Sustained treatment of PMA affects trafficking and signaling of EGFR

It has been shown previously that activation of protein kinase C (PKC) with phorbol 12‐myristate 13‐acetate (PMA) diverts internalized epidermal growth factor receptor (EGFR) from a degradative to a recycling pathway by phosphorylation of EGFR on Thr654. In this study, we evaluated if sustained treatment with PMA protects EGFR from degradation by sequestration of EGFR to a subset of Rab11‐positive recycling endosomes, named the pericentrion. Moreover, mechanisms of EGFR protection from degradation by sustained treatment of PMA were investigated. Treatment of HEK293 cells with PMA induced sequestration of EGFR in a PKC‐and PLD‐dependent manner, as shown using pharmacological inhibitors. Next, the molecular mechanisms of PMA‐induced EGFR protection from degradation were investigated. As shown previously, when cells were treated with EGF alone, there was significant loss of the EGFR protein. In striking difference to that, pretreatment with PMA prevented EGFR downregulation. We showed that this protection from degradation is phospholipase D (PLD)‐dependent by using 1‐‐butanol (PLD inhibitor) as well as PLD 1 and 2 specific siRNAs. Also our data shows that phosphorylation of Thr654 coincides with pericentrion formation and was pericentrion‐dependent. Taken together, these results strongly suggest that sustained treatment of PMA can protect EGFR from degradation by sequestrating EGFR to pericentrion.