Phosphorylation of the Autophagy Receptor Optineurin Restricts Salmonella Growth

Abstract

Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitincoated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin-or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might be a general mechanism for regulation of cargo-selective autophagy.Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process by which cells deliver bulk cytosolic components for degradation to the lysosome (1-4). Selectivity in cargo targeting is mediated via autophagy receptors that simultaneously bind cargoes and autophagy modifiers, autophagy-related protein 8 (ATG8)/ microtubule-associated protein light chain 3 (LC3)/γ-aminobutyric acid receptor-associated protein (GABARAP) proteins, which are conjugated to the autophagosomal membranes (5, 6). The regulatory mechanisms controlling the spatiotemporal dynamics of the autophagy receptor-target interaction in cells remain unclear (7). Multiple autophagy receptors have been identified with the yeast two-hybrid system (8, 9), which included an N-terminal fragment of optineurin (OPTN), a ubiquitin-binding protein also known as NF-κB essential modulator-related protein ( Fig. 1, A and B). The specific interactions between OPTN and LC3/GABARAP proteins were verified by pull-down assays in mammalian cells, directed yeast two-hybrid transformations, and in vitro using purified proteins ( Fig. 1C and fig. S1, A and B) (10). OPTN bound to ubiquitin chains and autophagy modifiers ATG8/LC3/GABARAP proteins but not to mono-ubiquitin or other ubiquitin-like proteins ( Fig. 1C and fig. S1C). Deletion mapping of the N-terminal region of OPTN identified an LC3 interacting motif (LIR), a linear tetrapeptide sequence present in known autophagy receptors that binds directly to LC3/GABARAP modifiers (9, 11, 12). The LIR was located between the coiled-coil domains of OPTN encompassing amino acids 169 to 209 (Fig. 1A) and was essential for in vitro and in vivo binding between OPTN and LC3/ GABARAP (Fig. 1, B and C, and figs. S1A and S2A). Single point mutations at either OPTN Phe 178 →Ala 178 (F178A) or I181A (13), corresponding to the WxxL of p62, were sufficient to abrogate the interaction with LC3/GABARAP proteins, whereas these mutants were still able to bind to linear ubiquitin chains fused to glutathione S-transferase (GST-4xUb) (...

Recruitment of TBK 1 to cytosol‐invading Salmonella induces WIPI 2‐dependent antibacterial autophagy

“…To further investigate the mechanism of how TBK1 recruits WIPI1 and WIPI2 to cytosol‐invading bacteria, we depleted cells of optineurin, the only known TBK1 substrate in anti‐bacterial autophagy (Wild et al , 2011). Cells lacking optineurin recruited WIPI1 and WIPI2B normally to S .…”

“…Typhimurium contains different linkage types; at minimum, M1‐ and K63‐linked chains are present but their functional contribution to anti‐bacterial autophagy remains unknown (van Wijk et al , 2012). NDP52 can bind M1‐, K48‐, and K63‐linked ubiquitin chains (Wild et al , 2011) with similar affinities for di‐ubiquitin molecules and tetra‐ubiquitin chains that are M1‐linked (Xie et al , 2015). To investigate whether NDP52 binds longer chains in a linkage‐specific manner, we exposed NDP52 to an equimolar mixture of M1‐, K48‐, and K63‐linked ubiquitin tetramers (Fig 7A).…”

“…Typhimurium by controlling autophagy. Consistent with TBK1 controlling anti‐bacterial autophagy is the enzyme's ability to phosphorylate functionally important sites in cargo receptors, namely the LC3‐interacting region (LIR) of optineurin (Wild et al , 2011) and the ubiquitin‐binding site of p62 (Pilli et al , 2012), thereby enhancing their affinity for LC3 and ubiquitin, respectively. While the spatial and temporal control of the tethering function of cargo receptors appears as an important contribution of TBK1 to anti‐bacterial autophagy, we found that in Tbk1 −/− MEFs, LC3 is still recruited to S .…”

“…VPS34 produces membrane patches rich in phosphatidylinositol 3‐phosphate (PI(3)P) that recruit PI(3)P‐binding proteins such as WIPI and DFCP1 to the site of phagophore formation (Axe et al , 2008). In contrast to the non‐selective engulfment of cytosol into starvation‐induced autophagosomes, anti‐bacterial autophagy is mediated by cargo receptors including NDP52, optineurin, and p62 (Thurston et al , 2009; Zheng et al , 2009; Wild et al , 2011). Cargo receptors bind members of the LC3/GABARAP family of ubiquitin‐like proteins on the autophagosomal membrane and specific “eat‐me” signals associated with cytosol‐invading bacteria, thereby selectively tethering bacteria to phagophore membranes (Weidberg et al , 2011; Rogov et al , 2014).…”

“…TBK1 accumulates in the vicinity of cytosol‐exposed bacteria together with its adaptor proteins Nap1, Sintbad, and their binding partner NDP52 (Fujita et al , 2003; Ryzhakov & Randow, 2007; Thurston et al , 2009; Verlhac et al , 2015). TBK1 also associates with optineurin and it has been reported to phosphorylate both optineurin and p62, thereby enhancing their affinity for LC3B and ubiquitin, respectively (Morton et al , 2008; Wild et al , 2011; Pilli et al , 2012; Heo et al , 2015; Richter et al , 2016). While these findings imply that TBK1 strengthens the tethering function of cargo receptors, TBK1 has also been suggested to promote autophagosome maturation (Pilli et al , 2012).…”