cortactin-F-actin assemblies. Thus, our findings indicate that p62 plays a key role in regulating the recruitment of F-actin network assemblies to the MTOC, a critical cellular function that is required for successful autophagic clearance of protein aggregates.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy/physiology , Histone Deacetylases/metabolism , Microtubule-Organizing Center/metabolism , Proteolysis , Actins/metabolism , Autophagy/genetics , Blotting, Western , Glutathione Transferase , HEK293 Cells , Histone Deacetylase 6 , Humans , Immunoprecipitation , Microscopy, Fluorescence , Protein Interaction Mapping , Sequestosome-1 Protein
3.
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
4.
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
5.
J Vis Exp ; (65)2012 Jul 26.
Article in English | MEDLINE | ID: mdl-22871921

ABSTRACT

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.


Subject(s)
Cytological Techniques/methods , Hippocampus/cytology , Hippocampus/embryology , Neurons/cytology , Animals , Cells, Cultured , Dissection/methods , Embryo, Mammalian/cytology , Female , Hippocampus/surgery , Mice , Mice, Inbred C57BL , Pregnancy , Rats
6.
BMC Proc ; 5 Suppl 2: S4, 2011 May 28.
Article in English | MEDLINE | ID: mdl-21554762

ABSTRACT

A new approach is described here to predict ubiquitinated substrates of the E3 ubiquitin ligase, TRAF6, which takes into account its interaction with the scaffold protein SQSTM1/p62. A novel TRAF6 ubiquitination motif defined as [-(hydrophobic)-k-(hydrophobic)-x-x-(hydrophobic)- (polar)-(hydrophobic)-(polar)-(hydrophobic)] was identified and used to screen the TRAF6/p62 interactome composed of 155 proteins, that were either TRAF6 or p62 interactors, or a negative dataset, composed of 54 proteins with no known association to either TRAF6 or p62. NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. Subsequent analyses revealed that this motif was biased to the C-terminal regions of the protein (nearly 50% the sites), and had preference for loops (~50%) and helices (~37%) over beta-strands (15% or less). In addition, the motif was observed to be in regions that were highly solvent accessible (nearly 90%). Our findings suggest that specific Lysines may be selected for ubiquitination based upon an embedded code defined by a specific amino acid motif with structural determinants. Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination. The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

7.
J Neurochem ; 111(4): 1062-73, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19765191

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Heat-Shock Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Superoxide Dismutase/metabolism , Ubiquitin/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cells, Cultured , Embryo, Mammalian , Heat-Shock Proteins/genetics , Humans , Immunoprecipitation , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/genetics , Motor Neurons/metabolism , Mutation/genetics , Protein Binding , Protein Structure, Tertiary/physiology , Sequestosome-1 Protein , Spinal Cord/cytology , Superoxide Dismutase/chemistry , Superoxide Dismutase/genetics , Transfection , Ubiquitin/genetics
8.
Neurobiol Dis ; 35(2): 302-10, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19481605

ABSTRACT

Recently we reported that declined SQSTM1/p62 expression in Alzheimer disease brain was age-correlated with oxidative damage to the p62 promoter. The objective of this study was to examine whether oxidative damage to the p62 promoter is common to DNA recovered from brain of individuals with neurodegenerative disease. Increased 8-OHdG staining was observed in brain sections from Alzheimer's disease (AD), Parkinson disease (PD), Huntington disease (HD), Frontotemporal dementia (FTD), and Pick's disease compared to control subjects. In parallel, the p62 promoter exhibited elevated oxidative damage in samples from various diseases compared to normal brain, and damage was negatively correlated with p62 expression in FTD samples. Oxidative damage to the p62 promoter induced by H2O2 treatment decreased its transcriptional activity. In keeping with this observation, the transcriptional activity of a Sp-1 element deletion mutant displayed reduced stimulus-induced activity. These findings reveal that oxidative damage to the p62 promoter decreased its transcriptional activity and might therefore account for decreased expression of p62. Altogether these results suggest that pharmacological means to increase p62 expression may be beneficial in delaying the onset of neurodegeneration.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Neurodegenerative Diseases/genetics , Oxidative Stress/genetics , Promoter Regions, Genetic , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Brain/drug effects , Brain/pathology , Brain/physiopathology , Cell Line , Dementia/genetics , Dementia/pathology , Female , Humans , Huntington Disease/genetics , Huntington Disease/pathology , Hydrogen Peroxide/pharmacology , Male , Middle Aged , Oxidants/pharmacology , Oxidative Stress/drug effects , Parkinson Disease/genetics , Parkinson Disease/pathology , Pick Disease of the Brain/genetics , Pick Disease of the Brain/pathology , Promoter Regions, Genetic/drug effects , Sequestosome-1 Protein , Transcription, Genetic/drug effects
9.
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
10.
Free Radic Biol Med ; 46(4): 492-501, 2009 Feb 15.
Article in English | MEDLINE | ID: mdl-19071211

ABSTRACT

The absence of the p62 gene in mouse brain leads to biochemical and cognitive deficits that resemble Alzheimer disease (AD). In this context, the objective of this study was to examine the relationship between age-induced oxidative damage to the p62 promoter and AD. Increased 8-OHdG staining, a marker of oxidative stress, was observed in brain sections from mice deficient in the p62 gene compared to control. Treatment of MEF cells deficient in p62 with H(2)O(2) resulted in decreased cell survival and an absence of Nrf2 nuclear translocation. The mouse p62 promoter exhibited elevated oxidative damage with increasing age, and the degree of p62 promoter damage was also age-correlated in human brain samples. In human subjects, the expression of p62 was decreased in AD brain relative to age-matched controls, and likewise decreased p62 expression correlated with oxidative damage to the promoter. Treatment of HEK cells with H(2)O(2) resulted in decreased p62 expression concomitant with increased promoter damage. Consistent with these findings, a transgenic AD mouse model also exhibited increased p62 promoter damage and reduced p62 levels in brain. Altogether, our results reveal that oxidative damage to the p62 promoter correlates with decreased expression of p62 and may contribute to age-associated neurodegenerative disease such as AD and others.


Subject(s)
Aging/physiology , Alzheimer Disease/genetics , DNA Damage/physiology , Oxidative Stress/physiology , Proto-Oncogene Proteins c-myc/metabolism , Active Transport, Cell Nucleus/physiology , Adolescent , Adult , Aged , Aged, 80 and over , Alzheimer Disease/enzymology , Alzheimer Disease/pathology , Animals , Cell Line , Cell Survival/physiology , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Knockout , Middle Aged , NF-E2-Related Factor 2/physiology , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/genetics , Risk Factors , Severity of Illness Index
11.
J Proteomics Bioinform ; 2: 316, 2009 Jul 24.
Article in English | MEDLINE | ID: mdl-20148194

ABSTRACT

It has been more than 30 years since the initial report of the discovery of ubiquitin as an 8.5 kDa protein of unknown function expressed universally in living cells. And still, protein modification by covalent conjugation of the ubiquitin molecule is one of the most dynamic posttranslational modifications studied in terms of biochemistry and cell physiology. Ubiquitination plays a central regulatory role in number of eukaryotic cellular processes such as receptor endocytosis, growth-factor signaling, cell-cycle control, transcription, DNA repair, gene silencing, and stress response. Ubiquitin conjugation is a three step concerted action of the E1-E2-E3 enzymes that produces a modified protein. In this review we investigate studies undertaken to identify both ubiquitin and SUMO (small ubiquitin-related modifier) substrates with the goal of understanding how lysine selectivity is achieved. The SUMOylation pathway though distinct from that of ubiquitination, draws many parallels. Based upon the recent findings, we present a model to explain how an individual ubiquitin ligase may target specific lysine residue(s) with the co-operation from a scaffold protein.

12.
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
13.
Biochem Biophys Res Commun ; 371(3): 521-4, 2008 Jul 04.
Article in English | MEDLINE | ID: mdl-18457658

ABSTRACT

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an ubiquitin ligase that regulates a diverse array of physiological processes via forming Lys-63 linked polyubiquitin chains. In this study, the lysine selection process for TRAF6/p62 ubiquitination was examined. The protein sequence of two characterized TRAF6/p62 substrates, NRIF and TrkA, revealed a conserved consensus pattern for the ubiquitination site of these two TRAF6 substrates. The consensus pattern established in the verified substrates was common to the other Trk receptor family members, TrkB and TrkC. Interestingly, Lysine 811 in TrkB was selected for ubiquination, and mutation of Lysine 811 diminished the formation of TRAF6/p62 complex that is necessary for effective ubiquination. Moreover, downstream signaling was affected upon binding of BDNF to the mutant TrkB receptor. These findings reveal a possible selection process for targeting a specific lysine residue by a single E3 ligase and underscore the role of the scaffold, p62, in this process.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Consensus Sequence , Polyubiquitin/metabolism , TNF Receptor-Associated Factor 6/metabolism , Ubiquitination , Amino Acid Motifs , Amino Acid Sequence , Cell Line , Humans , Lysine/genetics , Lysine/metabolism , Mutation , Receptor, trkA/genetics , Receptor, trkA/metabolism , Receptor, trkB/genetics , Receptor, trkB/metabolism , Receptor, trkC/genetics , Receptor, trkC/metabolism , Sequestosome-1 Protein
14.
Traffic ; 9(7): 1146-56, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18419753

ABSTRACT

Gaps in our knowledge exist regarding the degradation of the tropomyosin-regulated kinase A (TrkA) receptor after addition of neurotrophin, nerve growth factor (NGF). TrkA is rapidly and transiently ubiquitinated upon addition of NGF. Here, we demonstrate that the polyubiquitin tag plays a definitive role in receptor sorting. Treatment of PC12 cells with lactacystin prevented NGF-dependent deubiquitination and degradation of TrkA. However, treatment with methylamine, bafilomycin or leupeptin, did not prevent NGF-dependent deubiquitination but blocked the degradation of TrkA. Employing co-immunoprecipitation, biochemical fractionation and confocal microscopy, the kinetics of receptor trafficking post-internalization was observed to occur as a sequel from endosome/multivesicular body, proteasomes, culminating with degradation in the lysosomes. The trafficking of the polyubiquitin-deficient TrkA receptor mutant K485R was impaired and likewise failed to degrade revealing that the receptor escapes degradation. The interaction of TrkA with proteasomes was confirmed by purification and co-immunoprecipitation. We provide evidence that proteasomal deubiquitinating enzymes trim K63-ubiquitin chains from the TrkA receptor prior to its delivery to lysosomes for degradation. Taken together, our results reveal the existence of a novel proteasome-dependent step in receptor degradation.


Subject(s)
Endosomes/metabolism , Lysosomes/metabolism , Proteasome Endopeptidase Complex/chemistry , Receptor, trkA/metabolism , Ubiquitin/chemistry , Animals , Humans , Kinetics , Leupeptins/chemistry , Macrolides/pharmacology , Methylamines/chemistry , Mutation , PC12 Cells , Rats , Subcellular Fractions/metabolism
15.
J Neurochem ; 106(1): 107-20, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18346206

ABSTRACT

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 3beta, 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.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Brain/metabolism , Heat-Shock Proteins/genetics , Molecular Chaperones/genetics , Nerve Degeneration/genetics , Neurons/metabolism , tau Proteins/metabolism , Aging/genetics , Aging/metabolism , Animals , Brain/pathology , Brain/physiopathology , Brain-Derived Neurotrophic Factor/metabolism , Disease Models, Animal , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Metabolic Syndrome/complications , Metabolic Syndrome/genetics , Metabolic Syndrome/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/metabolism , Nerve Degeneration/metabolism , Nerve Degeneration/physiopathology , Neurofibrillary Tangles/genetics , Neurofibrillary Tangles/metabolism , Neurofibrillary Tangles/pathology , Neurons/pathology , Obesity/complications , Obesity/metabolism , Obesity/physiopathology , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Sequestosome-1 Protein , Signal Transduction/physiology , Solubility
16.
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
17.
Biochem Biophys Res Commun ; 361(3): 605-10, 2007 Sep 28.
Article in English | MEDLINE | ID: mdl-17669366

ABSTRACT

Fasciculation and elongation protein zeta-1 (FEZ1) promotes efficiently the neurite elongation of rat phaeochromocytoma PC12 cells. We here characterized FEZ1 in PC12 cells. Nerve growth factor (NGF) stimulation induces significant expression of endogenous FEZ1 in PC12 cells. Upon NGF stimulation FEZ1 localizes in both cytoplasm and neuritis, co-localizing with mitochondria. Silencing of FEZ1 by RNA interference efficiently reduces NGF-induced neurite elongation and the anterograde motility of mitochondria in PC12 cells. Immunoprecipitation and pulldown assay shows that FEZ1 interacts with kinesin superfamily protein 5 (KIF5) and tubulin. Thus, our results suggest that the FEZ1/kinesin complex functions for the transport of mitochondria along microtubules toward the extending neurites in differentiating PC12 cells.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Kinesins/metabolism , Mitochondria/metabolism , Neurites/metabolism , Tubulin/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Axons/metabolism , Biological Transport , Cell Differentiation , Cytoplasm/metabolism , Immunoprecipitation , Nerve Growth Factor/pharmacology , Neurites/drug effects , Neurons/metabolism , PC12 Cells , RNA Interference , RNA, Messenger/metabolism , Rats
18.
Proc Natl Acad Sci U S A ; 104(14): 5842-7, 2007 Apr 03.
Article in English | MEDLINE | ID: mdl-17389358

ABSTRACT

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.


Subject(s)
Axons/physiology , Endocytosis , Protein Serine-Threonine Kinases/metabolism , Pseudopodia/physiology , Animals , Autophagy-Related Protein-1 Homolog , Axons/drug effects , Cell Line , Embryo, Mammalian , Fluorescent Antibody Technique, Direct , Ganglia, Spinal/cytology , Ganglia, Spinal/enzymology , Green Fluorescent Proteins/metabolism , Growth Cones/enzymology , Growth Cones/physiology , Humans , Mice , Nerve Growth Factor/pharmacology , Neurites/physiology , Neurons, Afferent/enzymology , Protein Serine-Threonine Kinases/genetics , RNA Interference , RNA, Small Interfering/metabolism , Receptor Cross-Talk/drug effects , Receptor, trkA/metabolism , Transcription Factor TFIIH , Transcription Factors/metabolism , Ubiquitin/metabolism
19.
Trends Biochem Sci ; 32(2): 95-100, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17174552

ABSTRACT

Signal specificity of multifunctional enzymes is achieved through protein-protein interactions involving specific domains on scaffold proteins. p62 (also known as sequestosome 1) is such a scaffold protein that possesses PB1 and UBA domains, and the TRAF6 binding sequence. Proteins recruited to these domains enable p62 to integrate kinase-activated and ubiquitin-mediated signaling pathways. The biological function of p62 has been studied in diverse systems and processes such as osteoclastogenesis, inflammation, differentiation, neurotrophin biology and obesity. The availability of mice in which p62 has been genetically inactivated is providing new insight into the mechanism and function of p62 at a whole-organism level.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/metabolism , Signal Transduction , DNA-Binding Proteins , Humans , NF-kappa B/metabolism , Nuclear Proteins/metabolism , Sequestosome-1 Protein , TNF Receptor-Associated Factor 6/metabolism , Transcription Factors/metabolism , Ubiquitin/metabolism
20.
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
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