Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins

Selective macroautophagy (autophagy) of ubiquitinated protein is implicated as a compensatory mechanism of the ubiquitin-proteasome system. p62/SQSTM1 is a key molecule managing autophagic clearance of polyubiquitinated proteins. However, little is known about mechanisms controlling autophagic degradation of polyubiquitinated proteins. Here, we show that the specific phosphorylation of p62 at serine 403 (S403) in its ubiquitin-associated (UBA) domain increases the affinity between UBA and polyubiquitin chain, resulting in efficiently targeting polyubiquitinated proteins in "sequestosomes" and stabilizing sequestosome structure as a cargo of ubiquitinated proteins for autophagosome entry. Casein kinase 2 (CK2) phosphorylates S403 of p62 directly. Furthermore, CK2 overexpression or phosphatase inhibition reduces the formation of inclusion bodies of the polyglutamine-expanded huntingtin exon1 fragment in a p62-dependent manner. We propose that phosphorylation of p62 at S403 regulates autophagic clearance of ubiquitinated proteins and protein aggregates that are poorly degraded by proteasomes.     

PTK2/FAK regulates UPS impairment via SQSTM1/p62 phosphorylation in TARDBP/TDP-43 proteinopathies

ABSTRACT

TARDBP/TDP-43 (TAR DNA binding protein) proteinopathies are a common feature in a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and Alzheimer disease (AD). However, the molecular mechanisms underlying TARDBP-induced neurotoxicity are largely unknown. In this study, we demonstrated that TARDBP proteinopathies induce impairment in the ubiquitin proteasome system (UPS), as evidenced by an accumulation of ubiquitinated proteins and a reduction in proteasome activity in neuronal cells. Through kinase inhibitor screening, we identified PTK2/FAK (PTK2 protein tyrosine kinase 2) as a suppressor of neurotoxicity induced by UPS impairment. Importantly, PTK2 inhibition significantly reduced ubiquitin aggregates and attenuated TARDBP-induced cytotoxicity in a Drosophila model of TARDBP proteinopathies. We further identified that phosphorylation of SQSTM1/p62 (sequestosome 1) at S403 (p-SQSTM1 [S403]), a key component in the autophagic degradation of poly-ubiquitinated proteins, is increased upon TARDBP overexpression and is dependent on the activation of PTK2 in neuronal cells. Moreover, expressing a non-phosphorylated form of SQSTM1 (SQSTM1^S403A) significantly repressed the accumulation of insoluble poly-ubiquitinated proteins and neurotoxicity induced by TARDBP overexpression in neuronal cells. In addition, TBK1 (TANK binding kinase 1), a kinase that phosphorylates S403 of SQSTM1, was found to be involved in the PTK2-mediated phosphorylation of SQSTM1. Taken together, our data suggest that the PTK2-TBK1-SQSTM1 axis plays a critical role in the pathogenesis of TARDBP by regulating neurotoxicity induced by UPS impairment. Therefore, targeting the PTK2-TBK1-SQSTM1 axis may represent a novel therapeutic intervention for neurodegenerative diseases with TARDBP proteinopathies.Abbreviations: ALP: macroautophagy/autophagy lysosomal pathway; ALS: amyotrophic lateral sclerosis; ATXN2: ataxin 2; BafA1: bafilomycin A₁; cCASP3: cleaved caspase 3; CSNK2: casein kinase 2; FTLD: frontotemporal lobar degeneration; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; OPTN: optineurin; PTK2/FAK: PTK2 protein tyrosine kinase 2; SQSTM1/p62: sequestosome 1; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system.     

Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates

Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington’s disease.     

Dynamics of the degradation of ubiquitinated proteins by proteasomes and autophagy: association with sequestosome 1/p62

Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5(-/-) mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5(-/-) MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation.     

Size,organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

Selective macroautophagy/autophagy—with the help of molecular receptors—captures cargo for lysosomal degradation. Among the best-studied molecular receptors is SQSTM1/p62, a homo-oligomeric ubiquitin binding protein, which binds to both cargo and MAP1LC3B/LC3, a protein important for autophagosome biogenesis. Although the mechanisms underlying interaction of LC3 and SQSTM1 have been extensively studied, very little is known about the size or organization of soluble complexes formed between SQSTM1 and LC3 prior to phagophore (the autophagosome precursor) binding in live cells at the molecular level. To address this question, in the current study we use a combination of 2 microscopy-based approaches, FRET microscopy and confocal FRAP, to study the nanoscale properties of soluble SQSTM1 complexes and SQSTM1-LC3 complexes in living HeLa cells. We find that, independent of puncta, SQSTM1 oligomerizes to form very slowly diffusing complexes that contain multiple copies of SQSTM1 within FRET proximity of one another. Furthermore, we show that the interactions of soluble pools of LC3 and SQSTM1 can be readily detected by both FRAP and FRET. Finally, we uncover unexpected roles of SQSTM1's PB1 domain, a region of the protein involved in homo-oligomer formation, in complex formation. Taken together, these findings provide new insights into the nature of nanometer-sized protein complexes in the autophagy pathway.     

Modulation of SQSTM1/p62 activity by N-terminal arginylation of the endoplasmic reticulum chaperone HSPA5/GRP78/BiP

The N-end rule pathway is a proteolytic system, in which single N-terminal residues act as a determinant of a class of degrons, called N-degrons. In the ubiquitin (Ub)-proteasome system, specific recognition components, called N-recognins, recognize N-degrons and accelerate polyubiquitination and proteasomal degradation of the substrates. In this study, we show that the pathway regulates the activity of the macroautophagic receptor SQSTM1/p62 (sequestosome 1) through N-terminal arginylation (Nt-arginylation) of endoplasmic reticulum (ER)-residing molecular chaperones, including HSPA5/GRP78/BiP, CALR (calreticulin), and PDI (protein disulfide isomerase). The arginylation is co-induced with macroautophagy (hereafter autophagy) as part of innate immunity to cytosolic DNA and when misfolded proteins accumulate under proteasomal inhibition. Following cytosolic relocalization and arginylation, Nt-arginylated HSPA5 (R-HSPA5) is targeted to autophagosomes and degraded by lysosomal hydrolases through the interaction of its N-terminal Arg (Nt-Arg) with ZZ domain of SQSTM1. Upon binding to Nt-Arg, SQSTM1 undergoes a conformational change, which promotes SQSTM1 self-polymerization and interaction with LC3, leading to SQSTM1 targeting to autophagosomes. Cargoes of R-HSPA5 include cytosolic misfolded proteins destined to be degraded through autophagy. Here, we discuss the mechanisms by which the N-end rule pathway regulates SQSTM1-dependent selective autophagy.     

Role of autophagy,SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63^?/? mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.     

Regulation of SQSTM1/p62 via UBA domain ubiquitination and its role in disease

Macroautophagy/autophagy can be a selective degradative process via the utilization of various autophagic receptor proteins. Autophagic receptors selectively recognize ubiquitinated cargoes and deliver them to phagophores, the precursors to autophagosomes, for their degradation. For example, SQSTM1/p62 directly binds to ubiquitinated protein aggregates via its UBA domain and sequesters them into inclusion bodies via its PB1 domain. SQSTM1also interacts with phagophores via its LC3-interacting (LIR) motif. However, a regulatory mechanism for autophagic receptors is not yet understood.     

Cytosolic PINK1 promotes the targeting of ubiquitinated proteins to the aggresome-autophagy pathway during proteasomal stress

During proteasomal stress, cells can alleviate the accumulation of polyubiquitinated proteins by targeting them to perinuclear aggresomes for autophagic degradation, but the mechanism underlying the activation of this compensatory pathway remains unclear. Here we report that PINK1-s, a short form of Parkinson disease (PD)-related protein kinase PINK1 (PTEN induced putative kinase 1), is a major regulator of aggresome formation. PINK1-s is extremely unstable due to its recognition by the N-end rule pathway, and tends to accumulate in the cytosol during proteasomal stress. Overexpression of PINK1-s induces aggresome formation in cells with normal proteasomal activities, while loss of PINK1-s function leads to a significant decrease in the efficiency of aggresome formation induced by proteasomal inhibition. PINK1-s exerts its effect through phosphorylation of the ubiquitin-binding protein SQSTM1 (sequestosome 1) and increasing its ability to sequester polyubiquitinated proteins into aggresomes. These findings pinpoint PINK1-s as a sensor of proteasomal activities that transduces the proteasomal impairment signal to the aggresome formation machinery.     

ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase

ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is involved in the routing of misfolded glycoproteins for degradation in the cytoplasm. Previously, we reported that EDEM1 leaves the endoplasmic reticulum via non-COPII vesicles (Zuber et al. in Proc Natl Acad Sci USA 104:4407–4412, 2007) and becomes degraded by basal autophagy (Le Fourn et al. in Cell Mol Life Sci 66:1434–1445, 2009). However, it is unknown which type of autophagy is involved. Likewise, how EDEM1 is targeted to autophagosomes remains elusive. We now show that EDEM1 is degraded by selective autophagy. It colocalizes with the selective autophagy cargo receptors p62/SQSTM1, neighbor of BRCA1 gene 1 (NBR1) and autophagy-linked FYVE (Alfy) protein, and becomes engulfed by autophagic isolation membranes. The interaction with p62/SQSTM1 and NBR1 is required for routing of EDEM1 to autophagosomes since it can be blocked by short inhibitory RNA knockdown of the cargo receptors. Furthermore, p62/SQSTM1 interacts only with deglycosylated EDEM1 that is also ubiquitinated. The deglycosylation of EDEM1 occurs by the cytosolic peptide N-glycanase and is a prerequisite for interaction and aggregate formation with p62/SQSTM1 as demonstrated by the effect of peptide N-glycanase inhibitors on the formation of protein aggregates. Conversely, aggregation of p62/SQSTM1 and EDEM1 occurs independent of cytoplasmic histone deacetylase. These data provide novel insight into the mechanism of autophagic degradation of the ER-associated protein degradation (ERAD) component EDEM1 and disclose hitherto unknown parallels with the clearance of cytoplasmic aggregates of misfolded proteins by selective autophagy.     

Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation

Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.     

Research Article: Role of autophagy,SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63^−/− mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.     

p62/Sequestosome 1 regulates transforming growth factor beta signaling and epithelial to mesenchymal transition in A549 cells

Transforming growth factor beta (TGFβ) receptor trafficking regulates many TGFβ-dependent cellular outcomes including epithelial to mesenchymal transition (EMT). EMT in A549 non-small cell lung cancer (NSCLC) cells has recently been linked to the regulation of cellular autophagy. Here, we investigated the role of the autophagy cargo receptor, p62/sequestosome 1 (SQSTM1), in regulating TGFβ receptor trafficking, TGFβ1-dependent Smad2 phosphorylation and EMT in A549 NSCLC cells. Using immunofluorescence microscopy, p62/SQSTM1 was observed to co-localize with TGFβ receptors in the late endosome. Small interfering RNA (SiRNA)-mediated silencing of p62/SQSTM1 resulted in an attenuated time-course of Smad2 phosphorylation but did not alter Smad2 nuclear translocation. However, p62/SQSTM1 silencing promoted TGFβ1-dependent EMT marker expression, actin stress fiber formation and A549 cell migration. We further observed that Smad4-independent TGFβ1 signaling decreased p62/SQSTM1 protein levels via a proteasome-dependent mechanism. Although p62/SQSTM1 silencing did not impede TGFβ-dependent autophagy, our results suggest that p62/SQSTM1 may aid in maintaining A549 cells in an epithelial state and TGFβ1 decreases p62/SQSTM1 prior to inducing EMT and autophagy.     

TRAF6 mediates ubiquitination of KIF23/MKLP1 and is required for midbody ring degradation by selective autophagy

Upon completion of cytokinesis, the midbody ring is transported asymmetrically into one of the two daughter cells where it becomes a midbody ring derivative that is degraded by autophagy. In this study we showed that the ubiquitin-binding autophagy receptor SQSTM1/p62 and the interacting adaptor protein WDFY3/ALFY form a complex with the ubiquitin E3 ligase TRAF6 and that these proteins, as well as NBR1, are important for efficient clearance of midbody ring derivatives by autophagy. The number of ubiquitinated midbody ring derivatives decreases in TRAF6-depleted cells and we showed that TRAF6 mediates ubiquitination of the midbody ring localized protein KIF23/MKLP1. We conclude that TRAF6-mediated ubiquitination of the midbody ring is important for its subsequent recognition by ubiquitin-binding autophagy receptors and degradation by selective autophagy.     

SGPL1 (sphingosine phosphate lyase 1) modulates neuronal autophagy via phosphatidylethanolamine production

Macroautophagy/autophagy defects have been identified as critical factors underlying the pathogenesis of neurodegenerative diseases. The roles of the bioactive signaling lipid sphingosine-1-phosphate (S1P) and its catabolic enzyme SGPL1/SPL (sphingosine phosphate lyase 1) in autophagy are increasingly recognized. Here we provide in vitro and in vivo evidence for a previously unidentified route through which SGPL1 modulates autophagy in neurons. SGPL1 cleaves S1P into ethanolamine phosphate, which is directed toward the synthesis of phosphatidylethanolamine (PE) that anchors LC3-I to phagophore membranes in the form of LC3-II. In the brains of SGPL1^fl/fl/Nes mice with developmental neural specific SGPL1 ablation, we observed significantly reduced PE levels. Accordingly, alterations in basal and stimulated autophagy involving decreased conversion of LC3-I to LC3-II and increased BECN1/Beclin-1 and SQSTM1/p62 levels were apparent. Alterations were also noticed in downstream events of the autophagic-lysosomal pathway such as increased levels of lysosomal markers and aggregate-prone proteins such as APP (amyloid β [A4] precursor protein) and SNCA/α-synuclein. In vivo profound deficits in cognitive skills were observed. Genetic and pharmacological inhibition of SGPL1 in cultured neurons promoted these alterations, whereas addition of PE was sufficient to restore LC3-I to LC3-II conversion, and control levels of SQSTM1, APP and SNCA. Electron and immunofluorescence microscopy showed accumulation of unclosed phagophore-like structures, reduction of autolysosomes and altered distribution of LC3 in SGPL1^fl/fl/Nes brains. Experiments using EGFP-mRFP-LC3 provided further support for blockage of the autophagic flux at initiation stages upon SGPL1 deficiency due to PE paucity. These results emphasize a formerly overlooked direct role of SGPL1 in neuronal autophagy and assume significance in the context that autophagy modulators hold an enormous therapeutic potential in the treatment of neurodegenerative diseases.