PHOSPHATIDYLINOSITOL 3'-KINASE, BUT NOT P70 RIBOSOMAL S6 KINASE, IS INVOLVED IN MEMBRANE-PROTEIN RECYCLING - WORTMANNIN INHIBITS GLUCOSE-TRANSPORT AND DOWN-REGULATES CELL-SURFACE TRANSFERRIN RECEPTOR NUMBERS INDEPENDENTLY OF ANY EFFECT ON FLUID-PHASE ENDOCYTOSIS IN FIBROBLASTS

The exposure of 3T3-L1 fibroblasts to growth factors results in a 2- t o 3-fold increase in 2-deoxyglucose transport and a similar to 50% to 80% increase in cell-surface transferrin receptor levels. We sought to determine the role of phosphatidylinositol-3'-kinase and p70 ribosoma l S6 kinase in these stimulations, using selective inhibitors of these enzymes. Both basal and growth factor-stimulated deoxyglucose transpo rt are blocked by wortmannin, but with different IC50 values (65 nM vs . 15 nM, respectively), suggesting a functional difference between the se two states. This is accompanied by the accumulation of glucose tran sporters in Intracellular locations. Both basal and growth factor-stim ulated cell-surface transferrin receptor levels are downregulated by w ortmannin, but with identical IC50 values (similar to 15 nM). These tw o proteins are known to recycle between an intracellular site and the plasma membrane in these cells, thus implying a functional role for ph osphatidylinositol-3'-kinase in membrane recycling. In an effort to de termine whether the effect of wortmannin was selective for the protein component of this recycling, we examined fluid-phase endocytosis of r adiolabeled mannitol. Wortmannin was without effect on the fluid phase accumulation of mannitol, suggesting that the effects on membrane tra ffic are limited to the protein component of recycling membranes. Rapa mycin, an inhibitor of p70 ribosomal S6 kinase, was without effect on any of these parameters, but both rapamycin and wortmannin inhibit gro wth factor-stimulated p70 ribosomal S6 kinase activity. These data sup port an important role for phosphatidylinositol-3'-kinase, but not p70 ribosomal S6 kinase, in the regulation of membrane protein traffic. W e suggest that this enzyme may be involved in sorting of membrane prot eins during trafficking.