CSNK2 suppresses autophagy by activating FLN-NHL-containing TRIM proteins.

Macroautophagy/autophagy is a tightly regulated cellular process integral to homeostasis and innate immunity. As such, dysregulation of autophagy is associated with cancer, neurodegenerative disorders, and infectious diseases. While numerous factors that promote autophagy have been characterized, the key mechanisms that prevent excessive autophagy are less well understood. Here, we identify CSNK2/CK2 (casein kinase 2) as a negative regulator of autophagy. Pharmacological inhibition of CSNK2 activity or siRNA-mediated depletion of CSNK2 increased basal autophagic flux in cell lines and primary human lung cells. Vice versa, ectopic expression of CSNK2 reduced autophagic flux. Mechanistically, CSNK2 interacted with the FLN (filamin)-NHL domain-containing tripartite motif (TRIM) family members TRIM2, TRIM3 and TRIM71. Our data show that recruitment of CSNK2 to the C-terminal NHL domain of TRIM3 lead to its robust phosphorylation at serine 661 by CSNK2. A phosphorylation-defective mutant of TRIM3 was unable to reduce autophagosome numbers indicating that phosphorylation by CSNK2 is required for TRIM-mediated autophagy inhibition. All three TRIMs facilitated inactivation of the ULK1-BECN1 autophagy initiation complex by facilitating ULK1 serine 757 phosphorylation. Inhibition of CSNK2 promoted autophagy upon influenza A virus (IAV) and measles virus (MeV) infection. In line with this, targeting of CSNK2 or depletion of TRIM2, TRIM3 or TRIM71 enhanced autophagy-dependent restriction of IAV, MeV and human immunodeficiency virus 1 (HIV-1). Thus, our results identify the CSNK2-TRIM2, -TRIM3, -TRIM71 axis as a key regulatory pathway that limits autophagy. Targeting this axis may allow for therapeutic induction of autophagy against viral infections and in diseases associated with dysregulated autophagy.

An improved method for high-throughput quantification of autophagy in mammalian cells.

Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays.