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The epidermal growth factor receptor (EGFR) is a single-pass transmembrane protein with an extracellular ligand-binding region and a cytoplasmic tyrosine kinase. Ligand binding activates the tyrosine kinase, which in turn initiates signaling cascades that influence cell proliferation and differentiation. EGFR activity is essential for normal development of many multicellular organisms, and inappropriate activation of EGFR is associated with multiple human cancers. Several drugs targeting EGFR activity are approved cancer therapies, and new EGFR-targeted therapies are being actively pursued. Much of what is known about EGFR structure and function is derived from studies of soluble receptor fragments. We report here development of an approach to producing an active, membrane-spanning form of EGFR of suitable purity, homogeneity, and quantity for structural and functional studies. We show that EGFR is capable of direct autophosphorylation of tyrosine 845, which is located on its kinase activation loop, and that the kinase activity of EGFR is ~500-fold higher in the presence of EGF vs. the inhibitory anti-EGFR antibody Cetuximab. The potencies of the small molecule EGFR kinase inhibitors erlotinib and lapatinib for various forms of EGFR were measured, and the therapeutic and mechanistic implications of these results considered.
The epidermal growth factor receptor (EGFR) was the first cell-surface receptor shown to have intrinsic tyrosine kinase activity and is thus the archetype of a class of receptors, now numbering over 50 in humans, that includes receptors for insulin, VEGF, NGF, ephrins and FGF (1, 2). These receptors, known as receptor tyrosine kinases (RTKs), consist of an extracellular ligand binding region, a single membrane-spanning region, a cytoplasmic tyrosine kinase. EGFR and several other RTKs also include a C-terminal tail that harbors several autophosphorylation sites (3). RTKs transmit information across the cell membrane by adopting specific dimeric conformations in response to ligand binding, which in turn leads to activation of the intracellular kinase activity, autophosphorylation, and initiation of intracellular signaling cascades (4, 5).
Four EGFR homologs, EGFR (HER1/ErbB1), HER2 (ErbB2/Neu), HER3 (ErbB3), and HER4 (ErbB4), exist in humans and are collectively known as the EGFR, HER, or ErbB family of receptors (6). Each EGFR homolog mediates key cell proliferation and differentiation events, and loss of any family member results in severe developmental defects or embryonic lethality (7). In adults, inappropriate expression or activation of EGFR homologs has been associated with multiple human cancers (8), and drugs targeting ErbB activity have been approved for treatment of breast, colon, lung, and head-and-neck cancers. These drugs are of two types: monoclonal antibodies targeting ErbB extracellular regions, which include the anti-EGFR antibodies cetuximab (Erbitux®) and panitumumab (Vectibix®) and the anti-HER2 antibody trastuzumab (Herceptin®), and small molecule kinase inhibitors, which include erlotinib (Tarceva®), gefitinib (Iressa®), and lapatinib (Tykerb®) (9).
The extracellular regions of ErbBs comprise four independent domains identifiable in both primary and tertiary structures, and structural studies of active ErbB fragments have led to characterization of receptor conformations that appear correlated with specific functional states (10, 11). In the absence of ligand, the extracellular regions of EGFR, HER3, and HER4 adopt a “closed” structure in which an extended beta-hairpin from domain 2 is buried in a contact near the juxtamembrane region of domain 4 (10, 12–14). This contact constrains the extracellular region into an arrangement in which ligand-binding surfaces on domains 1 and 3 are too far apart to bind ligand simultaneously. When ligand is bound domains 1 and 3 become juxtaposed, the contact between domains 2 and 4 is broken, and the hairpin loop on domain 2 mediates receptor dimerization (10, 15, 16). Activation of the intracellular kinase activity relies on formation of a specific “asymmetric” dimer of the kinase domains (11), and formation of the extracellular dimer must promote formation of this asymmetric dimer. How the extracellular dimer promotes intracellular dimer formation and kinase activation is not apparent from studies with receptor fragments, however, and many outstanding questions concerning interactions and communication between different regions of the receptor remain.
Quantitative enzymological studies of ErbBs have also been primarily limited to soluble, active fragments of receptor intracellular domains or incompletely characterized whole receptor (11, 17–20). Although much has been learned from these studies, a complete picture of EGFR kinase activity is necessarily lacking. To enable structural and functional studies of an intact form of EGFR we have developed a strategy to produce a membrane-spanning form of EGFR that is of sufficient purity, homogeneity, and quantity for structural, biophysical, and enzymological studies. Our approach shares many features with one recently reported by Springer and colleagues (21). We have used our purified EGFR to demonstrate direct autophosphorylation of Y845 and make quantitative enzymological measurements of active and inhibited forms of EGFR.
Lapatinib was synthesized as previously described (20, 22). Pharmaceutical erlotinib was obtained from C. Rudin of Johns Hopkins and purified in the Johns Hopkins Synthetic Core facility using silica gel chromatography. Pharmaceutical cetuximab was obtained from C. Hann and C. Rudin (Erbitux lot# 18148, manufacture date 1/27/03), and the Fab portion of the cetuximab obtained by digestion of the antibody with papain followed by anion exchange chromatography using FPLC. PP2 was obtained from Calbiochem. Acetonitrile (MS grade, J.T. Baker), formic acid (98–100% “suprapure”, EMD), phosphoric acid (Puriss grade, Fluka), trifluoroacetic acid (neat, Supelco), Trypsin (sequencing grade, Promega). All aqueous solutions were prepared using Milli-Q water (Millipore). The anti-pY845 antibody (Life Sciences) was a gift from Mark Lemmon of the University of Pennsylvania.
The gene encoding human EGF was inserted into the pT7HT vector (27), transfected into E. coli, and expression induced by addition of isopropyl-beta-D-thiogalactopyranoside followed by growth for at least 3 hours at 37°C. Cells were lysed in 6 M guanidine hydrochloride, centrifuged, and the supernatant loaded onto a Ni-NTA column. After washing, the column was eluted with 200 mM imidazole, and 5 mM dithiothreitol was added to pooled EGF-containing elution fractions. EGF was refolded by rapid dilution into 0.1M Tris, pH 8.0, 50 mM NaCl, dialyzed overnight, and re-run on a Ni-NTA column. After elution from the Ni-NTA column, TEV protease was used to remove the histidine tag. EGF was then concentrated and chromatographed on a Superdex 75 size exclusion column (GE Healthcare). Peak fractions were pooled and concentrated.
A cDNA encoding human EGFR was mutagenized using the megaprimer method to introduce a hexahistidine tag encoding region and a stop codon immediately following the position encoding Gly 998 (numbering from the mature N-terminus) (23). The resulting cDNA, which encodes a truncated form of EGFR (tEGFR), was subcloned into the pLEXm expression vector using the NotI and XhoI cloning sites (24).
400 ml suspension cultures of HEK293 GnTi− cells were seeded with 0.2 × 106 cells/ml and allowed to grow to 2–2.5 × 106 cells/ml (3–4 days) in Freestyle 293 medium (Invitrogen 12338) supplemented with 1% FBS (Invitrogen 10437) and 2 mM Glutamine (Invitrogen 25030) (25). No antibiotics were used. Cells were incubated at 37°C, 8% CO2 and shaken at 130 rpm in square bottles (Fisher 033112E) (26). Cells were harvested at 4°C by centrifugation for 5 min at 1000 rpm and resuspended in 400 ml fresh medium for transfection. For transfection, a 1 mg/ml stock of linear PEI MAX (Polyscience 24765) that had been neutralized with NaOH and sterile filtered was combined in a 3:1 weight ratio with sterile filtered expression plasmid DNA. Typically, 1.2 ml of PEI stock solution was added to 8.8 ml of Hybridoma medium (Invitrogen 12045), and 400 μg of expression plasmid DNA added to a separate tube of 10 ml Hybridoma medium. The PEI and DNA containing media were combined (20 ml total), gently mixed, incubated for 20 min at room temperature, and added to the prepared cells. After 24 hours the cells are diluted 1:1 with fresh medium, split into two 400 ml bottles, incubated at 37°C with shaking for another 72 hours.
Transfected HEK293 GnTi− cells are harvested by centrifugation at 3000 rpm for 10 min, washed with phosphate-buffered saline (PBS), and either used directly or frozen at −20°C. Cells were lysed by addition of an equal volume of homogenization buffer (40 mM HEPES pH 7.4, 10 mM EGTA, 2% Triton X-100, 20% glycerol, and a protease inhibitor cocktail (Roche)). Cells are then sonicated in three 20-second bursts and centrifuged at 14,000 rpm. The supernatant is filtered, beads covalently modified with the anti-EGFR monoclonal antibody 528 (28) are added, and the slurry incubated overnight at 4°C with gentle shaking. The beads are then transferred to a column and washed in succession with (i) 25 ml receptor buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 10 % glycerol, and 0.03% Dodecylmaltoside), (ii) 25 ml receptor buffer/1 M NaCl, (iii) 25 ml receptor buffer, (iv) 25 ml receptor buffer/1 M NaCl, (v) 25 ml receptor buffer, (vi) 25 ml receptor buffer/1 M Urea, and (vii) 50 ml receptor buffer.
The column is then eluted with 5 successive additions of an equal volume of 25 μg/ml EGF or 0.1 mg/ml cetuximab Fab in receptor buffer that are incubated on the column for 30 minutes at room temperature. Each elution fraction is collected and pooled. A 1:1000 dilution by volume of 1 mg/ml stock solutions of Endoglycosidases H and F is added to the pooled fractions (10 μl of each stock solution per 10 ml protein solution) and incubated at 4°C for 1 hour. The protein solution is then concentrated to no more than 500 μl total volume using a 100 kD molecular weight cut off Amicon Ultra-4 (Millipore) and loaded onto a Superose 6 size-exclusion column and chromatographed in 20 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM DTT, 5% glycerol, and 0.03% dodecylmaltoside. Elution fractions containing tEGFR are identified by Western blot, pooled, and concentrated. Purified tEGFR was quantitated using UV Absorbance and comparison of bands of tEGFR and bovine serum albumin standards on Coomassie Blue-stained SDS-PAGE gels.
3 μM tEGFR/EGF complex was incubated in reaction buffer (20 mM Hepes, pH 7.4, 1 mM MnCl2, 5 mM MgCl2, 1 mM ATP, 1 mM Na3VO4) for 10 min at room temperature (~22°C). The reaction was stopped by the addition of SDS gel loading buffer and the proteins resolved on SDS-PAGE followed by Western blotting using either the anti-phosphotyrosine antibody 4G10 or a specific anti pY845 antibody (Life Sciences).
To determine the kinetic parameters for the EGF/tEGFR and cetuximab Fab/tEGFR complexes, radiometric kinase assays were carried out in 50 mM HEPES (pH 7.5), 37.5 mM NaCl, 1 mM DTT, 5% Glycerol, 125 μg/ml BSA with either 2 mM MnCl2 or 10 mM MgCl2 and Biotin-RAHEEIYHFFFAKKK-COOH as the peptide substrate in a 25 μl reaction volume. The peptide substrate was prepared using standard Fmoc solid-phase peptide synthesis followed by coupling of biotin to the N terminus using the biotin p-nitrophenyl ester (NovaBiochem) and purification by reverse phase HLC. Reactions were initiated by the addition of kinase, carried out at 30°C, and stopped by the addition of 10 μl of 100mM EDTA. To each sample, 10 μl of 10mg/ml avidin (Thermo Scientific) was added, and all samples were transferred to centrifugal filtration units with 30,000 nominal molecular weight limit membranes (Millipore) and washed three times with 100 ml wash solution (0.5 M Phosphate, 0.5 NaCl, pH 8.5). The limiting substrate turnover was less than 10% for all rate measurements. Duplicate measurements were generally within 20%.
The linear range for activity vs. time was established using multiple time points, 80 μM peptide, either 2 mM MnCl2 or 10 mM MgCl2, 100 μM ATP, and 25 nM enzyme (except for the cetuximab Fab/tEGFR complex with 10 mM MgCl2, which used 50 nM enzyme). The linear range for activity vs. enzyme concentration was established using various enzyme concentrations, 80 μM peptide, either 2 mM MnCl2 or 10 mM MgCl2, and 100 μM ATP.
The Kmapp for peptide substrate was determined with varying peptide concentrations and fixed ATP concentration (100 μM). Assays for EGF/tEGFR used 25 nM enzyme and the following peptide concentrations: 1.88, 3.75, 7.5, 15, 30, 60, and 120 μM. Assays for cetuximab Fab/tEGFR used 125 nM enzyme and the following peptide concentrations: 6.25, 12.5, 25, 50, 100, 200, 400, and 800 μM. The Kmapp for ATP was determined by using varying ATP concentrations and fixed peptide substrate concentration. Assays for EGF/tEGFR used 25 nM enzyme and the following ATP concentrations in the presence of 2 mM MnCl2: 0.78, 1.56, 3.13. 6.25, 12.5, 25, 50, and 100 μM. Assays for EGF/tEGFR used 25 nM enzyme and the following ATP concentrations in the presence of 10 mM MgCl2: 3.75, 7.5, 15, 30, 40, 60, 90, 120 μM. Assays for cetuximab Fab/tEGFR used 125 nM enzyme and the following ATP concentrations: 1.56, 3.13, 6.25, 12.5, 25, 50, 100 μM. Apparent Km and kcat values were obtained from nonlinear curve fits to the Michaelis-Menten equation.
For the IC50 determination for lapatinib, various concentrations of lapatinib (40, 10, 2.5, 0.625, 0.313, 0.159, and 0.078 μM) were pre-incubated with enzyme on ice for 10 min and the reactions run with 10 μM ATP, 30 μM peptide, 2 mM MnCl2, 25 nM enzyme and 0.15% DMSO for 6 min with EGF/tEGFR and for 40 min with cetuximab Fab/tEGFR. For the IC50 determination for erlotinib, the inhibitor erlotinib (160, 40, 10, 2.5, 0.625, 0.313, 0.159, and 0.078 μM) was pre-incubated with enzyme at 30°C for 10 min and the reactions were run with 10 μM ATP, 30 μM peptide, 2 mM MnCl2, 25 nM enzyme and 0.15% DMSO for 5 min with EGF/tEGFR and for 40 min with cetuximab Fab/tEGFR. IC50 values were obtained from fitting the isotherm from the dose-response plot.
The kinase reaction was carried out by incubating 3 μM tEGFR-EGF complex in 15μl reaction buffer (20 mM Hepes, pH 7.4, 1 mM MnCl2, 5 mM MgCl2, 1 mM ATP, 1 mM Na3VO4) for 30 min at 30°C. The reaction was stopped by the addition of SDS gel loading buffer, the proteins were resolved on SDS-PAGE. For the inhibitor experiment, 3 μM EGFR-EGF complex was preincubated with 200 μM erlotinib or PP2 for 30 min on ice before the addition of ATP-containing reaction buffer. The SDS-PAGE bands corresponding to EGFR at ~120kDa tEGFR were excised, destained and buffer exchanged by alternating acetonitrile and 50mM TEAB (triethylammonium bicarbonate) washes. Protein bands were then digested with 10 ng/μl sequencing grade trypsin (Promega) in 35 μl of 50mM TEAB for 16 hr at 37°C. Peptides were extracted with 55% acetonitrile containing 0.5%TFA (trifluoroacetic acid) and dried.
Extracted peptides were resuspended in 10 μl 0.5 M TEAB. Isopropanol (50 μl) was added to vials containing 113, 115, 117 or 121 iTRAQ-8plex reagent. Each sample was labeled for 2 h at room temperature by adding 25 μl of one iTRAQ reagent, maintaining the pH between 7.5–8.0 with 0.5 M TEAB. All four iTRAQ labeled samples were combined and dried. Approximately 8 μg of total labeled peptide (based on total protein loaded on the gel) was resuspended in 10μl 0.2%TFA, desalted using a C18 ziptip (Varian), eluted with 70% acetonitrile in 0.1%TFA and dried.
Desalted peptides were resuspended in 200 μl of SCX buffer A (10 mM potassium phosphate buffer, pH 2.85, 25% acetonitrile). The sample was adjusted to pH 2.5–2.8 with phosphoric acid and loaded onto a PolySulfoethyl A column (100 × 0.3 mm, 300Å, 5μm, PolyLC, Columbia, MD) at 5 μl/min for 40 min using an Agilent 1200 series capillary HPLC system. Peptides were fractionated using a 0–100% SCX buffer B (350 mM KCl in 10 mM potassium phosphate buffer, pH 2.85, 25% acetonitrile) linear gradient over 60 min at 5 μl/min. Fractions were collected manually (7 fractions, 40–100μL fraction), dried and stored at −80°C until LCMS/MS analysis.
Each SCX fraction was redissolved in 40 μl of 0.2% TFA and desalted on a C18 trap (75μm × 3 cm, 5–10μm, 120Å, YMC Gel) at 8 μl/min for 15 min with Buffer A (0.1% formic acid) using an Eksigent nano-2D LC system. After desalting, peptides were separated on a C18 column (75 μm × 10 cm, 5 μm, 120Å, YMC ODS-AQ, Waters, Milford, MA) with an 8 μm emitter tip (New Objective, Woburn, MA) using 5–40% B (90% acetonitrile in 0.1% formic acid) gradient over 60 min at 300 nl/min. Eluting peptides were sprayed directly into a QSTAR/Pulsar mass spectrometer (Applied Biosystems) and sequenced by tandem mass spectrometry. Survey scans were acquired from m/z 350–1300 with up to three precursors selected for MS/MS using a dynamic exclusion of 45 s. A rolling collision energy was used to promote fragmentation and the collision energy range was ~20% higher than that used for unlabeled peptides due to iTRAQ tags.
The MS/MS spectra were extracted and searched against SwissProt database (version 54.6), human species using ProteinPilot™ software (v2.0.1, Applied Biosystems) with Paragon™ search algorithm and with the following parameters: trypsin as enzyme (one missed cleavage allowed) and variable modifications for methionine oxidation of cysteine, phosphorylation of serine/threonine/tyrosine phosphorylation, and 8-plex-iTRAQ labeling of N-termini, lysine and tyrosine. The raw peptide identifications from the Paragon™ Algorithm (Applied Biosystems) searches were further processed by the Pro Group™ Algorithm (Applied Biosystems) within the ProteinPilot™ software set to identify peptides with confidence threshold 95% (“unused” confidence threshold Protscore >1.3). Identified phosphopeptides were verified by manual inspection. ProteinPilot was also used to calculate the protein and peptide ratios. For protein ratios, the contribution of each peptide ratio to the overall protein ratio is proportional to the confidence of individual peptide ratio.
The gene encoding the full-length human EGF receptor (EGFR) was stably transfected into Lec1 CHO cells, which lack the N-acetylglucosaminyltransferase I activity essential for synthesis of complex N-linked glycans (29). Following methotrexate amplification and selection for high levels of EGFR expression by fluorescence-activated cell sorting, transfected cells were estimated to express at least ~5 × 105 receptors per cell based on Western blots calibrated with known amounts of standard protein. Initial preparations of EGFR from these cells invariably included a proteolytic fragment of EGFR missing ~20 kD from the C-terminus (30, 31). Efforts to prevent proteolysis reduced but did not eliminate this fragment. A region encoding a hexahistidine tag followed by a stop codon was thus introduced into the EGFR gene following the position encoding Gly 998, which immediately follows the kinase region (32), and this truncated form of EGFR (tEGFR) was expressed in Lec1 cells.
Western blots carried out in reducing and nonreducing conditions showed that tEGFR formed disulfide-linked oligomers. All 50 conserved cysteines in the EGFR extracellular region are involved in disulfide bonds, implicating intracellular cysteines in mediating this oligomerization. To avoid formation of disulfide-linked tEGFR oligomers without using reducing agents and potentially destabilizing the disulfide-rich extracellular region, surface-exposed intracellular cysteines (Cys 751, Cys 757, and Cys 773) were substituted with serine by site-directed mutagenesis (32). A truncated EGFR bearing these cysteine substitutions was expressed in Lec1 cells and proved to be both relatively stable to proteolysis and monomeric in the absence of reducing agents as judged by size-exclusion chromatography. Addition of 0.5 mM dithiothreitol also disrupted unwanted disulfide-mediated oligomers without effect on the ability of tEGFR to bind either EGF or the Fab fragment of the anti-EGFR antibody cetuximab.
Attempts to scale up production of tEGFR to levels needed for structural and biochemical studies were hindered by difficulties growing transfected Lec1 cells above cell densities of ~5 × 105 cells/ml. HEK GnTi− cells, which reliably grow to 3–4 million cells/ml and also lack N-acetylglucosaminyltransferase I activity (25), were thus transfected with a gene encoding tEGFR and a C-terminal hexa-histidine tag inserted into the pLEXm expression vector (24). tEGFR expression levels in both transient- and stably-transfected HEK293 GnTi− cells were estimated to be at least 5 × 105 receptors per cell based on purification yields from known numbers of cells. tEGFR expression in stably-transfected cells decreased with time, but transient transfection using polyethylenimine (PEI) and square shaker bottles resulted in consistent yields of ~0.2 mg of purified tEGFR per liter of cells and was subsequently pursued (24, 26).
Ni-NTA resin failed to adsorb the bulk of tEGFR from large-scale cell lysates despite the C-terminal hexa-histidine tag. The anti-EGFR monoclonal antibody 528 (MAb528) was thus coupled to a matrix to create an affinity column, which efficiently adsorbed tEGFR from cell lysates (28). EGF and the anti-EGFR monoclonal antibody cetuximab both compete with MAb528 for EGFR binding, and both elute tEGFR from the MAb528 column. A combination of endoglycosidases H and F removed N-linked glycosylation (Figure 1A), and size-exclusion chromatography showed the EGF/tEGFR complex to elute at a volume consistent with a 2:2 heterotetramer (Figures 1B and S1). The tEGFR/cetuximab Fab eluted at a volume consistent with a 1:1 complex.
Western blot analysis of purified EGF/tEGFR complex using a phosphotyrosine-specific antibody (4G10) showed it to autophosphorylate following addition of ATP (Figure 2A). This autophosphorylation is inhibited by erlotinib, an ErbB inhibitor, but not PP2, a Src inhibitor (Figure 2A). tEGFR contains one of six phosphorylation sites (Y992) previously identified in the C-terminal tail of EGFR (7, 33). tEGFR also includes tyrosine 845 (Y845), which is situated in the activation loop and is known to become phosphorylated in an EGF-dependent manner following EGFR activation and to be phosphorylated by Src (34, 35). A pY845-specific antibody (Life Sciences) recognized tEGFR after incubation with ATP (Figure 2B), and tandem mass spectrometry of trypsinized tEGFR peptides following iTRAQ labeling demonstrates a ~17-fold increase in the amount of a pY845-containing EGFR peptide following addition of ATP to tEGFR (Table 1 and Figures S2–S6) (36). Autophosphorylation of tEGFR at Y845 was inhibited by erlotinib but not PP2, and the levels of threonine phosphorylation detected by mass spectrometry did not change significantly indicating that the changes in phosphotyrosine levels are not due to variability in protein amounts between samples (Table 1). PP2 appeared to inhibit EGFR activity to a greater extent in the Western blot vs. mass spectrometric analysis although the trends and direction were the same. A possible explanation for this difference is that the kinase reaction was carried out for a shorter time and at a lower temperature for the Western blot analysis.
For a quantitative analysis of the kinase activity of tEGFR, we used a radiometric assay to compare the effects of EGF and the Fab fragment cetuximab, an anti-EGFR antibody that blocks EGF binding and formation of extracellular EGFR dimers, on the ability of tEGFR to phosphorylate a biotinylated peptide substrate. Steady-state kinase assays were carried out using a ‘consensus’ peptide substrate with sequence RAHEEIYHFFFAKKK and repeated using either Mg2+ or Mn2+ as the divalent metal ion. Under the conditions of our assay, the activities of EGF/tEGFR and cetuximab/tEGFR were shown to be linear versus time and enzyme concentration, indicating that neither tEGFR oligomerization nor degradation was likely to be influencing activity in the ranges investigated. For EGF/tEGFR, the Kmapp for ATP was nearly ~13-fold lower with Mn2+ (3.6 μM) than Mg2+ (47 μM). Lower Kms for ATP in the presence of Mn2+ vs. Mg2+ are commonly observed with protein kinases, presumably owing to the increased nucleotide affinity of Mn2+ (37, 38). Mg2+ vs. Mn2+ had a smaller effect on the apparent kcat (24 vs. 18 min−1, respectively) and Km for peptide substrate (41 vs. 12 μM) values, which were within five-fold of one another.
The low activity of cetuximab/tEGFR precluded measurement of a full range of catalytic parameters with Mg2+ ion, but at fixed substrate concentrations the observed kinase activity was ~500-fold lower than that of EGF/tEGFR in the presence of Mg2+ and ~150-fold lower in the presence of Mn2+ (Table 2). This observation was in the range of the kcat/Km effects for ATP (920-fold) and peptide (260-fold). The major effect of ligand binding was on apparent kcat values, which were ~140-fold, but 2-fold (peptide) and 7-fold (ATP) increases in apparent Kms were observed in the presence of cetuximab (Table 3). Interestingly, addition of excess EGF to cetuximab/tEGFR did not significantly stimulate kinase activity, suggesting that exchange of these protein ligands is slow on the time scale of these assays.
We then evaluated the effects of EGFR kinase inhibitors erlotinib and lapatinib on the kinase activity of EGF and cetuximab complexed forms of tEGFR. Erlotinib showed a 10-fold enhanced potency against EGF/tEGFR compared to cetuximab/tEGFR (Table 4). Conversely, lapatinib inhibited cetuximab/tEGFR relative to EGF/tEGFR by a 4-fold margin. Taken together, these data are consistent with the prevailing view that erlotinib targets the active EGFR kinase conformation whereas lapatinib preferentially binds to the inactive conformation (32, 39).
We have developed a strategy to produce a form of the EGF receptor that is activated by ligand binding and suitable for structural, biophysical, and biochemical studies. Steps have been taken to maximize yields and minimize both the time and cost of production as well as heterogeneity arising from glycosylation and proteolysis. In particular, large-scale transient transfection of the EGFR gene into HEK293 GnTi− cells using PEI as a transfection reagent proved a cost- and time-effective approach that yields ~0.2 mg of purified EGFR per liter of transfected cells and enables efficient removal of N-linked glycosylation using Endoglycosidases H and F. Eliminating the structural and chemical heterogeneity of attached carbohydrates often proves beneficial to growth of diffraction-quality crystals (40). Heterogeneity in purified EGFR also arose from proteolysis of the C-terminal tail, which proved difficult to avoid completely, and a premature stop codon that eliminated the C-terminal tail was thus introduced immediately following the region encoding the EGFR kinase domain. Coupled with efficient deglycosylation of HEK293 GnTi− cell-expressed EGFR, truncation of EGFR following the kinase domain allowed purification of a relatively homogenous form EGFR that is activated by ligand binding. As also observed by Springer and colleagues (21), tEGFR is solubilized well in both Triton X-100 and dodecylmaltoside, is monomeric in the absence of ligand, and is dimeric in the presence of EGF as judged by size-exclusion chromatography.
Additional heterogeneity arose from the formation of unwanted disulfide-linked EGFR oligomers in mildly oxidizing initial purification conditions, presumably mediated by cytoplasmic cysteines that are reduced in normal cell environments. Two approaches, mutagenesis of exposed cytoplasmic cysteines and addition of a small amount of reducing agent, both successfully eliminated unwanted EGFR oligomers. Concerns that a small amount of reducing agent might disrupt the structure and function of the heavily disulfide-linked extracellular region were overcome when the inclusion of 0.5 mM dithiothreitol failed to diminish ligand binding activity or lead to oligomerization through transient reduction of extracellular disulfide bonds. To avoid complications owing to possible effects of cysteine mutations on EGFR enzymatic activity, 0.5 mM dithiothreitol was used during purification to produce native tEGFR for the studies reported here.
Preparation of purified tEGFR allowed quantitative characterization of its kinase activity. Early published reports identified six autophosphorylation sites in the EGFR C-terminal tail but no sites in the kinase region itself (7, 33). Later studies showed that a tyrosine on the kinase activation loop, Y845, is phosphorylated by the Src kinase and that phosphorylation of Y845 influences EGFR function (34, 35). Phosphoryation of Y845 is not required for EGFR activity, however (41). More recently, Arteaga and colleagues showed that Y845 becomes phosphorylated following TGFα stimulation or introduction of oncogenic EGFR mutations and that this Y845 phosphorylation is dependent on EGFR kinase activity (42). Using purified tEGFR and tandem mass spectrometry we now demonstrate that EGFR itself is capable of phosphorylating Y845. It is well established that phosphorylation of activation loop tyrosines increases the activity of several kinases (43), and phosphorylation of Y845 seems likely to promote or stabilize EGFR activity and modulate EGFR signaling.
Comparison of the kinase activities of EGF/tEGFR and cetuximab/tEGFR demonstrates that ligand stimulates a ~500-fold increase in catalytic activity (kcat) and that the effect of ligand binding to tEGFR is predominantly on kcat rather than substrate Kms. This observation suggests that substrate binding is likely to be only modestly perturbed in the cetuximab/tEGFR complex. Structural studies on the Abl tyrosine kinase support the idea that interactions with peptide substrate can be maintained despite kinase activation loop and C-lobe conformations that appear inconsistent with catalysis (44). In this case the orientation of the two substrates and/or placement of key catalytic residues appear distorted from active conformations. A similar mechanism may underlie our observations with EGFR.
Prior quantitative studies of EGFR/ErbB enzymatic activity have primarily utilized soluble intracellular domain fragments or incompletely characterized EGFR (11, 17–20). In particular, one recent approach mimicked the high local concentrations achieved in a membrane environment by tethering the EGFR kinase domain to lipid vesicles via a hexa-histidine tag (11). Stimulation of kinase activity by up to 20-fold were observed following vesicle targeting, consistent with a key role for kinase dimerization in activation.
To our knowledge, our study describes the first detailed kinetic analysis of near full-length EGFR in purified form. By comparing EGF and cetuximab-complexed forms of tEGFR, we have shown a 500-fold activation between autoinhibited and stimulated forms. Elimination of the C-terminal tail may have influenced our results, but this change in activity is some 25-fold greater than the stimulation observed with vesicle activation. This 500-fold differential is also considerably larger than measurements of kinase domains bearing activating mutations (11, 45, 46). Taken together, these results suggest that kinase-domain only assays incompletely recapitulate full activation/inhibition of the EGFR kinase. The differences observed between isolated kinase regions and a more intact form of the receptor are perhaps not surprising as the ligand-bound extracellular regions and the juxtamembrane regions must contribute to stronger dimerization if not also stereochemically favorable apposition of the kinase domains (47, 48).
Of potential clinical interest among the results reported here is the relative inhibitory potencies of the two anti-cancer agents erlotinib and lapatinib, which structural studies have shown bind to active and inactive EGFR kinase conformations, respectively (11, 32, 39). We observed the expected trend that erlotinib is more effective at inhibiting the ligand-activated EGFR kinase and lapatinib more effective at inhibiting inactive EGFR. This observation suggests the potential for therapeutic synergy between cetuximab and lapatinib. The relative selectivity of erlotinib and lapatinib for their preferred enzyme forms was only 5–10-fold, however, which was less than expected based on the much larger relative difference in kinase activity in the presence and absence of ligand. Either substantial crossover binding of these inhibitors for alternate EGFR kinase conformations occurs, or an unexpectedly high interconversion between active and inactive kinase conformations may occur irrespective of receptor dimerzation. Given the the conditions of our assay and the 7-fold higher Km for ATP of cetuximab complexed with tEGFR compared with EGF/tEGFR, the Ki values deduced for lapatinib blocking the inhibited and active enzyme forms are essentially identical. These findings run contrary to the concept of truly conformation-specific EGFR kinase inhibitors and suggest that further investigation of inhibitor-bound conformations of the EGFR kinase may prove illuminating.
A final observation to emerge from our results is that even the ‘inactive’ EGFR conformation observed crystallographically appears capable of catalyzing phosphoryl transfer, albeit at a significantly reduced rate. The differences in substrate Kms observed for the cetuximab versus EGF bound forms of tEGFR suggest that the residual kinase activity in the antibody-complexed EGFR arises from a different kinase conformation. Had the lower activity of the cetuximab-tEGFR complex resulted solely from shifting the conformational equilibrium of the kinase region away from the active conformation, one would expect to observe lower kcats but unaffected Kms. Although 500-fold lower than the EGF-tEGFR rate, the catalytic power of cetuximab-tEGFR is still more than 10,000-fold greater than the uncatalyzed phosphoryl transfer reaction (49), indicating substantial transition state-stabilization still occurs in this state.
Supported by NIH grants CA090466 (DJL) and CA74305 (PAC).
We thank members of the Cole and Leahy labs for helpful suggestions and discussions and M. Lemmon, C. Mukherjee, C. Hann, W. Bornmann, and D. Meyers for assistance and/or reagents.