Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria

Significance Selective autophagy of damaged mitochondria (mitophagy) requires protein kinases PINK1 and TBK1, ubiquitin ligase Parkin, and autophagy receptors such as OPTN, driving ubiquitin-labeled mitochondria into autophagosomes. Because all proteins have been genetically linked to either Parkinson’s disease (PINK1 and Parkin) or amyotrophic lateral sclerosis and frontotemporal lobar degeneration (TBK1 and OPTN), it is of great interest to understand their physiological functions. By utilizing quantitative proteomics we show that TBK1 phosphorylates four receptors on several autophagy-relevant sites. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and activation on mitochondria. TBK1-mediated phosphorylation of OPTN creates a signal amplification loop through combining recruitment and retention of OPTN/TBK1 on ubiquitinated mitochondria. Selective autophagy of damaged mitochondria requires autophagy receptors optineurin (OPTN), NDP52 (CALCOCO2), TAX1BP1, and p62 (SQSTM1) linking ubiquitinated cargo to autophagic membranes. By using quantitative proteomics, we show that Tank-binding kinase 1 (TBK1) phosphorylates all four receptors on several autophagy-relevant sites, including the ubiquitin- and LC3-binding domains of OPTN and p62/SQSTM1 as well as the SKICH domains of NDP52 and TAX1BP1. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and kinase activation on mitochondria. TBK1 in turn phosphorylates OPTN’s UBAN domain at S473, thereby expanding the binding capacity of OPTN to diverse Ub chains. In combination with phosphorylation of S177 and S513, this posttranslational modification promotes recruitment and retention of OPTN/TBK1 on ubiquitinated, damaged mitochondria. Moreover, phosphorylation of OPTN on S473 enables binding to pS65 Ub chains and is also implicated in PINK1-driven and Parkin-independent mitophagy. Thus, TBK1-mediated phosphorylation of autophagy receptors creates a signal amplification loop operating in selective autophagy of damaged mitochondria.

[1]  J. Harper,et al.  The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy. , 2015, Molecular cell.

[2]  I. Dikic,et al.  Expanding the ubiquitin code through post‐translational modification , 2015, EMBO reports.

[3]  D. Swaney,et al.  Phosphorylation of ubiquitin at Ser65 affects its polymerization, targets, and proteome‐wide turnover , 2015, EMBO reports.

[4]  J. Burman,et al.  The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy , 2015, Nature.

[5]  A. Prescott,et al.  Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation , 2015, EMBO reports.

[6]  J. Harper,et al.  Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response , 2015, Proceedings of the National Academy of Sciences.

[7]  Kevin F. Bieniek,et al.  Whole-genome sequencing reveals important role for TBK1 and OPTN mutations in frontotemporal lobar degeneration without motor neuron disease , 2015, Acta Neuropathologica.

[8]  Brittany N. Lasseigne,et al.  Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways , 2015, Science.

[9]  T. Wieland,et al.  Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia , 2015, Nature Neuroscience.

[10]  R. Youle,et al.  The Roles of PINK1, Parkin, and Mitochondrial Fidelity in Parkinson’s Disease , 2015, Neuron.

[11]  David Komander,et al.  Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis , 2014, The EMBO journal.

[12]  S. Gygi,et al.  Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. , 2014, Molecular cell.

[13]  E. Holzbaur,et al.  Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation , 2014, Proceedings of the National Academy of Sciences.

[14]  Shengdi Hu,et al.  An Efficient Genotyping Method for Genome-modified Animals and Human Cells Generated with CRISPR/Cas9 System , 2014, Scientific Reports.

[15]  D. Kirkpatrick,et al.  The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy , 2014, Nature.

[16]  A. Ernst,et al.  Cargo recognition and trafficking in selective autophagy , 2014, Nature Cell Biology.

[17]  T. Hirokawa,et al.  Ubiquitin is phosphorylated by PINK1 to activate parkin , 2014, Nature.

[18]  Soojay Banerjee,et al.  PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity , 2014, The Journal of cell biology.

[19]  K. Hofmann,et al.  Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65 , 2014, The Biochemical journal.

[20]  Y. Ohsumi.  Historical landmarks of autophagy research , 2013, Cell Research.

[21]  E. Dueber,et al.  Recent insights into the complexity of Tank‐binding kinase 1 signaling networks: The emerging role of cellular localization in the activation and substrate specificity of TBK1 , 2013, FEBS letters.

[22]  J. Terzic,et al.  Ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates , 2013, Journal of Cell Science.

[23]  Farren J. Isaacs,et al.  Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion , 2012, FEBS letters.

[24]  Steven B. Bradfute,et al.  TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. , 2012, Immunity.

[25]  R. Youle,et al.  Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin. , 2012, Developmental cell.

[26]  S. Ajroud‐Driss,et al.  SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis. , 2011, Archives of neurology.

[27]  N. Nukina,et al.  Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins. , 2011, Molecular cell.

[28]  G. Superti-Furga,et al.  Functional Dissection of the TBK1 Molecular Network , 2011, PloS one.

[29]  P. Cohen,et al.  Polyubiquitin Binding to Optineurin Is Required for Optimal Activation of TANK-binding Kinase 1 and Production of Interferon β* , 2011, The Journal of Biological Chemistry.

[30]  Sebastian A. Wagner,et al.  Phosphorylation of the Autophagy Receptor Optineurin Restricts Salmonella Growth , 2011, Science.

[31]  M. Mann,et al.  Mass Spectrometry-based Proteomics Using Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass Spectrometer* , 2011, Molecular & Cellular Proteomics.

[32]  M. Mann,et al.  Andromeda: a peptide search engine integrated into the MaxQuant environment. , 2011, Journal of proteome research.

[33]  H. Virgin,et al.  Autophagy in immunity and inflammation , 2011, Nature.

[34]  Takeo Kato,et al.  Mutations of optineurin in amyotrophic lateral sclerosis , 2010, Nature.

[35]  Ivan Dikic,et al.  A role for ubiquitin in selective autophagy. , 2009, Molecular cell.

[36]  Nobuhiro Suzuki,et al.  Specific Recognition of Linear Ubiquitin Chains by NEMO Is Important for NF-κB Activation , 2009, Cell.

[37]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[38]  Stephen S. Taylor,et al.  Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores , 2008, The Journal of cell biology.

[39]  V. Dötsch,et al.  Ubiquitin binding mediates the NF-κB inhibitory potential of ABIN proteins , 2008, Oncogene.

[40]  P. Cohen,et al.  Enhanced binding of TBK1 by an optineurin mutant that causes a familial form of primary open angle glaucoma , 2008, FEBS letters.

[41]  C. Klein,et al.  Alterations in the common fragile site gene Parkin in ovarian and other cancers , 2003, Oncogene.

[42]  J. Kong,et al.  Massive Mitochondrial Degeneration in Motor Neurons Triggers the Onset of Amyotrophic Lateral Sclerosis in Mice Expressing a Mutant SOD1 , 1998, The Journal of Neuroscience.

[43]  Feng Zhang,et al.  Genome engineering using CRISPR-Cas9 system. , 2015, Methods in molecular biology.