Abstract
Phosphoinositide 3-kinase (PI3K) family members are involved in diverse cellular fates including cell growth, proliferation, and survival. While many molecular details are known about the Class I and III PI3Ks, less is known about the Class II PI3Ks. To explore the function of all eight PI3K isoforms in autophagy, we knock down each gene individually and measure autophagy. We find a significant decrease in autophagy following siRNA-mediated PIK3C2A (encoding the Class 2 PI3K, PI3K-C2α) knockdown. This defective autophagy is rescued by exogenous PI3K-C2α, but not kinase-dead PI3K-C2α. Using confocal microscopy, we probe for markers of endocytosis and autophagy, revealing that PI3K-C2α colocalizes with markers of endocytosis. Though endocytic uptake is intact, as demonstrated by transferrin labeling, PIK3C2A knockdown results in vesicle accumulation at the recycling endosome. We isolate distinct membrane sources and observe that PI3K-C2α interacts with markers of endocytosis and autophagy, notably ATG9. Knockdown of either PIK3C2A or ATG9A/B, but not PI3KC3, results in an accumulation of transferrin-positive clathrin coated vesicles and RAB11-positive vesicles at the recycling endosome. Taken together, these results support a role for PI3K-C2α in the proper maturation of endosomes, and suggest that PI3K-C2α may be a critical node connecting the endocytic and autophagic pathways.
Highlights
Macroautophagy is an intracellular degradation pathway that targets cytosolic material for lysosomal degradation [1,2,3]
U2OS cells stably expressing LC3B fused to a tandem (EGFP and mRFP) fluorescent tag [56,57,58] were transfected with control siRNA and treated with rapamycin, an mTORC1 inhibitor and autophagy inducer, for 6 hours
We are interested in characterizing kinases and phosphatases that may serve as regulators of autophagy [57,58]
Summary
Macroautophagy (autophagy) is an intracellular degradation pathway that targets cytosolic material for lysosomal degradation [1,2,3]. Under conditions of stress, such as nutrient starvation, this process is used to produce amino acids and other biochemical building blocks to promote cell survival [4,5,6,7]. The importance of autophagy is underscored by its deregulation in a number of diseases, notably cancer [4,8,9]. While considerable progress has been made characterizing the mechanism of this process, many important issues, such as those concerning the incorporation of different membrane sources into this pathway, remain unaddressed. In response to nutrient stress, key cellular signals, such as the activation of AMP activated protein kinase (AMPK) and suppression of mechanistic target of rapamycin complex 1.
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