Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover

Abstract

The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. Here we show that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) and CryAB. NUAK biochemically and genetically interacts with Drosophila Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. We further demonstrate that Fil is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, our results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases.

Maintaining proteostasis under mechanical stress

“…In Drosophila, the serine/threonine kinase NUAK interacts with Starvin, the fly orthologue of BAG3 (Brooks et al , 2020 ). Starvin and NUAK mutant flies exhibit muscle degeneration and muscle contraction defects, and similar phenotypes are also observed following a depletion of Drosophila HSC70 and the autophagic membrane marker ATG8 (Arndt et al , 2010 ; Brooks et al , 2020 ). Moreover, muscle degeneration is accompanied by the accumulation of protein aggregates that contain filamin.…”

Drosophila p38 MAPK interacts with BAG‐3/starvin to regulate age‐dependent protein homeostasis

“…in Drosophila S2 cell culture and eye tissue, which consists mostly of photoreceptor neurons (Carra et al, 2010). Several studies on stv have focused on embryonic and larval muscle (Brooks et al, 2020;Coulson et al, 2005) and stv colocalizes with HspB8/Hsp67Bc in the larval muscle (Carra et al, 2010). However, in the adult flight muscle stv has only been characterized by an interaction with a CASA complex including HspB8/CG14207 (Arndt et al, 2010).…”

“…In adult Drosophila muscle, the CASA complex has been reported to consist of the Hsp70 homologue Hsc70-4, the HspB8 homologue CG14207 and the BAG-3 homologue starvin (stv) (Arndt et al, 2010). stv is a nucleotide exchange factor that binds to both Hsp70 and HspB8 as well as a variety of target proteins that need refolding (Brooks et al, 2020;Carra et al, 2008;Doong et al, 2002;Gamerdinger et al, 2011;Guilbert et al, 2018;Gupta et al, 2019;Ulbricht et al, 2013). As stv has been shown to be required for muscle functions (Arndt et al, 2010;Brooks et al, 2020;Coulson et al, 2005), we tested if p38Kb and stv colocalizes and find that they colocalize at both the Z-disk and M-line (Figure 3a,b).…”

“…stv is a nucleotide exchange factor that binds to both Hsp70 and HspB8 as well as a variety of target proteins that need refolding (Brooks et al, 2020;Carra et al, 2008;Doong et al, 2002;Gamerdinger et al, 2011;Guilbert et al, 2018;Gupta et al, 2019;Ulbricht et al, 2013). As stv has been shown to be required for muscle functions (Arndt et al, 2010;Brooks et al, 2020;Coulson et al, 2005), we tested if p38Kb and stv colocalizes and find that they colocalize at both the Z-disk and M-line (Figure 3a,b). We next tested if p38Kb physically interacts with stv in the adult muscle.…”

“…Those substrates that cannot be refolded by the complex are poly-ubiquitinated and targeted to the autophagosome through a handover between BAG-3/stv and the autophagy adaptor protein p62 (ref(2)p in flies), and subsequently degraded through the autophagosome-lysosome system (Behl, 2011;Carra et al, 2008;Gamerdinger et al, 2009;Kettern et al, 2010;Min et al, 2008;Selcen et al, 2009;Terman et al, 2007). Furthermore, stv has been shown to play a role in regulating protein homeostasis in fly muscle (Arndt et al, 2010;Brooks et al, 2020).…”