Potential sites of PI-3 kinase function in the endocytic pathway revealed by the PI-3 kinase inhibitor, wortmannin.
Previously we have shown that PDGF receptor mutants that do not bind PI-3 kinase internalize after ligand binding, but fail to downregulate and degrade. To define further the role of PI-3 kinase in trafficking processes in mammalian cells, we have investigated the effects of a potent inhibitor of PI-3 kinase activity, wortmannin. At nanomolar concentrations, wortmannin inhibited both the transfer of PDGF receptors from peripheral compartments to juxtanuclear vesicles, and their subsequent degradation. In contrast, the delivery of soluble phase markers to lysosomes, assessed by the accumulation of Lucifer yellow (LY) in perinuclear vesicles after 120 min of incubation, was not blocked by wortmannin. Furthermore, wortmannin did not affect the rate of transferrin uptake, and caused only a small decrease in its rate of recycling. Thus, the effects of wortmannin on PDGFr trafficking are much more pronounced than its effects on other endocytic events. Unexpectedly, wortmannin also caused a striking effect on the morphology of endosomal compartments, marked by tubulation and enlargement of endosomes containing transferrin or LY. This effect was somewhat similar to that produced by brefeldin A, and was also blocked by pre-treatment of cells with aluminum fluoride (AlF4-). These results suggest two sites in the endocytic pathway where PI-3 kinase activity may be required: (a) to sort PDGF receptors from peripheral compartments to the lysosomal degradative pathway; and (b) to regulate the structure of endosomes containing lysosomally directed and recycling molecules. This latter function could be mediated through the activation of AlFt4-)-sensitive GTP-binding proteins downstream of PI-3 kinase.
Shpetner
H
H
Program in Molecular Medicine and Department of Cell Biology, University of Massachusetts Medical School, Worcester, 01655, USA.
Joly
M
M
Hartley
D
D
Corvera
S
S
D000730:Androstadienes / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D006801:Humans; D007546:Isoquinolines; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000502:physiology*; D017479:Receptors, Platelet-Derived Growth Factor / Q000502:physiology; D014168:Transferrin / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007546:Isoquinolines; D014168:Transferrin; C017475:lucifer yellow; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D017479:Receptors, Platelet-Derived Growth Factor; D000077191:Wortmannin