The addition of phosphoryl groups to proteins can affect their activity and subcellular location. During the process of phosphorylation, enzymes known as kinases add these post-translational modifications (PTMs) in response to variety of stimuli, including nutrient depletion. While it has long been appreciated that glucose starvation induces autophagy, or the controlled degradation of our cells for energy, the individual elements of the signaling pathways involved in the process are unclear. A recent study by Karabiyik et al. combined a variety of complimentary techniques, including LC-MS/MS, to uncover key details related to individual kinases and cellular processes involved in this important phenomenon.
How was PEAKS used?
PEAKS was used to identify phosphorylation sites on the kinase PIKfyve. This was accomplished by first assigning phosphorylation (STY) as a variable modification during a PEAKS DB search, then searching for modifications in de novo sequencing results using PEAKS PTM, and finally identifying amino acid mutations in PEAKS SPIDER. Identification of the residues that were phophorylated meant that the researchers could swap out single amino acids (by site-directed mutagenesis), prevent phosphorylation at an individual site, and examine the functional consequences of individual modifications in a targeted manner.
Karabiyik, Cansu, et al. “Glucose Starvation Induces Autophagy via ULK1-Mediated Activation of PIKfyve in an AMPK-Dependent Manner.” Developmental Cell, Elsevier BV, June 2021. Crossref, doi:10.1016/j.devcel.2021.05.010.
Abstract
Autophagy is an essential catabolic process induced to provide cellular energy sources in response to nutrient limitation through the activation of kinases, like AMP-activated protein kinase (AMPK) and ULK1. Although glucose starvation induces autophagy, the exact mechanism underlying this signaling has yet to be elucidated. Here, we reveal a role for ULK1 in non-canonical autophagy signaling using diverse cell lines. ULK1 activated by AMPK during glucose starvation phosphorylates the lipid kinase PIKfyve on S1548, thereby increasing its activity and the synthesis of the phospholipid PI(5)P without changing the levels of PI(3,5)P2. ULK1-mediated activation of PIKfyve enhances the formation of PI(5)P-containing autophagosomes upon glucose starvation, resulting in an increase in autophagy flux. Phospho-mimic PIKfyve S1548D drives autophagy upregulation and lowers autophagy substrate levels. Our study has identified how ULK1 upregulates autophagy upon glucose starvation and induces the formation of PI(5)P-containing autophagosomes by activating PIKfyve.