Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover

Abstract

Author summary Non-dividing muscle and nerve cells have limited options to clear harmful biological insults. One such insult is the progressive accumulation of damaged and misfolded proteins that ultimately destroy cellular function and may result in cell or organismal death. Understanding how and why normal protein turnover occurs in healthy tissue is essential for the eventual treatment of age-related and/or degenerative diseases that result from abnormal protein accumulation. Using the large, easily manipulated muscles in fruit fly larvae, we find that loss of the evolutionarily conserved NUAK protein results in cellular degeneration due to the abnormal accumulation of certain proteins, including Fil and CryAB. Moreover, we identify Stv/BAG3 and its binding partner Hsc70-4 to be crucial for NUAK function as overexpression of Stv rescues the NUAK degenerative muscle phenotype. This work is the first to uncover NUAK as a key regulator of protein degradation through autophagy and may provide a novel therapeutic target for the treatment of protein aggregate myopathies. 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.