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. 2013 Aug 1;104(10):1315–1322. doi: 10.1111/cas.12225

Inverse correlation between Thr‐669 and constitutive tyrosine phosphorylation in the asymmetric epidermal growth factor receptor dimer conformation

Kanae Sato 1,†, Myoung‐Sook Shin 1,†, Ayaka Sakimura 2, Yue Zhou 2, Tomohiro Tanaka 2, Miho Kawanishi 1, Yuki Kawasaki 2, Satoru Yokoyama 1, Keiichi Koizumi 3, Ikuo Saiki 1, Hiroaki Sakurai 2,✉
PMCID: PMC7656534  PMID: 23822636

Abstract

We have recently identified tumor necrosis factor (TNF)‐α‐induced phosphorylation of epidermal growth factor receptor (EGFR) at Thr‐669 and Ser‐1046/1047 via ERK and p38 pathways, respectively. In the present study, we investigated the roles of ligand‐induced phosphorylation of serine and threonine residues in EGFR‐overexpressing MDA‐MB‐468 breast cancer cells. Epidermal growth factor and heregulin, an ErbB3 ligand, induced the phosphorylation of Thr‐669 and Ser‐1046/1047. Inversely, constitutive tyrosine phosphorylation of the C‐terminal domain, including Tyr‐1068, was significantly downregulated on ligand stimulation. Inhibition of the ERK pathway by U0126 blocked ligand‐induced Thr‐669 phosphorylation as well as Tyr‐1068 dephosphorylation. Downregulation of constitutive tyrosine phosphorylation of EGFR in HEK293 cells stably expressing the wild type was abolished by substitution of Thr‐669 for Ala. In an asymmetric EGFR homodimer structure, one Thr‐669 in the receiver kinase of the dimer was involved in downregulation. Similarly, Thr‐669 in an EGFR‐ErbB3 heterodimer also participated in tyrosine dephosphorylation. These results indicate that ERK‐mediated Thr‐669 phosphorylation suppresses constitutive tryrosine phosphosphorylation in the homo‐ and heterodimer asymmetric conformations of the EGFR.

Epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase (RTK) family and plays a critical role in a wide variety of cellular functions, including proliferation, differentiation and apoptosis.1, 2, 3, 4, 5 Recently, EGFR has been the focus of molecular‐targeted therapy for cancer, because overexpression, amplification and mutations are involved in carcinogenesis and the progression of several types of cancer.5, 6, 7, 8, 9

After EGFR homodimerization with extracellular ligands such as epidermal growth factor (EGF), the intracellular tyrosine kinase domain is activated and autophosphorylates multiple tyrosine residues in the C‐terminal tail domain.1 Several adaptor proteins, including Grb2 and Shc, are recruited to the phosphorylated tyrosine residues and trigger the activation of downstream mitogen‐activated protein kinases (MAPK) and Akt signaling pathways.10, 11 Recent crystallographic analyses have demonstrated a unique dimer conformation of EGFR intracellular domains. Asymmetric dimer conformation of the two tyrosine kinase domains is essential for activation of an EGFR homodimer.12, 13 This model establishes the concept that an EGFR homodimer consists of two functionally different EGFR proteins, with one kinase domain (called the activator) in the dimer activating the other (the receiver) through an allosteric mechanism. In addition, it has been demonstrated that the juxtamembrane (JM) domain of the receiver plays a critical role in activation of the EGFR via formation of a stable asymmetric dimer by direct association with the C‐terminal lobe of the tyrosine kinase domain of the activator EGFR.14, 15, 16

Recently, we and others have reported that tumor necrosis factor (TNF)‐α induces novel post‐translational modifications of EGFR, including phosphorylation of Thr‐669 and Ser‐1046/1047, which are located in the JM and C‐terminal tail domains, respectively.17, 18, 19 In addition, Thr‐669 and Ser‐1046/1047 are phosphorylated via extracellular signal‐regulating kinase (ERK) and p38 MAPK, respectively.19 TNF‐α and other cellular stress conditions, including ultraviolet and genotoxic agents, trigger p38‐mediated serine phosphorylation and subsequent endocytosis of EGFR in a tyrosine kinase‐independent manner.17, 18, 19, 20, 21, 22 It has been recently reported that Thr‐669 phosphorylation modulates ligand‐induced receptor signaling and subsequent degradation16, 23; however, little is known about the physiological functions of the phosphorylation of EGFR at serine/threonine residues.

In the present study, we investigated the effects of serine/threonine phosphorylation on ligand‐independent constitutive tyrosine phosphorylation in an EGFR‐overexpressing human breast cancer cell line, MDA‐MB‐468. It was found that Thr‐669 phosphorylation is involved in the negative feedback regulation of constitutive tyrosine phosphorylation in the asymmetric EGFR dimer conformation.

Materials and Methods

Antibodies and reagents

Phospho‐specific antibodies against p38 (Thr‐180 and Tyr‐182), JNK (Thr‐183 and Tyr‐185), ERK (Thr‐202 and Tyr‐204), EGFR (Tyr‐845, Tyr‐974, Tyr‐992, Tyr‐1045, Tyr‐1068, Tyr‐1173, Thr‐669 and Ser‐1046/1047), MET (Tyr‐1234/1235), and ErbB3 (Tyr‐1289) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against EGFR (1005), ErbB3 (C‐17), ERK (C‐16), p38 (C‐20), JNK (FL), α‐Tubulin (B‐7) and Actin (C‐11) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human TNF‐α, EGF and heregulin (HRG) were obtained from R&D Systems (Minneapolis, MN, USA). HGF was obtained from PeproTech (Rocky Hill, NJ, USA). SB203580, U0126 and PD153035 were from Merck Biosciences (Darmstadt, Germany). Gefitinib was purchased from Cayman Chemical (Ann Arbor, MI, USA). All chemical inhibitors were dissolved in dimethyl sulfoxide and the final concentration of dimethyl sulfoxide was <0.1%. TPA was obtained from WAKO Pure Chemical Industries (Osaka, Japan).

Cell culture

MDA‐MB‐468 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. HEK293 cells were maintained in DMEM (high glucose) supplemented with 10% fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin. PC‐9 cells were maintained in RPMI1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were incubated at 37°C in 5% CO2.

Transfection and establishment of stable cell lines

An expression plasmid for human EGFR was obtained as described previously.19 Human ErbB3 cDNA was amplified from HeLa cells by RT‐PCR and inserted into pcDNA3.1 vector. Deletion and amino acid substitution mutants were generated by a QuikChange site‐directed mutagenesis kit (Agilent, La Jolla, CA, USA) or PrimeSTAR HS Polymerase (Takara‐Bio, Shiga, Japan). The HEK293 cells were transfected using a Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Stable HEK293 cell lines were selected using G418 at 1 mg/mL and the cells were maintained in medium containing G418.

Immunoblotting

Whole cell lysates, prepared as described previously,24 were resolved using SDS‐PAGE and transferred to an Immobilon‐P nylon membrane (Millipore, Billerica, MA, USA). The membrane was treated with BlockAce (Dainippon Sumitomo Pharmaceutical, Osaka, Japan) and probed with primary antibodies, as described in the section of Materials and Methods. Antibodies were detected using horseradish peroxidase‐conjugated anti‐rabbit, anti‐mouse and anti‐goat IgG (Dako, Glostrup, Denmark) and visualized with the ECL system (GE Healthcare Bio‐Science, Piscataway, NJ, USA). Some antibody reactions were carried out in Can Get Signal solution (TOYOBO, Tokyo, Japan).

Flow cytometry

MDA‐MB‐468 cells were harvested in PBS, fixed with 2% paraformaldehyde for 20 min at room temperature, resuspended in 100 μL of FACS buffer (PBS containing 0.5% BSA) containing 1 μg of anti‐EGFR monoclonal antibody (clone LA1; Upstate Biotechnology, Waltham, MA, USA) and incubated on ice. After being washed with FACS buffer, cells were incubated with FITC‐conjugated anti‐mouse IgG antibody (Dako) on ice and analyzed with the FACSCalibur system (BD Biosciences, Bedford, MA, USA).

Results

Phosphorylation of Thr‐669 and Ser‐1046/1047 of EGFR in MDA‐MB‐468 cells

In the present study, we focused on the roles of ligand‐induced phosphorylation of Thr‐669 and Ser‐1046/1047 in an EGFR‐overexpressing human breast cancer cell line, MDA‐MB‐468. We first confirmed whether TNF‐α induces phosphorylation of Thr‐669 and Ser‐1046/1047 in this cell line. Both Thr‐669 and Ser‐1046/1047 were rapidly phosphorylated at 10–20 min and then dephosphorylated at 60 min (Fig. 1a). In addition, TNF‐α‐induced phosphorylation of Thr‐669 and Ser‐1046/1047 was selectively inhibited using a MEK inhibitor (U0126) and p38 inhibitor (SB203580), respectively (Fig. 1b). These phosphorylation patterns were quite similar to our previous observations in HeLa cells.19

Figure 1.

Figure 1

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Regulation of serine and threonine phosphorylation of epidermal growth factor receptor (EGFR). (a) MDA‐MB‐468 cells were treated with 20 ng/mL tumor necrosis factor (TNF)‐α or 10 ng/mL EGF for the indicated periods. Whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Ser‐1046/1047 and Tyr‐1068), EGFR, phospho‐mitogen‐activated protein kinase (MAPK), MAPK and Actin antibodies. (b,c) Cells were pretreated with gefitinib (GE; 10 μM), PD153035 (PD; 1 μM), SB203580 (SB; 10 μM) and U0126 (U; 5 μM) for 30 min, and then stimulated with TNF‐α (b), and EGF or heregulin (HRG) (c) for another 10 min. Whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Ser‐1046/1047 and Tyr‐1068), EGFR, phospho‐ErbB3, ErbB3 and Actin antibodies.

We then examined the effects of ErbB ligands, including EGF and HRG, on the phosphorylation of Thr‐669 and Ser‐1046/1047. Similar to TNF‐α stimulation, EGF also induced phosphorylation of the serine/threonine residues; however, the Ser‐1046/1047 phosphorylation level was weaker than that induced by TNF‐α (Fig. 1a,c). Heregulin also induced Thr‐669 and Ser‐1046/1047 phosphorylation of EGFR as well as tyrosine phosphorylation of its receptor ErbB3 (Fig. 1c). Ligand‐induced phosphorylation of the threonine and serine residues was completely inhibited by EGFR tyrosine kinase inhibitors (TKI), gefitinib and PD150305. Heregulin‐induced phosphorylation of ErbB3 was also abrogated by these EGFR‐TKI, indicating that HRG signaling was triggered by an EGFR‐ErbB3 heterodimer. Similar to TNF‐α signaling, ligand‐induced phosphorylation of Thr‐669 and Ser‐1046/1047 was selectively inhibited by U0126 and SB203580, respectively, indicating that EGFR‐mediated activation of ERK and p38 pathways controls Thr‐669 and Ser‐1046/1047, respectively, in feedback mechanisms via MAPK (Fig. 1c).

Ligand‐induced downregulation of tyrosine phosphorylation of EGFR

In the absence of exogenous ligands, multiple tyrosine residues in the C‐tail autophosphorylation domain of EGFR, including Tyr‐1068, were strongly and constitutively phosphorylated in MDA‐MB‐468 cells (Fig. 1a). The EGFR‐TKI were able to suppress tyrosine phosphorylation (Fig. 1c). In contrast to the increased phosphorylation of Thr‐669 and Ser‐1046/1047, it is interesting that Tyr‐1068 phosphorylation was significantly downregulated in cells stimulated with EGF and HRG (Fig. 1a,c). Similarly, constitutive tyrosine phosphorylation was slightly decreased at 20 min post‐TNF‐α stimulation (Fig. 1a).

Next we investigated the molecular mechanisms for the rapid dephosphorylation of constitutively phosphorylated tyrosine residues. First, phosphorylation levels at several tyrosine residues, including Tyr‐845, Tyr‐974, Tyr‐1045, Tyr‐1068 and Tyr‐1173, were determined at multiple time points within 10 min. Constitutive phosphorylation of all tyrosine residues gradually decreased in response to EGF stimulation in 3–10 min, whereas phosphorylation of Thr‐669 and Ser‐1046/47 was inversely increased (Fig. 2a). Heregulin rapidly induced Tyr‐1289 phosphorylation of ErbB3 within 1 min, which also led to the downregulation of tyrosine phosphorylation of EGFR as well as increases in the phosphorylation of Thr‐669 and Ser‐1046/1047 (Fig. 2b). In addition, Figure 2(c) clearly demonstrated an inverse correlation between tyrosine and serine/threonine phosphorylation. In the late phase of stimulation after 60 min, decreasing phosphorylation of Thr‐669 and Ser‐1046/1047 closely correlated with the restoration of tyrosine phosphorylation (Fig. 2c). Similar correlation was detected in HRG stimulation (Fig. 2c). In addition, the time‐course of MAPK activation was closely correlated with the inverse phosphorylation of tyrosine and serine/threonine residues, suggesting the involvement of feedback mechanisms from MAPK (Fig. 2c). Although EGF did not increase tyrosine autophosphorylation of EGFR (Fig. 2a,c), EGFR was slightly internalized in EGF‐treated cells, indicating activation of EGFR (Fig. 2d).

Figure 2.

Figure 2

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Inverse phosphorylation at serine/threonine and tyrosine residues. (a–c) MDA‐MB‐468 cells were treated with EGF or heregulin (HRG) for the indicated periods. Whole cell lysates were immunoblotted with phospho‐epidermal growth factor receptor (EGFR) (Thr‐669, Ser‐1046/1047, Tyr‐845, Tyr‐974, Tyr‐1045, Tyr‐992, Tyr‐1068 and Tyr‐1173), EGFR, phospho‐ErbB3, ErbB3, phospho‐mitogen‐activated protein kinase (MAPK) and Actin antibodies. (d) Cells were treated with EGF for 10 min and then cell surface EGFR expression was determined using a flow cytometer.

Negative feedback control of constitutive tyrosine phosphorylation of EGFR via the ERK pathway

We then focused on the ERK‐Thr‐669 pathway. To investigate the involvement of Thr‐669 phosphorylation, we examined the effect of U0126, a MEK inhibitor, on constitutive tyrosine phosphorylation of EGFR. Stimulation of MDA‐MB‐468 cells with EGF significantly induced the phosphorylation of Thr‐669 via phosphorylation of ERK 5–10 min after ligand stimulation, in which constitutive tyrosine phosphorylation of EGFR at multiple tyrosine residues was clearly downregulated (Fig. 3a). Pretreatment with U0126 blocked ERK activation and subsequent phosphorylation of Thr‐669 (Fig. 3a). It is interesting that U0126 treatment caused complete blockade of the downregulation of constitutive tyrosine phosphorylation (Fig. 3a). TPA, an activator of protein kinase C, is well known as a strong ERK activator, consequently, we examined the effect of TPA on constitutive EGFR phosphorylation. TPA strongly induced the phosphorylation of Thr‐669 and complete dephosphorylation of Tyr‐1068 (Fig. 3b). In addition, all TPA‐induced responses on EGFR were abrogated by U0126 (Fig. 3b). Similar results were also obtained in human lung adenocarcinoma PC‐9 cells harboring an active mutation in the EGFR tyrosine kinase domain. Pretreatment of PC‐9 cells with gefitinib inhibited constitutive phosphorylation of EGFR (both Thr‐669 and Tyr‐1068) and ERK, indicating that tyrosine kinase activity evoked Tyr‐1068 autophosphorylation and ERK‐mediated Thr‐669 phosphorylation. Even in constitutive Thr‐669 phosphorylation, TPA induced additional phosphorylation of Thr‐669, which was parallel to the decrease in Tyr‐1068 phosphorylation (Fig. 3c). The HGF‐induced activation of the MET receptor also induced Thr‐669 phosphorylation, which correlated with the downregulation of constitutive Tyr‐1068 phosphorylation of EGFR (Fig. 3d), indicating that inverse correlation between phosphorylation of Thr‐669 and Tyr‐1068 was also observed on EGFR with active mutations in lung cancer cells. Altogether, these findings demonstrate that the ERK pathway controls the negative feedback regulation of constitutive tyrosine phosphorylation of EGFR.

Figure 3.

Figure 3

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ERK‐mediated feedback control of constitutive tyrosine phosphorylation of epidermal growth factor receptor (EGFR). (a) MDA‐MB‐468 cells were pretreated with U0126 (5 μM) or vehicle (DMSO) for 30 min and then stimulated with EGF or heregulin (HRG) for another 10 min. (b) Cells were stimulated with TPA (100 ng/mL) for 10 min in the absence of presence of U0126. (c,d) PC‐9 cells were treated with gefitinib (1 μM) for 30 min and then stimulated with TPA for 10 min (c) or HGF (d) for 15–120 min. Whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Ser‐1046/1047, Tyr‐845, Tyr‐974, Tyr‐1045, Tyr‐1068 and Tyr‐1173), EGFR, phospho‐ERK, phosphor‐MET, Tubulin and Actin antibodies.

Thr‐669 phosphorylation is involved in negative feedback regulation

Experiments using U0126 suggested that Thr‐669 is involved in the negative feedback regulation of EGFR. To clarify the role of Thr‐669 phosphorylation, we established HEK293 cells stably expressing wild‐type and mutated EGFR. Overexpression of wild‐type EGFR resulted in constitutive Tyr‐1068 phosphorylation without exogenous ligand stimulation (Fig. 4a). Similar to the results in MDA‐MB‐468 cells (Fig. 2a), stimulation with EGF strongly induced phosphorylation of Thr‐669 in 3–10 min, which was parallel to the decrease in Tyr‐1068 phosphorylation (Fig. 4a). In contrast, phosphorylation of Ser‐1046/1047 was not significantly induced by EGF, which was correlated with marginal activation of p38. To elucidate the roles of serine and threonine, alanine substitution mutants were generated. Ser‐1046/1047 to Ala substitution (SS/AA) did not affect the negative feedback regulation of Tyr‐1068 phosphorylation as well as Thr‐669 phosphorylation, indicating that phosphorylation of Ser‐1046/1047 is dispensable for the negative regulation of tyrosine phosphorylation (Fig. 4a). This result correlated with the observation that p38 inhibitor did not affect the negative feedback inhibition of Tyr‐1068 autophosphorylation (Fig. 1c). We next examined the effect of amino acid substitution of Thr‐669 with Ala (T669A). In HEK293 cells stably expressing EGFR‐T669A, loss of Thr‐669 phosphorylation led to slight enhancement of Tyr‐1068 phosphorylation even in the absence of EGF (Fig. 4b). In addition, EGF stimulation did not cause significant downregulation of Tyr‐1068 phosphorylation of EGFR‐T669A, although ERK was strongly activated. TPA also induced Thr‐669 phosphorylation, which resulted in strong downregulation of C‐terminal tyrosine phosphorylation (Fig. 4c). These results clearly demonstrate that phosphorylation of Thr‐669 directly triggered tyrosine dephosphorylation in the feedback mechanism.

Figure 4.

Figure 4

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Negative feedback regulation of epidermal growth factor receptor (EGFR) via Thr‐669 phosphorylation. (a) HEK293 cells stably transfected with wild‐type or S1046/1047A mutant (SS/AA) were stimulated with EGF for the indicated periods. (b,c) HEK293 cells stably transfected with wild‐type or T669A mutant were stimulated with EGF (b) or TPA (c) for 10 min. Whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Ser‐1046/1047, Tyr‐845, Tyr‐974, Tyr‐1045, Tyr‐1068 and Tyr‐1173), EGFR, phospho‐ERK, phospho‐p38 and Actin antibodies.

Roles of Thr‐669 in the asymmetric EGFR homodimer

In the asymmetric homodimer model, the activator triggers tyrosine kinase activity of the counterpart receiver kinase.12 It has been reported that the JM region of the receiver, but not that of the activator, plays an essential role in EGFR activation.15, 16 As described in last section, we found that phosphorylation of Thr‐669 in the JM segment caused negative feedback regulation; therefore, we then tried to determine whether one or both Thr‐669 residues in the asymmetric dimer are necessary for the downregulation of constitutive tyrosine phosphorylation. To address this issue, we prepared expression vectors for two substitution mutants, Ile‐682‐Gln (I682Q; Act‐M) and Val‐924‐Arg (V924R; Rec‐M), which function as only the activator or receiver, respectively.15 As shown in Figure 5(a), although wild‐type EGFR induced its autophosphorylation in a transient overexpression experiment, Act‐M or Rec‐M alone showed no tyrosine kinase activity. In contrast, co‐expression of Act‐M and Rec‐M together reconstituted the constitutive tyrosine kinase activity of EGFR (Fig. 5a), indicating that the activator and receiver are functionally distinguishable by using these mutants. To investigate the role of Thr‐669 in the asymmetric dimer conformation, T669A mutants of Act‐M and Rec‐M were constructed. Although Act‐M‐T669A did not affect, but rather slightly decreased EGFR activity, it is interesting that Rec‐M‐T669A in combination with Act‐M or Act‐M‐T669A caused significant increases in constitutive tyrosine autophosphorylation (Fig. 5b). In contrast, Thr‐669 phosphorylation was decreased, but remained even with the T669A substitution of one EGFR, while it completely disappeared with substitution of both Thr‐669 residues (Fig. 5b). These results suggest the functional importance of the receiver's Thr‐669 in the negative feedback mechanism, although Thr‐669 residues in both kinases were equally phosphorylated.

Figure 5.

Figure 5

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Role of Thr‐669 in the asymmetric epidermal growth factor receptor (EGFR) homodimer. HEK293 cells were transiently transfected with (a) wild‐type or I682Q (Act‐M) and V924R (Rec‐M) mutants, (b) Act‐M and Rec‐M with or without additional T669A (TA) point mutation, (c) KK721/782AA (KK/AA) and Act‐M or Rec‐M with or without TA mutation, (d) ErbB3 and Act‐M or Rec‐M with or without TA mutation. At 24 h post‐transfection, whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Tyr‐974, Tyr‐1045, Tyr‐1068 and Tyr‐1173), EGFR and ErbB3 antibodies.

It has been demonstrated that kinase activity is not required for the activator function of the EGFR dimer.12 We then confirmed the role of receiver Thr‐669 in the feedback mechanism using kinase‐dead EGFR‐KK/AA (Fig. 5c) or ErbB3 (Fig. 5d) as an activator. EGFR‐KK/AA was able to activate Rec‐M, but not Act‐M, confirming that the KK/AA mutant functioned only as the activator (Fig. 5c). It is interesting that EGFR‐KK/AA‐induced phosphorylation of Tyr‐1068 was also enhanced in co‐expression with Rec‐M‐T669A (Fig. 5c). Moreover, ErbB3, which functions only as an activator in EGFR‐ErbB3 heterodimer, tyrosine phosphorylation of EGFR Rec‐M was also enhanced with T669A substitution (Fig. 5d). These results strongly support our understanding that phosphorylation of Thr‐669 in the receiver kinase negatively regulates tyrosine phosphorylation of EGFR.

Last, we investigated the role of stimulus‐induced phosphorylation of the receiver Thr‐699 in the feedback control. HEK293 cells transfected with Act‐M and Rec‐M were stimulated with TPA for 10 min and then analyzed for tyrosine autophosphorylation. As observed in MDA‐MB‐468 cells, TPA strongly induced ERK‐mediated Thr‐669 phosphorylation, while phosphorylation of Tyr‐1068 and Tyr‐1173 was significantly reduced (Fig. 6). In contrast, tyrosine phosphorylation was largely maintained by mutation of Thr‐669 in Rec‐M, but not in Act‐M, indicating loss of feedback regulation by the mutation in only the receiver kinase (Fig. 6). Collectively, these results indicate that although both Thr‐669 residues in the activator and receiver are phosphorylated, the receiver's Thr‐669 participates in the negative feedback regulation of EGFR tyrosine kinase activity in the asymmetric EGFR dimer (Fig. 7).

Figure 6.

Figure 6

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Role of Thr‐669 in the asymmetric epidermal growth factor receptor (EGFR) homodimer. HEK293 cells were transiently transfected with I682Q (Act‐M) and V924R (Rec‐M) with or without TA mutation. At 24 h post‐transfection, cells were stimulated with TPA for 10 min. Whole cell lysates were immunoblotted with phospho‐EGFR (Thr‐669, Tyr‐1068 and Tyr‐1173), EGFR, phospho‐ERK and Actin antibodies.

Figure 7.

Figure 7

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Schematic diagram of Thr‐669‐mediated inhibition of the asymmetric epidermal growth factor receptor (EGFR) dimer. Formation of an asymmetric dimer structure by docking the N‐lobe of the receiver to the C‐lobe of the activator induces EGFR autophosphorylation of tyrosine residues in the C‐terminal tail. ERK‐mediated Thr‐669 phosphorylation, especially in the receiver, induces conformational change to disrupt the active dimer structure, which leads to a decrease in C‐terminal autophosphorylation.

Discussion

There is accumulating evidence of ligand‐induced activation of EGFR via its intracellular tyrosine kinase domain. Recent crystallographic analyses made great strides toward structural insights into the activation mechanisms of the EGFR homodimer. New post‐translational modifications have been recently identified that include phosphorylation at serine and threonine residues; therefore, to fully understand the physiological roles of EGFR, it is essential to explore the functional interactions between tyrosine phosphorylation and serine/threonine phosphorylation, taking into account the asymmetric structure.

In the present study, we used MDA‐MB‐468 cells, a triple‐negative (ErbB2, estrogen receptor and progesterone receptor) breast cancer cell line as an EGFR overexpression model.25 Constitutive phosphorylation of EGFR maintained basal MAPK activity in this cell line; however, treatment with EGF strongly activated downstream MAPK within 1 min (data not shown). Nevertheless, the phosphorylation levels of tyrosine residues in western blot analysis were not significantly increased before MAPK activation, although a small part of EGFR was internalized. These results suggest that constitutively phosphorylated EGFR is involved in basal MAPK activity, but not ligand‐induced MAPK activation. In addition, it is possible that ligand binding to EGFR triggers MAPK activation through its conformational changes of intracellular domains without affecting tyrosine phosphorylation. Conversely, the tyrosine phosphorylation level is not always a marker of the activation status of EGFR. These results suggest that it is necessary to functionally distinguish these different conformations of EGFR in cancer cells overexpressing the receptor.

The main purpose of the present study was to understand the role of serine/threonine phosphorylation in the maintenance of tyrosine phosphorylation of EGFR. We first confirmed our previous findings in HeLa cells.18, 19 TNF‐α induced phosphorylation of Thr‐669 and Ser‐1046/1047 of EGFR through MAPK in a tyrosine kinase‐independent manner (Fig. 1a,b), indicating that the serine/threonine residues are general targets of MAPK signaling pathways. We also detected phosphorylation of Thr‐669 and Ser‐1046/1047 on EGF and HRG stimulation, where Thr‐669 plays a role in the negative feedback control of constitutive tyrosine phosphorylation (Fig. 2). These results raise the possibility that the basal tyrosine phosphorylation level is controlled by ERK activity in EGFR‐overexpressing cancer cells. In contrast, we could not detect any tyrosine and threonine phosphorylation in non‐transfected HEK293 cells, which express a physiological level of EGFR (data not shown); therefore, further study in normal epithelial and cancer cells expressing different levels of EGFR protein are needed to understand additional physiological roles of the negative feedback regulation of EGFR.

Li et al. previously reported that ERK‐mediated Thr‐669 phosphorylation is involved in the stabilization of EGFR at 1–4 h after ligand stimulation in CHO‐GHR and human sarcoma C14 (a derivative of HT1080) cells.23 In contrast, we could not detect the significant effect of T669A substitution on ligand‐induced degradation of EGFR in HEK293 cells (data not shown). In addition, as shown in Figure 2(c), EGF stimulation for 2 h did not induce EGFR degradation in MDA‐MB‐468 cells, which is supported by the result in Figure 2(d) that only a small part of EGFR was internalized. However, it is important to clarify the role of Thr‐669 phosphorylation in the modulation of EGFR protein stability in other human carcinoma cells, including non‐small‐cell lung cancer cells with activating EGFR mutation.

From a clinical point of view, EGFR‐overexpressing colorectal tumors without a KRAS mutation are treated with anti‐EGFR neutralizing antibodies, including cetuximab and panitumumab.26, 27, 28 In contrast, these antibodies have no therapeutic potential for colorectal cancers harboring KRAS mutation, indicating EGFR‐independent survival and proliferation.29, 30 It is well known that mutated KRAS activates the ERK pathway;31, 32 therefore, it is possible that Thr‐669 phosphorylation suppresses constitutive activation of EGFR in KRAS mutant cells. This hypothesis can support mechanisms for EGFR‐independent survival and cetuximab resistance of colorectal cancers harboring the KRAS mutation. It has been demonstrated recently that feedback activation of EGFR is the main cause in unresponsiveness of colon cancer cells harboring BRAF‐V600E active mutation to vemurafenib, a BRAF kinase inhibitor.33 In untreated BRAF active cells, the MEK‐ERK pathway contributes to the downregulation of EGFR through inactivation of the CDC25 family of protein tyrosine phosphatases. In the present study, we demonstrated that ERK‐mediated Thr‐669 phosphorylation of EGFR is another mechanism for the negative feedback inhibition. These observations suggest a functional correlation between CDC25 and Thr‐669 phosphorylation; therefore, future characterization using colon cancer cells harboring BRAF mutation will provide more information for the inverse correlation between Thr‐669 and tyrosine phosphorylation of EGFR in clinical settings of natural resistance to vemurafenib.

The JM region of the receiver kinase functions as an activation domain by docking on the C‐lobe of the activator kinase, in which several hydrogen bonds and hydrophobic interactions are involved.15 In particular, a small segment from Leu‐664 to Ser‐671 of the receiver kinase (called a JM latch) is important for interaction with the activator kinase.15, 16 The hydroxyl group of Thr‐669 is not directly involved in this interaction; rather, it is located on the surface of the dimer structure to be easily phosphorylated. In the present study, we confirmed the previous observation that phosphorylation of Thr‐669 is involved in a negative regulatory role in EGFR activation. In addition, using activator and receiver mutants, we identified that only Thr‐669 in the receiver EGFR contributed to the feedback control, although Thr‐669 in the activator kinase was also phosphorylated. These findings strongly raise the possibility that the addition of an acidic charge to the JM segment induces its conformational change and subsequent disruption of the docking on the C‐lobe of the activator kinase. This idea is supported by the fact that Thr‐669 is conserved in both ErbB2 and ErbB4. In contrast, the corresponding threonine residue in kinase dead ErbB3 is substituted by acidic amino acid (aspartic acid), possibly preventing the formation of ErbB dimers consisting of ErbB3 as the receiver kinase.

In summary, we found that Thr‐669 in the JM region, especially on the receiver side, contributes to negative feedback inhibition of the constitutive tyrosine phosphorylation of the receptor. In the databases of phosphorylation sites, multiple uncharacterized serine and threonine residues are deposited as possible phosphorylation sites; therefore, it is essential to investigate further the functional interactions between tyrosine phosphorylation and serine/threonine phosphorylation of the EGFR and other ErbB receptors. These characteristics, especially in cancer cells with overexpression or activating mutation, will provide new information for establishing new anti‐EGFR therapeutics, including overcoming acquired drug resistance.

Disclosure Statement

The authors have no conflict of interest.

Acknowledgments

This work was supported in part by Grants‐in‐Aid for Scientific Research on Innovative Areas and the Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and grants from Naito Foundation and the Japan Foundation for Applied Enzymology.

(Cancer Sci 2013; 104: 1315–1322

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