Regulation of EGFR Signal Transduction the plasma membrane was fractionated into 3 distinct fractions, high density membrane (HDM), intermediate density membrane (IDM), and lipid raft enriched membrane (LR) by gradient ultracentrifugation. Fractions were collected and an equal amount of protein from each fraction was analyzed by Western blotting using antibodies against EGFR, caveolin-1, and clathrin or using peroxidase conjugated cholera toxin B subunit (for GM-1). Quantification of band intensity was performed, and data are presented as the relative amount of EGFR in each fraction, with the sum of each fraction equaling 100. Western blots are representative of 2 independent experiments. C, control; LA, linoleic acid; DHA, docosahexaenoic acid; PM, plasma membrane; HDM, high density membrane; IDM, intermediate density membrane; LR, lipid raft enriched membrane. B) YAMC cells were treated with 50 mM BSA-complexed fatty acids for 72 h. Twenty-four h after initiating fatty acid treatment, cells were co-transfected with RFP-tH and EGFR-mGFP. Approximately 32 h after transfection, cells were incubated in low serum media (0.5% FBS) overnight prior to imaging. Images are representative of 4 independent experiments. Whole cell images of each individual channel and the merged images are shown on the left. High magnification images of the plasma membrane are shown on the right. Mander’s colocalization coefficient was calculated at the plasma membrane for the amount of EGFR- mGFP (green) colocalizing with RFP-tH (red) using Nikon Elements AR 3.2. The coefficient is the mean of n = 30–40 cells per treatment. Statistical significance between treatments (*P,0.05) was determined using ANOVA and Tukey’s test of contrast. Bars,10 mm. doi:10.1371/journal.pone.0039682.g001 Subsequently, cells were stimulated with 25 ng/mL EGF for 10 min followed by isolation of cell lysates. To assess the effects of fatty acid treatment on EGF-induced EGFR phosphorylation, lysates were probed for EGFR phosphorylated on Tyr1068, one of the major sites of EGFR phosphorylation that is involved in activation of downstream signaling. DHA treatment resulted in a greater than two-fold increase in EGFR phosphorylation compared to untreated control or LA treated cells (Figure 2A). EGFR was also immunoprecipitated from cell lysates followed by immunoblotting for total phosphorylated tyrosine residues. Consistent with the initial results, DHA enhanced total EGFR tyrosine phosphorylation by greater than two-fold (Figure 2A). Since EGFR phosphorylation is characteristically associated with activation of downstream signaling, we further probed cell lysates for phosphorylation of downstream effector proteins, including ERK1/2, STAT3, and Akt. DHA treatment resulted in a ,50% reduction in the activation of ERK1/2 and STAT3 in DHA treated cells compared to untreated control and LA treated cells (Figure 2B). DHA had no effect on Akt phosphorylation at Ser473 (Figure 2B). The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that is often hyperactivated in cancer, and stimulation of both Akt and ERK1/2 can lead to activation of a protein complex containing mTOR, i.e., mTORC1. To assess mTORC1 activity, we measured phos- phorylation of ribosomal S6 kinase (S6K), a kinase that is directly phosphorylated by mTOR at Thr389. We found that DHA significantly suppressed EGF-induced phosphorylation of S6K, indicating reduced activation of mTOR (Figure 1C). Together, these data suggest that DHA disrupts the EGFR signaling cascade in colonocytes by disengaging EGFR phosphorylation from signal transduction. We also investigated the effect of DHA on EGFR phosphor- ylation and signaling at multiple time points. Untreated control and DHA treated cells were stimulated with 25 ng/mL EGF from 0–30 min. Consistent with initial results, EGFR phosphor- ylation was significantly increased by DHA treatment at 2, 5, and 10 min following stimulation (Figure 3A). EGFR phosphor- ylation peaked in DHA treated cells at 5 min and started decreasing, whereas EGFR phosphorylation was highest at 10 min in control samples, suggesting a dynamic alteration in EGFR regulation. Downstream signaling from EGFR through ERK1/2 and STAT3 was suppressed by DHA at each time point assessed (Figure 3B,C). Effect of Fatty Acid on EGFR is DHA Specific Since DHA and EPA are both long chain omega-3 fatty acids that have been shown to reduce colon tumor development, we examined whether each of these fatty acids cause an increase in EGFR phosphorylation or if the effect is specific to DHA. We also assessed the effects of arachidonic acid (AA; 20:4 n26) on EGFR activation as a control, long-chain n-6 PUFA. Lysates were probed for phosphorylated EGFR. Neither AA nor EPA treatment resulted in an increase in EGFR phosphorylation compared to control (Figure 3D). Only DHA treatment increased the phosphorylation status of EGFR. Mechanism of DHA-induced Increase in EGFR Phosphorylation EGFR phosphorylation is controlled by a variety of factors. Therefore, we next determined the mechanism by which DHA increases EGFR phosphorylation. Previous studies have suggested a number of possible contributing mechanisms. For example, EGFR phosphorylation has been shown to be increased by an adaptive mechanism in response to suppression of downstream signaling through ERK1/2 [61]. Another study demonstrated that the lipid environment directly influences EGFR dimerization, which results in increased EGFR phosphorylation [62]. Since we demonstrated that DHA both alters the membrane environment of EGFR and suppresses ERK1/2 phosphorylation, we next assessed whether either of these two mechanisms contributed to the observed DHA-induced increase in EGFR phosphorylation. First, we utilized a specific inhibitor of ERK1/2, U0126, to recapitulate the suppressive effect of DHA on ERK1/2 activation. We then assessed the effect of this inhibitor on EGFR activation status. We found that treatment of cells with 10 mM U0126 for 2 h prior to stimulation completely inhibited EGF-induced activation of ERK1/2 (Figure 4A). However, this inhibition had no effect on overall EGFR phosphorylation, suggesting that a downstream feedback-mediated increase in EGFR phosphorylation induced by ERK1/2 suppression is not a mechanism by which DHA enhances EGFR phosphorylation. We subsequently measured the effect of DHA on EGFR dimerization. Following stimulation with EGF, EGFR dimers were linked using bis[sulfosuccinimidyl] suberate (BS3), a non-perme- able crosslinking reagent. DHA treated cells exhibited a greater than three-fold increase in dimerization compared to control and LA treated cells (Figure 4B). This is consistent with previous studies indicating that the lipid environment affects EGFR dimerization [62], suggesting that the effect of DHA on EGFR is due to the alteration in the lipid environment of the receptor, resulting in increased EGFR dimerization and, therefore, phos- phorylation. We postulated that if the DHA-induced adaptive phosphoryla- tion of EGFR is a result of membrane enrichment causing disruption of lipid rafts, then the effect should be reversible by depleting the cells of DHA. To test this hypothesis, cells were treated with DHA, followed by a washout period wherein the cells were either treated with no fatty acid or with LA. As expected, the washout period resulted in significantly reduced levels of DHA in the plasma membrane (Figure 4C), and ligand-induced EGFR phosphorylation was normalized back to the same level as control (Figure 4D). These data indicate that the inhibition of EGFR PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39682