Lysyl-tRNA synthetase modulates autophagy in a Lysine- and phosphorylation- dependent manner

Abstract Background tRNA synthetases are ancient proteins which have been shown to exhibit multiple non-canonical functions including key functions in metabolic regulation. We have previously shown that Lysyl-tRNA synthetase (KARS) Ser207 phosphorylation provokes a new conformer of that protein (p- KARS), inactivates its translational function and allows it to act as a transcriptional regulator of MITF, a transcription factor known to be an autophagy regulator. This has raised the question whether p-KARS has an effect on autophagy. Methods We compared autophagy flux and nuclear p-KARS levels in various cell lines under starvation (EBSS, 4–5 h) versus control conditions. Using lung cancer cells transduced with Tet-On systems for non-phosphomimetic (A KARS) and phosphomimetic (D KARS) variants, we analyzed differences in autophagy flux, autophagy index, and mTORC1 activity. We also added Lysine to the cells in order to assess the possible role of KARS as a Lysine sensor. We further evaluated the nuclear translocation of MiT/TFE transcription factors and their interaction with p-KARS under varying nutrient conditions. Finally, we assessed the autophagy-related transcriptome, the specific binding affinities of KARS variants to MITF, and the impact of p-KARS on extracellular Lysine sensing and downstream autophagy regulation. Results We show that the ratio of the nuclear levels of p-KARS is inversely correlated to the autophagic flux in 14 out of 15 different cell lines. Furthermore, in transduced lung cancer cells expressing a pseudophosphorylated form of KARS (D KARS) there was lower levels of autophagy flux compared with those expressing non-phosphorylatable form, A KARS. Mechanistically, we noted lower expression levels of MITF- regulated autophagy genes in D KARS expressing cells. We also provided evidence that p-KARS may act as a sensor for Lysine since high dose Lysine added to nutrient-depleted media resulted in higher nuclear p-KARS and reduced autophagy, a phenomenon which is recapitulated in D KARS transduced cells. Conclusion All together our data points towards Lysyl-tRNA synthetase and its phosphorylated as having a possible important metabolic regulatory function.