Abstract B20: STK3/4-mediated phosphorylation of LC3B regulates directional intracellular transport of autophagic vesicles

Abstract

Abstract Autophagy is a fundamental catabolic process that allows the degradation of cytosolic components to supply the cell with nutrients in response to starvation, among other functions. Recently, our lab discovered that the Hippo pathway members, STK3 and STK4, phosphorylate the autophagy protein LC3B, a key component of autophagic vesicles that participates in specifying their content and their intracellular transport. Depletion of STK3/4 kinases or generation of an LC3B phospho-null mutant (T50A) causes a block of the autophagy process evidenced by an accumulation of autophagic vesicles throughout the cell. However, these studies did not clarify which step of the autophagy process was impaired. To address this question, we employed proteomics to analyze LC3B-binding partners affected by LC3B phosphorylation, and identified multiple proteins implicated in intracellular vesicle transport. Accordingly, we hypothesized that LC3B phosphorylation may affect transport of autophagic vesicles. We tested this hypothesis directly by live-cell imaging in multiple cell types and found that the absence of LC3B phosphorylation was accompanied by an aberrantly high transport of LC3B-positive autophagic vesicles toward the periphery of the cell, and a low transport of the vesicles toward the perinuclear area, where the autophagy process is normally completed. Moreover, starvation-induced perinuclear positioning of autophagic vesicles was prevented in the absence of LC3B phosphorylation. Collectively, these findings indicate that STK3/4-mediated phosphorylation of LC3B regulates the directional transport of autophagic vesicles and this may promote autophagy completion. Notably, this regulatory axis may be triggered when the nutrient availability is low and autophagy is induced. Understanding how input signals regulate STK3/4, and in turn control autophagy, may prove important to develop therapies against diseases such as cancer. Citation Format: Jose L. Nieto-Torres, Sean-Luc Shanahan, Romain Chassefeyre, Sara Landeras-Bueno, Lee R. Lee, Sviatlana Zaretski, Sandra Encalada, Malene Hansen. STK3/4-mediated phosphorylation of LC3B regulates directional intracellular transport of autophagic vesicles [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B20.