Regulation of EGFR Signal Transduction Figure 2. DHA increases EGFR phosphorylation but suppresses activation of downstream signaling. YAMC cells were untreated (control) or treated with 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 either unstimulated or stimulated for 10 min with 25 ng/mL EGF and subsequently harvested. A) Equal amounts of protein from whole cell lysates were Western blotted for total and phosphorylated (Tyr1068) EGFR. Additionally, EGFR was immunoprecipitated from total cellular lysates prior to Western blotting for EGFR and phosphorylated tyrosine resides. B) Whole cell lysates were analyzed by Western blotting for total and phosphorylated downstream mediators of EGFR signaling, including ERK1/2, STAT3, S6kinase, and Akt. Each blot is representative of 3–4 independent experiments with 3 replicates of each treatment per experiment. Quantification of band volume was performed and data are presented as mean6SEM. Data are expressed as the ratio of the phosphorylated protein to total protein and normalized to control. Statistical significance between treatments (*P,0.05; **P,0.01) was determined using ANOVA and Tukey’s test of contrast. C, control; LA, linoleic acid; DHA, docosahexaenoic acid. doi:10.1371/journal.pone.0039682.g002 signaling by DHA is dependent on its presence in the plasma membrane. DHA Inhibits EGF-stimulated Ras GTP-binding Due to the fact that DHA treatment increased EGFR phosphorylation while concurrently suppressing activation of downstream mediators, we attempted to pinpoint the site of perturbation in the signaling cascade. Since the EGFR-Ras- ERK1/2 pathway is well documented, we examined the components of this pathway downstream of the receptor. Following EGF stimulation, growth receptor-bound protein 2 (Grb2) is recruited to the plasma membrane and binds to phosphorylated tyrosine residues of EGFR (Tyr1068 and Tyr1086). To assess recruitment of this immediate proximal signal downstream of EGFR phosphorylation, cells were transfected with fluorescently-tagged SH2 domain of Grb2 (Grb2-YFP), capable of binding to the phosphorylated tyrosine residues of EGFR. Cells were subsequently imaged using total internal reflective fluores- cence (TIRF) microscopy to assess Grb2-YFP translocation to the plasma membrane in response to EGFR activation. Following stimulation with EGF, Grb2-YFP was rapidly recruited to the plasma membrane (Figure 5A). We observed a significant increase in EGF-stimulated Grb2-YFP plasma membrane translocation in DHA treated cells compared to control and LA treated cells. Grb2 recruits Son of Sevenless (Sos), a guanine nucleotide exchange factor (GEF), to activate Ras. Therefore, we assessed Ras activation status by pulling-down GTP-bound Ras followed by Western blotting for Ras. DHA-treated cells had significantly PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39682