Recent studies have suggested that the serine-threonine kinase Ulk1, a mammalian homologue of the yeast autophagy regulator, Atg1, plays an important role in mitochondrial clearance during reticulocyte maturation. In order to gain insight into the regulation and activity of Ulk1, we used an unbiased proteomics approach to identify Ulk1-interacting proteins. Here, we demonstrate that Ulk1 interacts with heat shock protein 90 (Hsp90) and Cdc37, and is among a growing list of proteins whose steady state levels are regulated by this kinase-specific chaperone complex. Treatment of murine embryonic fibroblasts (MEFs) with the HSP90 inhibitor, 17AAG, inhibits the AKT/Tor pathway and induces autophagy despite the decreased steady state levels of Ulk1. By contrast, Ulk1-deficient MEFs are defective in autophagy induction, as measured by LC3 puncta formation (but not LC3 conversion) and ultrastructural analysis. Exposure of terminally differentiating erythroid cells to HSP90 inhibitors prevents the normal accumulation of Ulk1 protein and results in impaired autophagic clearance of organelles, similar to the effect of Ulk1 gene deletion. These findings highlight the importance of Ulk1 protein levels in terminal erythroid maturation and induction of autophagy and its regulation by the Hsp90/Cdc37 chaperone complex.

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