Merrill Nathan M, Schipper Joshua L, Karnes Jonathan B, Kauffman Audra L, Martin Katie R, MacKeigan Jeffrey P
Van Andel Institute Graduate School, Grand Rapids, Michigan, United States of America.
Center for Cancer Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, United States of America.
PLoS One. 2017 Sep 14;12(9):e0184909. doi: 10.1371/journal.pone.0184909. eCollection 2017.
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.
磷酸肌醇3激酶（PI3K）家族成员参与多种细胞命运，包括细胞生长、增殖和存活。虽然关于I类和III类PI3K的许多分子细节已为人所知，但关于II类PI3K的了解较少。为了探究所有八种PI3K异构体在自噬中的功能，我们分别敲低每个基因并测量自噬。我们发现，在通过小干扰RNA介导敲低PIK3C2A（编码2类PI3K，PI3K-C2α）后，自噬显著减少。这种有缺陷的自噬可通过外源性PI3K-C2α挽救，但不能通过激酶失活的PI3K-C2α挽救。使用共聚焦显微镜，我们检测内吞作用和自噬的标志物，发现PI3K-C2α与内吞作用的标志物共定位。尽管转铁蛋白标记显示内吞摄取完整，但敲低PIK3C2A会导致回收内体处的囊泡积累。我们分离出不同的膜来源，并观察到PI3K-C2α与内吞作用和自噬的标志物相互作用，特别是与ATG9相互作用。敲低PIK3C2A或ATG9A/B，而不是PI3KC3，会导致回收内体处转铁蛋白阳性网格蛋白包被囊泡和RAB11阳性囊泡的积累。综上所述，这些结果支持PI3K-C2α在内涵体正常成熟中的作用，并表明PI3K-C2α可能是连接内吞途径和自噬途径的关键节点。
