Physiological role of Vps34 phosphatidylinositol 3-kinase in mammalian cells.

摘要

The human Class III phosphatidylinositol 3-kinase (PI3K), hVps34, converts phosphatidylinositol (PI) to phosphatidylinositol 3-phosphate (PI3P). Previous studies using PI3K inhibitors have indicated that production of PI3P is important for vesicle-mediated trafficking events, including endocytosis, sorting of receptors in multivesicular bodies (MVBs), transport of lysosomal enzymes from the trans-Golgi network (TGN), and autophagy. This study utilizes siRNA-mediated gene silencing to define the specific trafficking pathways in which hVps34 functions in human U-251 glioblastoma cells. Suppression of hVps34 expression caused the accumulation of large, acidic phase-lucent vacuoles that contain lysosomal membrane proteins. Analysis of these structures by electron microscopy suggests that they represent swollen late endosomes that have lost the capacity for inward vesiculation but retain the capacity to fuse with lysosomes. In contrast to the effects on late endosomes, suppression of hVps34 expression did not inhibit trafficking of cathepsin D from the TGN to late endosomes, endocytic uptake of fluid-phase markers, recycling of transferrin receptors, degradation of activated epidermal growth factor (EGF) receptors, or association of a PI3P-binding protein, EEA1, with early endosomes. Nevertheless, EGF receptor phosphorylation and signaling were enhanced in the absence of hVps34, consistent with the retention of the EGF receptor on the limiting membranes of the enlarged late endosomes prior to degradation. These findings indicate that hVps34 plays a major role in generating PI3P required for internal vesicle formation in late endosomes and that, unexpectedly, other mechanisms may exist to generate the PI3P required for vesicular trafficking in the early endocytic pathway or the TGN. Additionally, suppression of hVps34 expression in U-251 cells resulted in a marked reduction in cell growth accompanied by a block in DNA synthesis. Furthermore, investigation of the subcellular distribution of hVps34 in human HEp-2 laryngeal carcinoma cells revealed that hVps34 localizes to the nucleus. The nuclear pool of hVp34 is resistant to detergent extraction but can be partially released upon treatment with RNase. Overall, this suggests a role for hVps34 in nuclear regulatory events at the level of RNA processing and/or transport.