Regulation of epidermal growth factor receptor endocytosis by wortmannin through activation of Rab5 rather than inhibition of phosphatidylinositol 3‐kinase

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Abstract
The involvement of phosphatidylinositol 3‐kinase (PI3K) in membrane trafficking in mammalian cells has largely come from experiments with wortmannin. This compound inhibits endosome fusion in vitro , possibly by inhibiting the production of phosphatidylinositol (PtdIns)‐3‐P, which co‐regulates EEA1 with Rab5. However, the results from wortmannin inhibition experiments performed in vivo differ significantly. We have recently shown that wortmannin enlarges endosomes containing the epidermal growth factor receptor (EGFR) and enhances the lysosomal degradation of EGFR. In this report, we demonstrate that addition of the PI3K reaction products does not suppress wortmannin‐induced enlargement of EGFR‐containing endosomes and enhancement of EGFR degradation. Moreover, the effects of wortmannin on the intracellular trafficking of EGFR mimic those of the permanently activated Rab5 mutant, Rab5 Q79L, which stimulates endosome fusion. We also found that an inactive Rab5 mutant, Rab5 S34N, blocks wortmannin‐induced endosome enlargement and that wortmannin stimulates the activation of Rab5. We further showed that wortmannin reduced the membrane association of p120 Ras GTPase‐activating protein (GAP) and inhibited the interaction between Rab5 and p120 Ras GAP. We conclude that wortmannin alters intracellular trafficking of EGFR by activating Rab5 rather than by inhibiting PI3K.