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NIMA-related kinase 9-mediated phosphorylation of the microtubule-associated LC3B protein at Thr-50 suppresses selective autophagy of p62/sequestosome 1.

ABSTRACT: Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein kinase C? (PKC?) and never in mitosis A (NIMA)-related kinase 9 (NEK9). All three kinases phosphorylated LC3B Thr-50 in vitro A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKC? knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.

SUBMITTER: Shrestha BK

PROVIDER: S-EPMC6996884 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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NIMA-related kinase 9-mediated phosphorylation of the microtubule-associated LC3B protein at Thr-50 suppresses selective autophagy of p62/sequestosome 1.

Shrestha Birendra Kumar BK Skytte Rasmussen Mads M Abudu Yakubu Princely YP Bruun Jack-Ansgar JA Larsen Kenneth Bowitz KB Alemu Endalkachew A EA Sjøttem Eva E Lamark Trond T Johansen Terje T

The Journal of biological chemistry 20191219 5

Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactio ...[more]

PMID: 31857374

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Project description:Diffuse myofiber necrosis in the context of inflammatory myopathy is the hallmark of immune-mediated necrotizing myopathy (IMNM). We have previously shown that skeletal muscle fibers of IMNM patients may display nonrimmed vacuoles and sarcoplasmic irregularities. The dysfunctional chaperone activity has been linked to the defective assembly of skeletal muscle proteins and their degradation via lysosomes, autophagy and the proteasomal machinery. This study was undertaken to highlight a chaperone-assisted selective autophagy (CASA) pathway, functionally involved in protein homeostasis, cell stress and the immune response in skeletal muscle of IMNM patients. Skeletal muscle biopsies from 54 IMNM patients were analyzed by immunostaining, as well as by qPCR. Eight biopsies of sIBM patients served as pathological controls, and eight biopsies of nondisease control subjects were included. Alteration of autophagy was detectable in all IMNM biopsy samples highlighted via a diffuse sarcoplasmic staining pattern by p62 and LC3 independent of vacuoles. This pattern was at variance with the coarse focal staining pattern mostly confined to rimmed vacuoles in sIBM. Colocalization of p62 with the chaperone proteins HSP70 and αB-crystalline points to the specific targeting of misfolded proteins to the CASA machinery. Bcl2-associated athanogene 3 (BAG3) positivity of these fibers emphasizes the selectivity of autophagy processes and these fibers also express MHC class I sarcolemma. Expression of genes involved in autophagy and endoplasmic reticulum (ER) stress pathways studied here is significantly upregulated in IMNM. We highlight that vacuoles without sarcolemmal features may arise in IMNM muscle biopsies, and they must not be confounded with sIBM-specific vacuoles. Further, we show the activation of selective autophagy and emphasize the role of chaperones in this context. CASA occurs in IMNM muscle, and specific molecular pathways of autophagy differ from the ones in sIBM, with p62 as a unique identifier of this process.

Project description: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.

Dataset Information

NIMA-related kinase 9-mediated phosphorylation of the microtubule-associated LC3B protein at Thr-50 suppresses selective autophagy of p62/sequestosome 1.

Publications

NIMA-related kinase 9-mediated phosphorylation of the microtubule-associated LC3B protein at Thr-50 suppresses selective autophagy of p62/sequestosome 1.

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