Regulation of EGFR Signal Transduction Figure 6. DHA mediates increased EGFR internalization and degradation. YAMC cells were incubated with untreated media or media supplemented with 50 mM BSA-complexed DHA for a total of 72 h. For the final 16–18 h, cells were incubated with low serum media (0.5% FBS). A) Cell surface proteins were labeled with EZ-Link Sulfo-NHS-SS-Biotin followed by stimulation with 25 ng/mL EGF for 0–30 min. After stimulation, cells were washed and biotin remaining on the cell surface was cleaved. Cell lysates were harvested, and biotinylated EGFR was quantified by ELISA using streptavidin coated plates and anti-EGFR antibody. B) Cell surface EGFR was assessed by treating cells the same as in A, and harvesting without stimulating with EGF or cleaving cell surface biotin. C) Cells were stimulated with 25 ng/mL EGF for 0–30 min and harvested. EGFR was immunoprecipitated from the total cell lysate, assessed by Western blotting for ubiquitin, and quantification of band intensity was performed. All results are representative of at least 3 independent experiments. Data represent mean6SEM. In (A) and (C), data are normalized to time 0. In B), data are normalized to control (no fatty acid). Statistical significance between treatments (*P,0.05. **P,0.01) was determined using Student’s t-test. C, control; DHA, docosahexaenoic acid. doi:10.1371/journal.pone.0039682.g006 (Figure 6B). Additionally, concomitant with increased EGFR phosphorylation, DHA treated cells exhibited enhanced EGFR endocytosis following receptor stimulation (Figure 6A). Because a majority of signaling cascades are activated by EGFR at the plasma membrane, receptor endocytosis and degradation play important roles in terminating EGFR signaling. Therefore, the DHA-mediated enhancement of EGFR internalization is an additional mechanism by which DHA can suppress the ability of EGFR to activate downstream signaling. Once internalized, EGFR can continue signaling from endosomes, recycle back to the plasma membrane, or traffic for degradation. This is noteworthy because DHA increased EGFR ubiquitination and degradation (Figure 6C). Interestingly, although DHA treatment did not affect the steady-state levels of EGFR, following ligand- induced activation, it significantly increased EGFR ubiquitinata- tion and degradation. Since receptor endocytosis and degradation can function to mitigate signal transduction, we propose that this is an additional mode by which DHA modulates the signaling capacity of EGFR. To further understand the mechanism by which DHA suppresses EGFR signal transduction, we attempted to identify the exact locus of the DHA-induced perturbation in EGFR signal transduction. Therefore, we assessed the individual components of the EGFR-Ras-ERK1/2 signaling axis. Grb2 is an immediate proximal downstream mediator of EGFR signaling and directly binds to phosphorylated tyrosine residues of EGFR, including Tyr1068, which we showed here to be increased by DHA treatment. Grb2 mediates multiple responses to EGFR phosphor- ylation, including propagation of intracellular signaling and receptor endocytosis. We observed that DHA treatment increased PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39682