Regulation of EGFR Signal Transduction Figure 4. DHA alterations to EGFR function require enrichment of DHA in the plasma membrane. A) Control treated YAMC cells were incubated with low serum media overnight. Select cultures were then treated with 10 mM U0126 for 2 h. Untreated and U0126 treated cultures then underwent stimulation with 25 ng/mL EGF for 10 min. Lysates were assessed by Western blotting for phosphorylation of Erk1/2 and EGFR (Tyr 1068). B) Following stimulation with EGF, cells were subjected to chemical crosslinking prior to harvesting cell lysates. Cell lysate was assessed by Western blotting for EGFR. EGFR dimers were identified as bands with twice the molecular weight of EGFR monomers. C-D) For ‘‘wash out’’ experiments, YAMC cells were either untreated or treated with 50 mM DHA for 72 h. Select DHA treated cultures were then washed and incubated for an additional 40 h with either untreated media or 50 mM LA. For the final 16–18 h, cells were incubated with low serum media (0.5% FBS) followed by stimulation with 25 ng/mL EGF then harvested. C) Membrane lipids were extracted and enrichment of membrane phospholipids with DHA was quantified by capillary gas chromatography/mass spectrometry. D) Lysates were assessed by Western blotting for phosphorylated EGFR (Tyr1068). The blot is representative of 3 independent experiments. Data are presented as mean6SEM of phosphorylated EGFR, normalized to control. Statistical significance between treatments as indicated by different letters (P,0.05) was determined using ANOVA and Tukey’s test of contrast. C, control; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; LA, linoleic acid. doi:10.1371/journal.pone.0039682.g004 Recent evidence suggests that lipid rafts may play a role in mediating EGFR endocytosis [65,66]. Additionally, EGFR phosphorylation and Grb2 recruitment to EGFR are intimately linked to EGFR internalization [67,68,69,70]. Therefore, we next assessed the effect of DHA on EGFR endocytosis using a surface biotinylation assay. Interestingly, internalization of EGFR oc- curred more rapidly in DHA treated cells compared to untreated (control) cells (Figure 6A). In addition, consistent with data in Figure 4, A and B, DHA reduced the steady-state plasma membrane localization of EGFR in unstimulated cells (Figure 6B). Following internalization, EGFR can either be recycled back to the cell surface or be targeted to the lysosome for degradation. Ubiquitin acts as a sorting signal to down-regulate the functions of plasma membrane proteins. EGFR internalization and degradation are facilitated by ubiquitin [71]. Receptor endocytosis stimulated by ubiquitination is considered to be crucial to prevent oncogenesis because it sorts the receptor to the lumen of multivesicular bodies and terminates growth factor signaling [72]. This is especially crucial for EGFR since it has been shown to continue to signal from endosomes after it is internalized [67]. Therefore, we assessed ubiquitination of the receptor in order to determine whether the DHA-induced increase in EGFR internalization was associated with increased receptor ubiquitina- tion and degradation. Immunoprecipitation of EGFR followed by Western blotting for ubiquitin to assess EGFR ubiquitination was utilized. We found that EGFR ubiquitination was significantly PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39682