Phosphorylation of serine 349 of p62 in Alzheimer’s disease brain
© TANJI et al.; licensee BioMed Central Ltd. 2014
Received: 27 February 2014
Accepted: 22 April 2014
Published: 3 May 2014
Extensive research on p62 has established its role in oxidative stress, protein degradation and in several diseases such as Paget’s disease of the bone, frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Importantly, previous studies showed that p62 binds directly to Keap1, which is a ubiquitin E3 ligase responsible for degrading Nrf2. Indeed, colocalisation of p62 and Keap1 occurs in tumorigenesis and neurodegeneration. A serine (S) residue in the Keap1-interacting region of p62 is phosphorylated in hepatocellular carcinoma, and this phosphorylation contributes to tumour growth through the higher affinity of p62 to Keap1. However, it remains largely unknown whether p62 is phosphorylated in the Keap1-interacting region under neurodegenerative conditions.
To answer this question, we generated an antibody against phosphorylated S349 (P-S349) of p62 and showed that S349 is phosphorylated following disruption of protein degradation. In particular, the ratio of P-S349 to total p62 levels was significantly increased in the brains with Alzheimer’s disease (AD) compared with controls. We also compared the reactivity of the P-S349 antibody with P-S403 of p62 and showed that these two phosphorylated sites on p62 cause different responses with proteasome inhibition and show distinct localisation patterns in AD brains. In addition to disruption of protein degradation systems, activation of oxidative stress can induce P-S349.
These results support the hypothesis that disruption of protein degradation systems and sustained activation of the Keap1-Nrf2 system occur in the brains with AD.
Accumulation of misfolded or abnormally modified proteins is a major characteristic of many neurodegenerative diseases and is largely attributed to aging, oxidative stress, and genetic and environmental factors. Additionally, protein aggregates can occur in any situation causing intracellular disruption of the protein degradation system. Two major systems for protein degradation exist in mammals, the autophagy-lysosome system and the ubiquitin-proteasome system. Both systems cooperatively play an important role in intracellular protein degradation in the brain. Further studies using a brain-specific deletion of each system have shown that mice exhibit neurological deficits with age and that misfolded proteins are accumulated in neurons [1, 2].
p62/SQSTM1/sequestosome 1 (referred to as p62) is a multifunctional protein that is highly involved in protein degradation. p62 contains a ubiquitin-associated (UBA) domain at the C-terminus, thereby interacting with ubiquitinated and misfolded proteins [3, 4]. Additionally, p62 binds to one of the proteasomal subunits, regulatory particle 1 (Rpt1), through Phox and Bem1p (PB1) domains at the N-terminus . In addition, p62 interacts with the autophagy-related gene (ATG) 8 family , which is essential to autophagosomal formation . Because of its unique properties, it has been suggested that p62 functions as an adaptor protein to transport ubiquitinated and misfolded proteins for proteasomal and autophagic degradation. Importantly, because p62 itself is degraded by autophagy , increased levels of the p62 protein suggests that autophagic flux is impaired.
Recently, we assessed the level of p62 in the brains of patients with neurodegenerative dementia, Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB), and showed that the level of p62 was significantly increased in the brains of patients with AD relative to controls [9, 10]. Furthermore, consistent with previous reports [11–13], several genes related to the stress response and detoxification were also increased in the brains with AD compared with controls. Interestingly, recent studies have shown that p62 binds directly to Keap1 [14–17], which functions as a stress sensor through regulation of NF-E2 related factor 2 (Nrf2) . p62 is reported to be one of the Nrf2-target genes and was also identified as an antioxidant-responsive gene [15, 19]. These findings suggest a tight relationship between stress responses and protein degradation dysfunction.
In this study, we focused on the binding region of p62 with Keap1 (amino acids 344–356 of human p62). Notably, Hancock et al. and Ichimura et al. demonstrated that phosphorylation of serine 349 (S349) enhanced the binding affinity between Keap1 and p62 [20, 21]. However, it remains unclear whether this phosphorylation occurs in neurodegenerative conditions. Here, we generated an antibody specific to S349 of p62 and demonstrated that S349 was phosphorylated in the brains of patients with AD, with levels significantly higher in AD relative to controls. Further studies showed that S349 on p62 was phosphorylated upon disruption of the protein degradation systems and exposure to sustained oxidative stress.
Materials and methods
For generation of antibodies against phosphorylated p62 at S349, rabbits were immunised with a synthetic peptide based on residues 344–354 of human p62 with phosphorylated S349 (P-S349), conjugated at the amino terminus by an additional cysteine to keyhole limpet hemocyanine together with adjuvants. Antisera were purified by obtaining flow-through fractions from a column conjugated with p62 peptide. Phosphorylated p62 was characterised by ELISA by plating immunogen peptide in multiwell plates. To demonstrate specificity, the rabbit antisera against P-S349 were pre-absorbed with phosphorylated peptide. After centrifugation, the supernatant was filtered and used for experiments. For immunohistochemical studies, treatment with alkaline phosphatase (NEB, Beverly, MA, USA) was also performed before incubation with the primary antibody. An anti-p62 antibody specific to phosphorylation at S403 of human p62 was purchased from Millipore (Bedford, MA, USA) .
Summary of human subjects
Cell cultures and treatment
SH-SY5Y human neuroblastoma (ECACC, Salisbury, Wiltshire, UK) and HeLa (JCRB, Osaka, Japan) cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and antibiotics. Cells were treated with epoxomicin (Calbiochem, San Diego, CA, USA), MG132 (Calbiochem), bortezomib (Cell Signaling Technology, Inc., Danvers, MA, USA), bafilomycin A (Wako, Osaka, Japan), and amyloid β peptide 1–40 or 1–42 (Aβ40 or Aβ42) (Peptide Institute Inc., Osaka, Japan). For activation of the Nrf2 system, diethyl maleate (DEM) was used at 100 μM. At the indicated times, the cultures were washed with phosphate-buffered saline (PBS) (pH 7.4), harvested and used as samples for further studies.
Human p62 cDNA was prepared as previously described . p62 cDNA was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA, USA) tagged with hemagglutinin (HA) or pEGFP-N1 (Invitrogen). Mutagenesis was performed according to the manufacturer’s instructions (Takara, Otsu, Japan), followed by sequencing to confirm the mutation site. Serine (S) was changed into glutamic acid (E) for a phosphorylation-mimetic mutant, or into alanine (A) for a phosphorylation-deficient mutant. Cells were transfected using Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN, USA) or Lipofectamine 2000 (Invitrogen). siRNAs were purchased from Dharmacon (Lafayette, CO, USA). The siRNAs (final concentration 20 nM) for p62 (5’-GCA TTG AAG TTG ATA TCG A-3’), Keap1 (M-012453-00-0005), and Nrf2 (M-003755-02-0005) were performed using Lipofectamine RNAi MAX (Invitrogen). After 24 h, the cells were treated with an inhibitor for an additional 24 h. Cells were subsequently harvested and lysed with lysis buffer [75 mM Tris–HCl, pH 6.8, 4% sodium dodecyl sulphate (SDS), 25% glycerol, 5% β-mercaptoethanol].
Staining for tissues and cultured cells
For routine histological examination, the brains were fixed with 10% buffered formalin for 3 weeks. Blocks were cut from various cortical and subcortical regions, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin. For immunohistochemistry, 4-μm-thick sections were cut from the frontal and temporal neocortex and hippocampus of patients with AD and controls. The sections were dehydrated and pretreated with 0.25% potassium permanganate for 15 min, then 2% oxalic acid for 3 min, followed by formic acid for 10 min for rabbit anti-P-S349 and anti-P-S403 antibodies. The sections were then subjected to immunohistochemical processing using the avidin-biotin-peroxidase complex method with diaminobenzidine (Sigma, Saint Louis, MO, USA). The sections were counterstained with hematoxylin.
Double immunofluorescent staining was performed to detect overlapping expression of phosphorylated p62 and phosphorylated tau or Keap1. For this purpose, the sections were blocked with donkey serum and then incubated overnight at 4°C with a mixture of rabbit anti-phosphorylated p62 and mouse anti-phosphorylated tau (AT8; Innogenetics, Ghent, Belgium) or rat anti-Keap1. On the next day, the sections were washed 5 times for 3 min each with PBS, followed by an incubation for 1 h with Alexa Fluor 488- and 594-conjugated secondary antibodies (Invitrogen). After a rinse with PBS, the sections were mounted with Fluoromount G (Southern Biotechnology Inc., Birmingham, AL, USA) and examined using a confocal microscope (EZ-Ci; Nikon, Tokyo, Japan). Adobe Photoshop CS5 software (Adobe systems, San Jose, CA, USA) was used for image processing.
After the cells were transfected with p62-EGFP wild-type or mutants for 24 h, cultured cells were double-immunolabelled with rabbit anti-phosphorylated p62 and mouse anti-p62 antibodies. Alexa Fluor 594- and 680-conjugated secondary antibodies (Invitrogen) were used.
Fractionation of brain extracts
For biochemical analysis, brain tissues were dissected at autopsy and rapidly frozen at -70°C. Frozen tissues from the middle temporal cortex of patients with AD (n = 3) and control subjects (n = 3) were weighed and sequentially extracted with buffers of increasing detergent strength using a previously described protocol . Briefly, samples were homogenised with 10 volumes of buffer A (10 mM Tris–HCl, pH 7.5, 1 mM EGTA, 10% sucrose, 0.8 M NaCl) and centrifuged (fraction 1). Afterwards, an equal volume of buffer A containing 2% Triton X-100 was added. The samples were then incubated for 30 min at 37°C and centrifuged at 100,000 × g for 30 min at 4°C (fraction 2). The resultant pellet was homogenised in 5 volumes of buffer A with 1% Sarkosyl and incubated for 30 min at 37°C. The homogenate was then centrifuged at 100,000 × g for 30 min at room temperature (fraction 3). The Sarkosyl-insoluble pellet was homogenised in 4 volumes of buffer A containing 1% 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS) (Sigma) and centrifuged at 100,000 × g for 20 min at room temperature (fraction 4). The pellet was sonicated in 0.2 volumes of 8 M urea buffer (fraction 5). The CHAPS-soluble fraction (fraction 4) usually contains less total protein than other fractions by this method. We applied a constant volume of extract in each fraction to an SDS-polyacrylamide gel electrophoresis (PAGE).
Frozen tissues form the middle temporal cortex of patients with AD (n = 8) and normal controls (n = 8) were used in this study. Each tissue was weighed and homogenised with a 20-fold volume of lysis buffer described above.
An equal amount of elution was subjected to SDS-PAGE, and Western blot analysis was performed as previously described . Horseradish peroxidase-conjugated anti-mouse, anti-rat or anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a secondary antibody. Detection was performed according to the protocol provided with the ECL or ECL prime detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The data were quantified as described below and statistically analysed. Rabbit polyclonal antibodies against Keap1 (ProteinTech Group, Inc., Chicago, IL, USA), p62 (MBL, Nagoya, Japan), β-actin (Sigma), GFP (Invitrogen), acetylated histone (Millipore) and LC3 (Sigma), and mouse monoclonal antibodies against ubiquitin (FK1; Millipore) and Aβ oligomer (6E10; Covance, Richmond, CA, USA) were used in this study.
Proteasomal activity assay
Chymotryptic, tryptic, and caspase proteasome activities were measured as described previously  with minor modifications. SH-SY5Y neuroblastoma cells were washed with PBS and pelleted by centrifugation. Cell pellets were sonicated in homogenisation buffer [25 mM Tris (pH 7.5), 100 mM NaCl, 5 mM ATP, 0.2% (v/v) NP-40 and 20% glycerol], and cell debris was removed by centrifugation at 4°C. Protein concentration in the resulting crude cellular extracts was determined by the bicinchoninic acid method (Pierce, Rockford, IL, USA). One hundred micrograms of protein from crude cellular extracts of each sample was diluted with buffer I [50 mM Tris–HCl (pH 7.4), 2 mM dithiothreitol, 5 mM MgCl2, 2 mM ATP] to a final volume of 0.5 mL (assayed in quadruplicate). Fluorogenic proteasome substrates were purchased from Boston Biochem (Cambridge, MA, USA): Suc-LLVY-7-amido-4-methylcoumarin (AMC) (chymotrypsin-like peptidase activity), Ac-RLR-AMC (trypsin-like peptidase activity), and Z-LLE-AMC (caspase-like or peptidylglutamyl peptide-hydrolysing activity). Each was dissolved in DMSO and brought to a final concentration of 80 μM. Proteolytic activities were assessed in 2 h at 25°C by measuring the release of the fluorescent group AMC using a fluorescence plate reader (Fluoroskan Ascent, Thermo Scientific, Waltham, MA, USA) with excitation and emission wavelengths of 380 and 460 nm, respectively.
Dot blot analysis
We modified the filter-trap analysis as described previously . Briefly, phosphorylated or non-phosphorylated peptides were diluted with TBS (Tris–HCl, pH 7.5, 150 mM NaCl), and applied to a 0.22-μm cellulose acetate membrane (Millipore) on a slot blot apparatus (Bio-Rad, Hercules, CA, USA) using a vacuum manifold. After washing with TBS containing 0.1% Triton X-100, the membrane was incubated with anti-phosphorylated p62 and pan-p62 antibodies and detected using the ECL detection system as described above.
Quantitative analysis and statistical analysis
A semi-quantitative analysis of protein detection was performed by image analysis using the Image J software provided by the NIH. All of the values were represented as the means + standard deviation (SD). The statistical significance was evaluated using Student’s t-test when comparing two conditions. A probability value of less than 0.05 (p < 0.05) was considered to be significant.
Specificity of antibodies against p62 phosphorylated at serine 349
S349 is phosphorylated following proteolysis disruption
S349 phosphorylation induces efficient formation of cytoplasmic inclusions
We next examined the effect of disruption of the p62-Keap1-Nrf2 complex on the phosphorylation of p62 (Figure 4d). Epoxomicin treatment enhanced the P-S349 signal in cells treated with control siRNA, whereas no P-S349 signal was observed in cells subjected to sip62 or siKeap1, and a weaker signal in cells treated with siNrf2 compared to controls. Knockdown experiments using siRNA suggested that this phosphorylation is sensitive to alteration of the tertiary structure of the p62-Keap1 complex. Collectively, these results indicate that p62 is phosphorylated at S349 under several pathological conditions and that phosphorylation at S349 efficiently forms cytoplasmic inclusions, leading to a higher affinity for Keap1.
S349 is phosphorylated in the brains with alzheimer’s disease
Further studies were performed to determine whether the phosphorylation of p62 alters its solubility. Frozen samples were fractionated with detergents of increasing strength and subsequently analysed by immunoblotting. The P-S349 signal was primarily detected in detergent-insoluble fractions (fraction 5) in controls and AD (top panel in Figure 5f). In addition, detergent-soluble P-S349 was also present in fraction 3 in controls. However, it was shifted to fraction 1 in AD. P-S403 signal was distributed to fractions 1, 3 and 5 in AD and fractions 1 and 5 in controls (the second top panel in Figure 5f). Because total p62 is primarily detected in fractions 3 and 5 in controls and AD (the third top panel in Figure 5f), this suggests that P-S349 is easily solubilised and its level is increased in AD brains.
Neurofibrillary tangles are positive for P-S349
Under oxidative stress and certain pathological conditions, the binding affinity of Keap1 for Nrf2 is decreased, leading to the intranuclear shuttling of Nrf2 and the subsequent increase of several types of genes, including antioxidant and detoxifying enzymes. In our study, exposure to oxidative stress (DEM) induced the phosphorylation of p62 at S349 within KIR, which is the binding region for Keap1. Total p62 levels are known to increase by oxidative stress . Because the phosphorylation of S349 on p62 strengthens its affinity for Keap1 [20, 21], higher p62 levels and an increased affinity for Keap1 simultaneously occur under oxidative stress. In addition, we showed that disruption of protein degradation systems can also induce the phosphorylation of S349. Although S403 on p62 is also known to be phosphorylated by inhibition of autophagic flux, proteasome inhibition had less of an effect on the phosphorylation of S403 than on S349. This result was also supported by immunofluorescence showing that P-S349 signals were clearly observed in the cytoplasmic inclusions of cultured cells. In contrast, P-S403 signals were weakly detected in cytoplasmic inclusions. Because the phosphorylation of S403 likely enhances the affinity to ubiquitinated molecules and helps degrade them by autophagy, it is possible that P-S403 signals physiologically occur, but may be relatively undetectable due to rapid self-degradation. By contrast, p62 S394 is efficiently phosphorylated in response to proteasome inhibition and exposure to oxidative stress. Furthermore, even transfection reagents can induce P-S394 immunoreactivity. These data suggest that the ratio of the P-S394 level to total p62 may be an indicator for noxious stimuli to cells.
Phosphorylation-mimetic mutations have been commonly used to investigate the role of phosphorylation. Our specific antibody against P-S349 recognised the phosphorylation-mimetic mutant p62 S349E. Immunofluorescence showed that the S349E mutant had a higher number of inclusions than p62 wild-type and S349A, the latter being a phosphorylation-deficient mutant. An anti-phosphorylated p62 antibody strongly detected signals in cells with S349E, whereas it showed partial reactivity in cellular inclusions with p62 wild-type and S349A. Thus, it is likely that the phosphorylation of S349 enhances the formation of inclusions. Because Nrf2 target genes are activated through the higher affinity of Keap1 for p62 with P-S349, inclusion formation itself exerts cyto-protective roles in the early stages of this process.
Importantly, we showed that S349 on p62 is in fact phosphorylated in the brains of patients with AD and that the level was significantly increased in AD compared with controls. Consistent with previous results [9, 32], we also demonstrated that the total p62 level was significantly increased in AD relative to controls. The ratio of P-S349 levels to total p62 is significantly increased in AD relative to controls. This suggests a disruption of protein degradation systems and/or the occurrence of oxidative stress in the brains with AD, and the data also support previous evidence demonstrating decreased proteasome activity [11–13] and sustained oxidative stress occur in AD compared with controls [33–35].
On the basis of these findings, we hypothesise that Aβ deposition can induce phosphorylation of S349. Unexpectedly, however, we failed to find any significant changes induced by Aβ40 or Aβ42 in our system. Anti-Aβ oligomer antibody clearly showed positive signals in cells treated with Aβ42, but not with Aβ40, and we confirmed that only Aβ42 was accumulated in lysosomes using confocal microscopy as previously described [36–38]. Therefore, our experimental system works, but further studies will be needed to clarify the relationship between Aβ deposition, phosphorylation, and protein degradation using primary neuronal cells or an in vivo system.
p62 is physiologically expressed in most organs, and can be incorporated into a wide spectrum of ubiquitin-positive inclusions in neurodegenerative diseases [39–41]. In AD, p62 is extensively accumulated in NFTs . Similarly, we showed that an anti-P-S349 antibody specifically reacted with NFTs, whereas an anti-P-S403 antibody recognised dystrophic neurites in amyloid plaques. Both pathological lesions (NFTs and dystrophic neurites) are composed of hyper-phosphorylated tau. Babu et al. reported that p62 interacts with ubiquitinated tau through its UBA domain and helps degrade tau by a ubiquitin-proteasome process . As described above, phosphorylated p62 at S403 is highly involved in the degradation of ubiquitinated molecules by the autophagy-lysosome system . Taken together, phosphorylated tau is potentially delivered to the proteasome as well as the autophagy-lysosome system together with p62. Importantly, NFTs were immunonegative for P-S403. These results suggest that P-S349 or P-S403 on p62 is able to distinguish hyper-phosphorylated tau in NFTs from that in dystrophic neurites.
In conclusion, we provide evidence that S349 on p62 is efficiently phosphorylated following inhibition of the proteasome and of autophagy. Because p62 is highly involved in protein degradation systems as well as the pathogenesis of several diseases, these results suggest that post-translational modifications of p62 have pivotal roles in the progression of neurodegeneration in AD.
This work was supported by JSPS KAKENHI Grant Number 23500425 (KT), 23500424 (FM), 24300131 (KW), Priority Research Grant for Young Scientists Designated by the President of Hirosaki University (KT, TO, JM, TM), Hirosaki University Institutional Research Grant (TI, KI, KW), the Collaborative Research Project (2013–2508) of the Brain Research Institute, Niigata University (FM), Grants-in Aid from the Research Committee for Ataxic Disease, the Ministry of Health, Labor and Welfare, Japan (KW), an Intramural Research Grant (24–5) for Neurological and Psychiatric Disorders of NCNP (KW) and Adaptable and Seamless Technology transfer Program through Target-driven R & D, JST (KT). The authors wish to express their gratitude to M. Nakata, K. Saruta and A. Ono for their technical assistance.
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