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 P I-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 po tent inhibitor of PI-3 kinase activity, wortmannin. At nanomolar conce ntrations, wortmannin inhibited both the transfer of PDGF receptors fr om peripheral compartments to juxtanuclear vesicles, and their subsequ ent degradation. In contrast, the delivery of soluble phase markers to lysosomes, assessed by the accumulation of Lucifer yellow (LY) in per inuclear vesicles after 120 min of incubation, was not blocked by wort mannin. Furthermore, wortmannin did not affect the rate of transferrin uptake, and caused only a small decrease in its rate of recycling. Th us, the effects of wortmannin on PDGFr trafficking are much more prono unced than its effects on other endocytic events. Unexpectedly, wortma nnin also caused a striking effect on the morphology of endosomal comp artments, 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 al uminum fluoride (AlF4-). These results suggest two sites in the endocy tic pathway where PI-3 kinase activity may be required: (a) to sort PD GF receptors from peripheral compartments to the lysosomal degradative pathway; and (b) to regulate the structure of endosomes containing ly sosomally directed and recycling molecules. This latter function could be mediated through the activation of AlF4--sensitive GTP-binding pro teins downstream of PI-3 kinase.