Regulation of EGFR Signal Transduction Figure 3. DHA uniquely alters EGFR function. YAMC cells were treated with control or 50 mM BSA-complexed fatty acids (LA or DHA) for 72 h. For the final 16–18 h, cells were incubated in low serum media (0.5% FBS) with the same concentration of fatty acids. Cells were stimulated for 0– 30 min with 25 ng/mL EGF and subsequently harvested. Equal concentrations of protein from the whole cell lysates were analyzed by Western blotting for A) phosphorylated EGFR, B) phosphorylated ERK1/2, and C) phosphorylated STAT3. Each immunoblot is representative of 3 independent experiments. Quantification of band volume was performed and data are presented as mean6SEM and normalized to time 0 (n = 3). Statistical significance between treatments (*P,0.05; **P,0.01) was determined using Student’s t-test. D) Additionally, YAMC cells were treated with 50 mM BSA-complexed fatty acids (AA, EPA, or DHA) for a total of 72 h. For the final 16–18 h, cells were incubated in low serum (0.5% FBS) then stimulated for 10 min with 25 ng/mL EGF. Whole cell lysates were separated assessed by Western blotting for phosphorylated (Tyr1068) EGFR. A representative blot from 3 independent experiments is presented. Data are expressed as mean6SEM of phosphorylated EGFR, normalized to control (n = 3). Statistical significance between treatments (P,0.05) is indicated by different letters and was determined using ANOVA and Tukey’s test of contrast. C, control; LA, linoleic acid; DHA, docosahexaenoic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid. doi:10.1371/journal.pone.0039682.g003 lower levels of GTP-bound Ras compared to control and LA- treated cells (Figure 5B). This observation indicates that the DHA- induced perturbation in the EGFR-Ras-ERK1/2 pathway occurs at the site of Ras activation. Ras is comprised of three distinct isoforms, including H-Ras, K- Ras, and N-Ras. Isoform-specific signaling is regulated by differential compartmentalization within cell surface microdo- mains and intracellular compartments [60]. H-Ras is enriched in lipid rafts, both caveolar and noncaveolar, whereas K-Ras is exclusively located in the bulk membrane [59,60]. Additionally, N- Ras is mainly segregated into noncaveolar lipid rafts [63]. Furthermore, Ras proteins have been shown to signal from the Golgi complex, the endoplasmic reticulum, and endomembranes, e.g., endosomes (9, 26, 32). The effect of this distinctive segregation on the signals generated by Ras is just beginning to unfold. Therefore, we assessed the effect of DHA on activation of each Ras isoform to provide further clarity into the mechanism by which DHA suppressed EGF-induced Ras activation. We found that DHA suppressed activation of all three isoforms of Ras, indicating a common mechanism by which DHA suppresses EGF- induced activation of Ras. Furthermore, activation of H-Ras was almost entirely inhibited by DHA, suggesting a distinct mechanism of regulation for this specific isoform. DHA Induces Increased EGFR Internalization and Degradation Endocytosis is a major mechanism of EGFR signal attenuation by targeting the activated receptor for lysosomal proteolysis [64]. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39682