ULK1-Hsp90-Cdc37 and AMPK: Novel Insight Into the Regulation of Mitophagy

Abstract 987

Autophagy plays an important role in maintaining mitochondrial integrity, directing lysosome-mediated destruction of cellular cargo, including damaged or dysfunctional mitochondria. Flux through the autophagy pathway is rapidly induced to promote survival in response to metabolic or proteotoxic stress resulting from exposure to noxious environmental cues, such as starvation, hypoxia or heat stress and likely contributes to the increase in mitochondrial turnover observed under each of these conditions. Dysregulation of this process has been linked to the pathogenesis of diseases, including anemia, diabetes, neurodegeneration and cancer.

Atg1 is a serine-threonine kinase that directs the autophagy machinery to appropriate cargo in responses to changes in the availability of carbon and nitrogen in yeast. Ulk1, one of the mammalian homologues of Atg1, is required for starvation-induced autophagy and clearance of mitochondria in terminally differentiating erythroid cells. The function of Ulk1 is also regulated by AMP dependent Kinase (AMPK)-mediated phosphorylation, however, the precise molecular consequence of this post-translational modification has not been explored. Our preliminary findings indicate that AMPK phosphorylates Ulk1 during red blood cell maturation and in response to mitochondrial uncoupling, and that this phosphorylation is critical for mitochondrial clearance. Therefore, we sought to use these systems to explore the mechanism by which AMPK phosphorylation regulates Ulk1 function.

We previously demonstrated that the stability and kinase activity of Ulk1 depends on its physical interaction with Hsp90 and the kinase-specific co-chaperone, Cdc37. Hsp90 is an abundant chaperone that directs the maturation and activation of a restricted group of metastable proteins, typically kinases and signaling molecules, and orchestrates a broad response to cellular stress. Here, we demonstrate that AMPK phosphorylation of Ulk1 does not affect Ulk1 kinase activity, but instead promotes its release from Hsp90 and its localization to damaged mitochondria. Preliminary studies indicate that the serine-proline rich domain of Ulk1, which contains at least 4 residues that are phosphorylated by AMPK, is an intrinsically disordered domain. We hypothesize that phosphorylation of Ulk1 by AMPK stabilizes a predicted alpha-helical structure within this domain and contributes to release of Hsp90. These findings are important because they provide significant insight into the regulation and function of Ulk1, a protein involved in mitochondrial turnover during red blood cell maturation and in proliferating cells.