A conserved STRIPAK complex is required for autophagy in muscle tissue

Abstract

Autophagy is important for cellular homeostasis and to prevent the abnormal accumulation of proteins. While many proteins that comprise the canonical autophagy pathway have been characterized, the identification of new regulators may help understand tissue and/or stress-specific responses. Using an in silico approach, we identified Striatin interacting protein (Strip), MOB kinase activator 4 (Mob4), and Fibroblast growth factor receptor 1 oncogene partner 2 (Fgop2) as conserved mediators of muscle tissue maintenance. We performed affinity purification mass spectrometry (AP-MS) experiments with Drosophila melanogaster ( D. melanogaster) Strip as a bait protein and co-purified additional Striatin Interacting Phosphatase and Kinase (STRIPAK) complex members from larval muscle tissue. NUAK and Starvin (Stv) also emerged as Strip-binding proteins and these physical interactions were verified in vivo using Proximity Ligation Assays (PLA). To understand the functional significance of the STRIPAK-NUAK-Stv complex, we employed a sensitized genetic assay combined with RNA interference (RNAi) to demonstrate that both NUAK and stv function in the same biological process with genes that encode for STRIPAK complex proteins. RNAi-directed knockdown of Strip in muscle tissue led to the accumulation of ubiquitinated cargo, p62, and Atg8a, consistent with a block in autophagy. Indeed, autophagic flux was decreased in Strip RNAi muscles, while lysosome biogenesis and activity were unaffected. Our results support a model whereby the STRIPAK-NUAK-Stv complex coordinately regulates autophagy in muscle tissue.

Protocol for affinity purification-mass spectrometry interactome profiling in larvae of Drosophila melanogaster

“…The selection of individual STRIPAK components in the LFQ Results Table allows for a comparison of protein abundance in either bait or control pull-down datasets ( Figure 3 F). Both physical and genetic interactions among STRIPAK complex members are well validated in multiple systems and across organisms, 1 , 16 , 17 , 18 , 19 , 20 , 21 thus demonstrating the robustness of our approach.…”

“…We recently used this strategy to identify muscle-enriched proteins that function with Strip in the STRIPAK complex. 1 In addition to the FLAG-Strip pull-down experiments, we also performed AP-MS pull-downs using transgenic flies that express a V5 tag on the C-terminus of Strip (Strip-V5). Proteins common in both pull-downs using a Venn diagram were considered for further analysis.…”

Cornerstone Cellular Pathways for Metabolic Disorders and Diabetes Mellitus: Non-Coding RNAs, Wnt Signaling, and AMPK

“…Autophagy is another programmed cell death pathway that is involved in the sequestration of cytoplasmic proteins and organelles for recycling and tissue remodeling [59,246,274,275,[354][355][356][357][358]. Most descriptions of autophagy involve macroautophagy, rather than microautophagy or chaperone-mediated autophagy [59,359], which can form autophagosomes for combining into lysosomes.…”

“…The processing of circulating oxidized fatty acids during DM may require autophagy activation [18,165], and mitochondrial homeostasis can be dependent upon the activation of autophagy pathways [46]. Maintenance of autophagic flux during DM also may be one factor in preserving cognition [77,371], maintaining muscle integrity [354], fostering the function of pancreatic β-cells [372], decreasing insulin resistance in models of autophagy Atg7 gene deletion and obesity [373], blocking nephropathy during DM with maintenance of autophagy Atg7, Atg5, and microtubule-associated protein 1A/1B-light chain 3 (LC3) proteins [374], and controlling the development of pancreatic β-cells [375]. Yet, a balance in autophagy activation is necessary since autophagy also can lead to detrimental effects.…”

Cellular Impacts of Striatins and the STRIPAK Complex and Their Roles in the Development and Metastasis in Clinical Cancers (Review)

“…A recent study has revealed interaction between STRN1 and ULK1 (Unc-51 Like Autophagy Activating Kinase 1) in a positive-feedback manner via PP2A, promoting cell autophagy [ 75 ]. The implication of STRIPAK in autophagy within muscle tissues has additionally been demonstrate in cancer research [ 76 , 77 ]. Although MAP4K2, a GCKI family member, is not considered a STRIPAK member, the depletion of STRN4 induces hyperphosphorylation and activation of MAP4K2, suggesting a crucial regulatory role of STRIPAK upstream to MAP4K2.…”