Epidermal growth factor receptor (EGFR) can undergo post-translational modifications, including phosphorylation, glycosylation and ubiquitylation, leading to diverse physiological consequences and modulation of its biological activity. There is increasing evidence that methylation may parallel other post-translational modifications in the regulation of various biological processes. It is still not known, however, whether EGFR is regulated by this post-translational event. Here, we show that EGFR Arg 1175 is methylated by an arginine methyltransferase, PRMT5. Arg 1175 methylation positively modulates EGF-induced EGFR trans-autophosphorylation at Tyr 1173, which governs ERK activation. Abolishment of Arg 1175 methylation enhances EGF-stimulated ERK activation by reducing SHP1 recruitment to EGFR, resulting in augmented cell proliferation, migration and invasion of EGFR-expressing cells. Therefore, we propose a model in which the regulatory crosstalk between PRMT5-mediated Arg 1175 methylation and EGF-induced Tyr 1173 phosphorylation attenuates EGFR-mediated ERK activation.

R e s e a R c h a R t i c l e4 5 3 0 jci.org Volume 125 Number 12 December 2015 ing activation of HER2 or MET signaling, mutation of PIK3CA and BRAF, or expression status of PTEN, but retrospective analyses revealed inconsistent and controversial findings (16). So far, the most accepted predictive marker for poor cetuximab response is mutant KRAS status, due to its association with poor survival rate under cetuximab treatment in colorectal cancer clinical trials (17)(18)(19)(20). Therefore, American Society of Clinical Oncology recommended cetuximab treatment for only patients with WT KRAS (21). However, there is increasing evidence showing that WT KRAS is not sufficient to confer sensitivity to cetuximab (22)(23)(24), and some patients with mutant KRAS are still sensitive to cetuximab (16,(25)(26)(27)(28). These findings suggest that further investigation into the underlying mechanisms of cetuximab resistance and identification of a better predictor for cetuximab response are ing of chemotherapeutic agents have improved the response and survival rate of colorectal patients. Currently, rational targeting of molecular signaling pathways that are involved in the etiology of malignancies is one of the most promising strategies in novel anticancer drug development (13). Owing to the role of EGFR in tumorigenesis, new classes of drugs that target EGFR are among the most clinically advanced molecular-targeted therapies. Although EGFR tyrosine kinase inhibitors combined with chemotherapy presented severe toxicity and limited effect (14), the combination of EGFR monoclonal antibody, such as cetuximab and panitumumab, with chemotherapy has shown efficacy in colorectal cancer treatment (15). Unfortunately, resistance to EGFR-targeted therapy has recently been observed. Many mechanisms have been proposed to explain the poor response to cetuximab, includ-

Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH 2 -terminus resides within the lumen of Golgi/ ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with γ-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.

Accumulating evidence indicates that endocytosis plays an essential role in the nuclear transport of the ErbB family members, such as epidermal growth factor receptor (EGFR) and ErbB-2. Nevertheless, how full-length receptors embedded in the endosomal membrane pass through the nuclear pore complexes and function as non-membrane-bound receptors in the nucleus remains unclear. Here we show that upon EGF treatment, the biotinylated cell surface EGFR is trafficked to the inner nuclear membrane (INM) through the nuclear pore complexes, remaining in a membrane-bound environment. We further find that importin ␤ regulates EGFR nuclear transport to the INM in addition to the nucleus/nucleoplasm. Unexpectedly, the well known endoplasmic reticulum associated translocon Sec61␤ is found to reside in the INM and associate with EGFR. Knocking down Sec61␤ expression reduces EGFR level in the nucleoplasm portion and accumulates it in the INM portion. Thus, the Sec61␤ translocon plays an unrecognized role in the release of the membrane-anchored EGFR from the lipid bilayer of the INM to the nucleus. The newly identified Sec61␤ function provides an alternative pathway for nuclear transport that can be utilized by membrane-embedded proteins such as full-length EGFR.Receptor tyrosine kinases (RTKs), 3 including insulin-like growth factor 1 receptor, c-Met, fibroblast growth factor receptor, vascular endothelial growth factor receptor, and the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus (1-6). Among these, both EGFR and ErbB-2 are suggested to be involved in transcriptional regulation, cell proliferation, DNA repair, DNA replication, and chemo and radio resistance (7-14). Nuclear EGFR is associated with poor clinical prognosis for breast cancer, ovarian cancer, and in oropharyngeal and esophageal squamous cell carcinomas (15-19). In addition, nuclear EGFRvIII, a constitutively activated EGFR variant, is also correlated with poor patient outcome in prostate cancer (20). In the canonical model of nuclear import, nuclear localization signal (NLS)-bearing molecules form a complex with importin ␣/␤ or importin ␤ alone. Importin ␤ is responsible for nuclear translocation through nuclear pore complexes (NPCs) by directly associating with the nucleoporins (21). Several studies have shown that importin ␤ and NLS are involved in the nuclear transport of many cell surface RTKs, including EGFR (14, 22-26), ErbB-2 (27, 28), and fibroblast growth factor receptor (29). In addition, nuclear transport of RTKs is mediated by the mechanisms involving endocytosis and endosomal sorting by associating with early endosomal proteins in the nucleus (24, 27). However, the exact mechanisms by which RTKs embedded in the endosomal membrane translocate into the nucleus through NPCs and exist as nonmembrane-bound receptors in the nucleus are still largely unknown.In eukaryotes, the membrane system of the endoplasmic reticulum (ER) is contiguous with the nuclear envelope (NE), a lipid bilayer that forms the bound...