Ligand binding to the extracellular domain of EGFR triggers the autophosphorylation of multiple intracellular tyrosine residues by the formation of an asymmetric dimer, with one kinase domain in the EGF-mediated dimer activating the other through an allosteric mechanism (52
). Several adaptor proteins, including Grb2 and Cbl, bind to phosphorylated tyrosines and evoke intracellular signals, including MAPK activation. EGFR has recently been shown to be phosphorylated under cellular stress conditions, including TNF-α, high levels of osmotic stress, anisomycin, UV, and cisplatin through p38. In the present study, we identified two independent signaling pathways from the TNF-α receptor to EGFR through ERK and p38 MAPK pathways, which caused the phosphorylation of EGFR at Thr-669 and Ser-1046/1047, respectively (Fig. ). Adachi et al. recently reported that anisomycin induces Ser-1046/1047 in colon cancer cells (1
). These findings emphasize the functional importance of these residues; however, an effort to search for other unidentified residues is necessary to completely elucidate the role of TNF-α-induced modification of EGFR. Upon EGF stimulation, the serine and threonine were also phosphorylated through the same MAPKs; however, duration and frequency were considerably different. Ligand-induced phosphorylation of Thr-669 was sustained until 60 min, while it had largely disappeared at 60 min in TNF-α-treated cells, although comparable phosphorylation was induced by these treatments at 30 min. It is interesting that TNF-α, but not EGF, induced strong Ser-1046/1047 phosphorylation, although p38 was comparably activated by these stimuli. The marked structural difference of EGFR in cells treated with TNF-α or EGF is whether it is naked (monomer) or phosphorylated (dimer occupied with ligands). Multiple tyrosine residues, including Tyr-1045, the next residue of Ser-1046/1047, are primarily phosphorylated in the process of ligand-induced EGFR activation. Subsequently, MAPKs are activated through Grb2 binding to phosphorylated tyrosines, such as Tyr-1068 and Tyr-1086, and then activated EGFR are targeted by MAPKs for Ser/Thr phosphorylation as a feedback control. This supports the hypothesis that earlier Tyr-1045 phosphorylation causes a conformational change around Ser-1046/1047 that interferes with access of the kinase. In accordance with this hypothesis, it is possible that EGF-induced Ser-1046/1047 phosphorylation occurred only on inactive and monomeric EGFR remaining on the plasma membrane without Tyr-1045 phosphorylation. In contrast, no tyrosine phosphorylation site has been reported around Thr-669, which may allow later phosphorylation by ERK at this site even in activated EGFR. It has been reported that MEK and ERK are recruited to endosomes to evoke sustained signals from intracellular compartments, suggesting that ligand-induced Thr-669 phosphorylation occurred after internalization. Similarly, TNF-α-induced Thr-669 phosphorylation was delayed by Ser-1046/1047 phosphorylation and inhibited by disruption of clathrin-coated pits. These findings raise the possibility that the different mechanisms of TNF-α- and ligand-induced Ser/Thr phosphorylation of EGFR are reflected by conformational changes, subcellular localization, and signaling complex formation, which influence the access of the corresponding kinases.
FIG. 8. Schematic diagram of TNF-α-induced antiapoptotic signals. TNF-α binding to TNF-R1 rapidly induces the activation of TAK1. TAK1 induces two independent signaling pathways to EGFR through ERK and p38. TAK1 regulates two independent antiapoptotic (more ...)
Another feature of TNF-α-induced Ser/Thr phosphorylation is that it is rapidly dephosphorylated within 60 min due to the rapid turnover of ERK and p38 activation. In contrast, osmotic stress caused sustained MAPK activation, leading to continuous EGFR phosphorylation. We have previously demonstrated that the rapid turnover of Ser/Thr phosphorylation is associated with EGFR recycling to the cell surface, as discussed below; therefore, identification of Ser/Thr phosphatases is essential to understand the function and postendocytic trafficking of the EGFR.
The molecular mechanisms underlying ligand-mediated intracellular trafficking of EGFR have been extensively studied. Ligand binding induces multiple autophosphorylation and ubiquitination of EGFR. The modified EGFR dimer is rapidly internalized with ligands via clathrin-coated pits and subsequently sorted in early endosomes. In the present study, we found that TNF-α induces EGFR endocytosis through the TAK1-p38 pathway in a Ser-1046/1047 phosphorylation-dependent manner, in which ERK-mediated Thr-669 phosphorylation and TK activity of EGFR were dispensable. In contrast, EGF slightly induced p38-mediated phosphorylation of Ser-1046/1047, while it was also involved in the endocytosis of EGFR. Since ligand-induced p38 activation is dependent on EGFR TK activation, p38-mediated endocytosis is also dependent on its TK activity. Based on these observations, we proposed the following two different mechanisms of the ligand-mediated endocytosis: the major route in a primary TK-dependent fashion and a secondary p38-mediated feedback mechanism on inactive EGFR proteins which remains on the cell surface as a monomer. The secondary mechanism is similar to the stress-induced phosphorylation and endocytosis of EGFR, raising the possibility that TNF-α amplifies a ligand-induced minor reaction to overcome cellular stress conditions.
In ligand-induced internalization, a large part of EGFR is transported into multivesicular bodies and late endosomes and finally degraded in lysosomes; however, a small number of receptors are incorporated into recycling endosomes. We have shown that internalized EGFR mediated by TNF-α-induced p38 activation is efficiently recycled to the plasma membrane after dephosphorylation, which is similar to the proposed secondary mechanism of ligand-mediated endocytosis; therefore, it is possible that internalized EGFR with no phosphorylated tyrosine via the secondary Ser-1046/1047-dependent mechanism escapes from degradation. This possibility is supported by the finding that phosphorylated Tyr-1045 is a Cbl-binding site for ubiquitination that regulates subsequent degradation in lysosomes.
It should be emphasized that preservation of EGF-receptor association in endosomes is essential for sorting EGFR to the degradation pathway. Some ligands of EGFR other than EGF, including TGF-α, have weaker affinities to the receptor and dissociate from the receptor in the acidic environment of endosomes. For instance, TGF-α is released from the receptor in early endosomes, leading to receptor dephosphorylation and recycling back to the plasma membrane (8
); therefore, TGF-α does not cause significant degradation of EGFR. Similarly, it has been shown that EGFR is colocalized with Rab5 and EEA1, early endosomal markers, and internalized EGFR is not occupied by the ligand under stress. These observations point out the significance of comparative analyses of Ser/Thr/Tyr phosphorylation, endocytosis, dephosphorylation, and postendocytic trafficking by some ligands and TNF-α to understand a variety of physiological functions of the EGFR.
Thr-669 is a unique site commonly and efficiently phosphorylated by both ligand and TNF-α stimulation; however, rapid turnover of TNF-α-induced phosphorylation was well contrasted by the sustained phosphorylation by EGF. This shows a good correlation with the duration of ERK activation (Fig. ). Figure shows that phosphorylation of this residue was dispensable for ligand- and TNF-α-induced endocytosis. In addition, the phosphorylation status at Thr-669 did not affect the dephosphorylation of Ser-1046/1047 and recycling to the cell surface upon stimulation of TNF-α. Li et al. recently demonstrated that Thr-669 negatively regulates EGF-induced EGFR kinase activity by promoting EGFR degradation in overexpression of the mutated EGFR (17
); however, TNF-α-induced Ser/Thr phosphorylation does not direct EGFR toward degradation; rather, these sites are rapidly dephosphorylated during recycling. Moreover, Thr-669 phosphorylation is not required for TNF-α-induced antiapoptotic cellular responses. The role of stress-induced Thr-669 phosphorylation is still unknown; therefore, study of this site will shed light on the common physiological functions of the ERK pathway in EGFR regulation.
TNF receptor 1 (TNF-R1) is a death receptor that transduces both death and survival signals, but the molecular mechanisms via which TNF-R1 mediates these signals are not yet fully understood. NF-κB is a ubiquitously expressed transcription factor that plays a pivotal role in antiapoptotic functions in TNF-R1 signaling pathways. TAK1 is well known as an NF-κB-regulating kinase, and TAK1-deficient mouse embryonic fibroblasts, keratinocytes, and intestinal epithelial cells are sensitive to TNF-α-induced apoptosis (12
). In addition, it has been reported that RNAi-mediated knockdown of TAK1 in cancer cells, including HeLa and A549 cells, results in enhanced apoptotic cell death upon stimulation with TNF-α or TRAIL (6
). In the present study, we identified the EGFR pathway via TAK1 as a novel survival signal from TNF-R1 in a ligand- and TK-independent manner. This signal is independent of the TAK1-NF-κB pathway. These findings suggest that TAK1 controls two independent survival pathways to protect apoptosis in a stress condition, especially in EGFR-overexpressing epithelial cancer cells. It has recently been demonstrated that the expression of EGFR, a receptor TK associated with cell proliferation and survival, is overactive in many tumors of epithelial origin, and anti-EGFR agents, including neutralizing antibodies and TK inhibitors, have been used for cancer therapy. Investigations into functional interactions between TNF-R1 and the EGFR signaling pathway have recently been extensively studied. These experiments showed that cancer cell resistance to the cytotoxic effects of TNF-α could be induced by EGFR ligands EGF and TGF-α in a TK-dependent manner (50
). On the other hand, TK activity was not necessary for the phosphorylation of EGFR at Ser-1046/1047 to prevent TNF-α-induced proapoptotic signals. A recent report by Weihua et al. adds a new wrinkle to the role of EGFR in cancer: it demonstrated that kinase-inactive EGFR facilitates glucose transport into cells by associating with and stabilizing a sodium/glucose cotransporter (SGLT1) (48
). We observed that the sensitivity of EGFR-knocked down cells to TNF-α-induced cell death was significantly enhanced under low-glucose culture conditions (data not shown), raising the possibility that the TAK1-EGFR pathway regulated the nutritional environment. Furthermore, we are also interested in another unknown function of the Ser/Thr phosphorylation of EGFR in the IL-1 signaling pathway, since IL-1 does not control apoptosis as TNF-α. Identification of EGFR-targeted genes in TNF-α and IL-1 signaling pathways will help our understanding of the TK-independent function of the EGFR.
It has been well known that TNF-α and EGF colocalize in tumor microenvironments and inflamed tissues. We have reported that TNF-α suppresses extracellular EGF responses through internalization of the EGFR (40
). Similarly, Fig. demonstrated that overexpression of TAK1 inhibited phosphorylation of multiple tyrosine residues on EGFR. We recently reported the pathway of intracellular signaling in the opposite direction. EGFR activation interferes with TNF-α-induced TAK1 activation via p38-mediated phosphorylation of TAB1 (37
). Collectively, these results indicate that the TNF-α and EGFR signaling pathways interfere with each other. The cross-interference suggests that a balance between TNF-α and EGF stimuli determines which signal is dominant in cells that face both EGFR and TNFR activation at the same time.
In summary, we identified novel signaling pathways to EGFR via TAK1 and MAPKs. Study of the molecular mechanisms of the novel survival function of EGFR will be an attractive subject to understand functional interactions between cellular stress factors and growth/survival factors in cancer cells. It is also interesting whether cytokine-induced phosphorylation of EGFR occurs in lung adenocarcinoma cells with activating mutations in the TK domain. These analyses will contribute to the establishment of a more effective therapeutic strategy with anti-EGFR agents.