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Science 333 (6039): 228-233

Copyright © 2011 by the American Association for the Advancement of Science

Phosphorylation of the Autophagy Receptor Optineurin Restricts Salmonella Growth

Philipp Wild1, Hesso Farhan2, David G. McEwan1, Sebastian Wagner3, Vladimir V. Rogov4,5, Nathan R. Brady6, Benjamin Richter1, Jelena Korac7, Oliver Waidmann1, Chunaram Choudhary3, Volker Dötsch4, Dirk Bumann2, and Ivan Dikic1,7,*

1 Frankfurt Institute for Molecular Life Sciences and Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, D-60590 Frankfurt (Main), Germany.
2 Infection Biology, Biozentrum, University Basel, Klingelbergstr. 50/70, CH-4056 Basel, Switzerland.
3 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.
4 Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Marie Curie Strasse 9, D-60439 Frankfurt (Main), Germany.
5 Institute of Protein Research, 142290, Pushchino, Russia.
6 Systems Biology of Cell Death Mechanisms, German Cancer Research Center, Bioquant, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany.
7 University of Split, School of Medicine, Department of Immunology and Medical Genetics, Soltanska 2, 21 000 Split, Croatia.


Figure 1
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Fig. 1. OPTN is an autophagy receptor. (A) Schematic representation of OPTN’s domain architecture. An alignment of known LIR motifs is shown underneath, with the tetra-peptide LIR highlighted in bold. N-terminal end of the LIR, acidic residues (green), and potential phosphorylation sites (red) are shown and are considered part of an extended LIR. CC, coiled-coil; aa, amino acids; ZnF, zinc finger. (B) Directed yeast two-hybrid of bait proteins (pYTH9: scAtg8, GABARAPL-1, LC3A, and SUMO1) and prey OPTN variants [pACT2: full-length OPTN WT, LIR mutant (F178A) or ubiquitin binding mutant (E478G)]. p62 LIR (aa 311 to 444) was also included. Interaction was assessed using a β-galactosidase assay. (C) GST pull-down assay of EGFP-OPTN from stable MCF-7 cells using GST or GST-ubiquitin like proteins. IB, immunoblot. (D) Representative confocal images of HeLa cells overexpressing EGFP-OPTN and treated with nutrient deprivation plus lysosomal bafilomycin A1 (ND/BafA1) localized to endogenous LC3B and ubiquitin (inset). Scale bars, 10 μm. (E) Colocalization quantification of ≥1000 cells expressing mCherry-LC3B and EGFP-OPTN [WT and deletion; {Delta}LIR ({Delta}178-181) or point mutation F178A] within single cells by ImageStream analysis. Error bars indicate mean ± SD; n = 3 independent experiments. *P < 0.05; one-tailed, paired t test.

 

Figure 2
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Fig. 2. TBK1 phosphorylates OPTN within the extended LIR motif. (A) MEFs were stimulated with 1 μg/ml LPS in the presence or absence of 1 μM BX795 for the indicated time points. Cell lysates were analyzed with antibodies against OPTN, phospho-OPTN (Ser177), total IRF3, and phospho-IRF3 (pSer396). pSer177 OPTN was lost after inhibition of TBK1 with BX795. (B) MEFs were left untreated or stimulated with LPS for 30 min, lysed, and endogenous OPTN precipitated with GST-LCB. (C) SILAC-labeled untreated or 30-min LPS-stimulated MEF cells and OPTN precipitated with GST-GABARAPL1 and analyzed by mass spectrometry (MS). MS spectrum showed enriched phosphor-Ser177 OPTN in LPS-stimulated cells compared with control. (D) ITC titration of LC3B with OPTN peptides—namely LIR_P0 (no phosphorylation), LIR_P1 (pSer177) (middle), and LIR_Ptot (all serines phosphorylated, left)—corresponding to human OPTN amino acids 169 to 184 (right). Raw data (upper boxes) and the integrated heat per titration step (points) and best-fit curves (lower panels) are shown. Calculations assume a one-site binding model. The dissociation constant (Kd), in the presence of phosphorylated OPTN serine residues showed a 5- to 13-fold decrease indicating enhanced affinity of LC3B to OPTN LIR. Ka, acid constant; {Delta}H, change in enthalpy; {Delta}S, change in entropy.

 

Figure 3
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Fig. 3. Phosphorylated OPTN colocalizes with ubiquitin- and LC3B-positive cytosolic Salmonella. (A) Confocal images of HeLa cells expressing EGFP-OPTN and mCherry-LC3B 4 hours post infection (hpi) with S. Typhimurium (SL1344). EGFP-OPTN colocalizes with LC3B (upper panel) on a fraction of Salmonella. Phospho-Ser177 OPTN localizes to EGFP-expressing Salmonella (MW57) with Ub at 1 hpi. Scale bar, 10 μm. (B) EGFP-OPTN (green) and pS177 OPTN (purple) colocalized with TBK1 (upper panel), mCherry-LC3B (middle panel), and ubiquitin (lower panel) at 4 hpi with cytosolic Salmonella sifA (blue). (C) Quantification of colocalization of cytosolic Salmonella sifA with EGFP-OPTN, ubiquitin, and pSer177 OPTN from cells represented in (B), as compared with cells treated with 1 μM BX795. Error bars indicate mean ± SEM.

 

Figure 4
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Fig. 4. Salmonella proliferation is enhanced in the absence of OPTN in vivo. (A) HeLa cells transfected with indicated siRNAs were infected with Salmonella (SL1344) and lysed at the indicated time points post-infection, and bacterial colonies were counted on selective agar plates. (B) Numbers of bacteria recovered from HeLa cells transfected with the indicated siRNAs and infected with Salmonella for 8 hpi. Intracellular Salmonella replication was calculated as fold increase at the 2-hour time point. Depletion of OPTN, NDP52, or TBK1 resulted in increased Salmonella intracellular replication. Error bars indicate mean ± SD of n = 3 independent experiments. *P < 0.002, two-tailed t test. CFU, colony-forming units. (C) ShRNA OPTN-depleted HeLa cells were transiently reconstituted with shRNA-resistant OPTN WT, shR-OPTN F178A, and shR-OPTN E478G. Both LIR mutants and Ub-binding mutants of OPTN failed to rescue OPTN-depleted HeLa cells 8 hpi compared with OPTN WT. Error bars indicate mean ± SD of n = 3 independent experiments. (D) Three-dimensional reconstitution of confocal image z-stacks of Salmonella-infected HeLa cells at 4 hpi. EGFP-OPTN WT (green), NDP52 (red, left panel), and endogenous p62 (red right panel) form distinct "patches" on the surface of cytosolic Salmonella.

 


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