EGFR Variants Lacking Tyr(P)998 and Ser(P)991 Are Competent for EGFR → ERK Signaling but Defective for Endocytosis
The accumulation of phosphorylation at EGFR residue Tyr998
is documented to be relatively slow compared with other EGFR Tyr(P) sites when measured in HeLa cells (5
), and our own experiments with HEK-EGFR cells are in agreement with these kinetics (data not shown). Similarly the kinetics of EGF-stimulated accumulation of a doubly phosphorylated EGFR peptide containing Ser(P)991
was reportedly slow (10
). In our experiments, the singly phosphorylated peptide containing Ser(P)991
was the most efficiently detected phosphoisomer of the MHLPSPTDSNFYR peptide.
The MS method of multiple reaction monitoring or SRM has been applied to quantify protein phosphorylation (32
), including temporal analysis of Tyr(P) modifications associated with EGF treatment (8
). The neutral loss of phosphoric acid from Ser(P) and Thr(P) sites is a frequent event during tandem MS of phosphopeptides (35
), and the MS/MS spectrum of the EGFR tryptic peptide containing Ser(P)991
contained two prominent product ions representing loss of phosphoric acid from the parent ion and from the y10 fragment (A
, see arrows
). These neutral loss transitions were monitored as a means to measure Ser(P)991
following EGF stimulation of HEK-EGFR cells, and the duration of the EGF treatment was extended to 90 min (, B
), which is longer than previous studies in which EGF-stimulated phosphorylation was monitored for up to 20 min post-EGF treatment (10
). SRM analysis of neutral loss for phosphorylation measurement has been described previously (33
). The SRM analysis indicated that the Ser(P)991
peptide increased ~10-fold after 90 min of EGF treatment and did not appear to reach a plateau or maximum by this time (C
As a control, EGF-stimulated phosphorylation at EGFR Tyr1092
, which is associated with GRB2 binding and activation of RAS-ERK signaling, was monitored by Western immunoblotting (38
). As expected, phosphorylation at this position was rapid and reached an apparent maximum by 1 min (D
). It was observed that even 90 min after EGF stimulation there was not yet appreciable down-regulation of EGFR (E
), which is typically more apparent by 4 h post-EGF (21
). The relatively slow accumulation of phosphorylation at Ser991
following EGF treatment was pronounced and differs from the kinetics of the doubly phosphorylated isomer (Ser(P)991
) described by Olsen et al.
) that reached a maximum abundance near 10 min post-EGF and then receded to basal levels by 20 min. However, it is possible that the kinetics of these phosphorylations differ as a function of cell type and receptor densities, which are parameters that are different in our study and the comprehensive study by Olsen et al.
The SRM measurements of the Ser(P)991
peptide were normalized to the amount of a non-phosphorylated EGFR peptide YSFGATCVK (residues 285–293) from the same sample. The signal for this control peptide was generated by summation of transitions precursor → y6 and precursor → y7 at corresponding time points of EGF stimulation (supplemental Fig. 1
). The EGFR control peptide produced a high signal (intensity >106
units) and low coefficient of variation (CV = 8.3 ± 3.3%, n
= 9) in test samples. Measurements of this peptide, therefore, were used as an indicator of EGFR amount in all SRM experiments in this study.
The relatively slow kinetics of phosphorylation at Ser991 and Tyr998 suggested that these modifications might not be involved in rapid signaling events associated with EGFR activation. To test this, site-directed mutagenesis was used to remove the side chain hydroxyl groups at these positions to prevent phosphorylation. Transfected HEK293 cell lines stably expressing variant proteins containing Tyr998-to-Phe (EGFR Y998F) and Ser991-to-Ala (EGFR S991A) at levels comparable to wild type (WT) EGFR in HEK-EGFR were established. These cell cultures were monitored for EGF-induced phosphorylation at Tyr1092 and for activation (phosphorylation) of the downstream MAP kinase ERK1/2. Phosphorylation of EGFR at Tyr1092 was unaffected by either Y998F or S991A substitution (, A and B), and the two mutant receptors did not show statistically significant changes in phosphorylated ERK1/2 following EGF treatment when compared with WT EGFR (, C and D).
Fig. 2. Phosphorylation of EGFR Tyr1092 and ERK in EGF-stimulated HEK cells expressing wild type EGFR and the mutant receptors Y998F and S991A. A, EGFR-FLAG proteins from HEK cells stably expressing wild type receptor (EGFR) or the indicated variants (Y998F and (more ...)
To examine endocytosis of WT EGFR and the phosphorylation-defective receptor variants, localization of chimeric receptors containing carboxyl-terminal green fluorescent protein (GFP) was monitored by fluorescence microscopy. In serum-starved HEK-EGFR, HEK-S991A, and HEK-Y998F cells, the receptors were concentrated near the cell periphery consistent with plasma membrane localization (, left panels
, long arrows
). After exposure to 100 ng/ml EGF and incubation at 37 °C for 5 and 15 min, WT EGFR staining at the cell margins was diminished and became internalized and concentrated into intracellular aggregates characteristic of endosomes (, upper row
, short arrows
). By contrast, Y998F and S991A mutant receptors displayed far less internalization/aggregation and remained largely concentrated at the cell periphery (, middle
and lower rows
, long arrows
). These observations demonstrate defective endocytosis of the mutant receptors. The endocytosis defect of EGFR Y998F is expected based on Sorkin et al.
). The cell staining was categorized into two types: WT-like, typified by internalized and aggregated receptors and little or no staining at the cell margins, and mutant-like, which lacked intracellular aggregates and retained concentrated staining at the cell margins. After tabulating these patterns in 100 cells in three separate experiments, ~60% of WT receptors displayed the WT-like pattern of internalized receptors (diminished peripheral staining accompanied by internalized aggregates), whereas only ~30% of Y998F and 25% of S991A receptors showed this pattern.
Fig. 3. Spatial and temporal dynamics of wild type EGFR and phosphorylation-deficient variants. HEK293-based cells stably expressing WT, S991A, and Y998F EGFR proteins (as indicated) fused to GFP were treated with EGF (100 ng/ml) for 0, 5, and 15 min at 37 °C (more ...)
To further examine and to quantify the extent of EGF-stimulated receptor internalization, cells were treated with or without EGF for 30 min, and then receptors exposed on the cell surface were covalently labeled with biotin. Receptors were then purified by immunoprecipitation and quantified for biotinylation by Western blotting with a streptavidin probe (A). After EGF treatment, the amount of surface-exposed, biotinylated WT EGFR was decreased by ~50%, whereas the amounts of surface exposed Y998F and S991A receptors were almost equivalent to levels in untreated cells (A). As an additional measure of receptor endocytosis, receptor-mediated internalization of rhodamine-labeled EGF was measured by flow cytometry. As shown in B, ligand internalization by Y998F and S991A was less than that observed for WT EGFR. Therefore, by three different measures, the Y998F and S991A variants were defective for receptor endocytosis.
EGFR-associated Proteins and Phosphorylation
To further address the function of the kinase domain proximal phosphorylation sites at Ser991
, receptor-associated proteins and receptor phosphorylation were examined by using LC-MS/MS and in some instances quantified by SRM. Tandem MS analysis served three purposes. First, it enabled the identification of receptor-associated proteins, which could then be compared with a reference set of EGFR-associated proteins. Second, it facilitated the measurement of spectral counts as a first approximation of protein abundance (39
). Third, it provided information on peptides and their fragmentations, which were useful in the design of SRM methods for the quantification of receptor-associated proteins in repeated experiments.
EGFR immunoprecipitates from EGF-stimulated cells expressing WT and variant EGFR proteins were prepared by using anti-FLAG antibodies and analyzed by LC-MS/MS for associated proteins similar to previously published methods involving gel-free, LC-MS/MS analysis of trypsin-digested, anti-FLAG immunoprecipitates (22
). In aggregate 444 proteins were identified in the anti-FLAG immunoprecipitates of WT and mutant receptors by using criteria described under “Experimental Procedures” (i.e.
at least two distinct peptides and 95% probability scores by Peptide Prophet and Protein Prophet algorithms (27
)). Not surprisingly, this list (supplemental Table 1
) contains proteins commonly known to associate with FLAG immunoprecipitates (40
). To focus the analysis of receptor-associated proteins, the list was compared with data from the manually curated Human Protein Reference Database, which lists 144 EGFR-binding proteins that have experimental validation in the literature. shows the 17 proteins, in addition to the EGFR, that intersect these two data sets including the ligand EGF; proteins involved in EGFR signaling such as SHC1, GRB2, and SOS1; and others associated with receptor endocytosis, including the ubiquitin E3 ligases CBL and CBLB and ubiquitin ().
The spectral counts of these EGFR-associated proteins were noted as a crude measure of their abundance in the IP samples. Spectral counts were normalized by adjusting for the number of spectral counts from non-phosphorylated EGFR peptides. EGFR peptides, as expected, were by far the most abundant class of peptide in the samples (), and this observation served to validate that the IP method was specific for this antigen. For each of the proteins mentioned above, VAV2, and the phosphatidylinositol 3-kinase p85 subunits (PIK3R1 and PIK3R2), spectral counts associated with WT EGFR increased as a consequence of EGF treatment of cells. Furthermore with the exception of EGF and CBLB, the spectral counts for these proteins measured with EGF-stimulated Y998F and S991A receptors were less than those observed for WT ().
To quantify the receptor-associated proteins, including changes due to EGF stimulation and as a consequence of the Y998F and S991A mutations, SRM analysis was applied to the receptor·immunoprecipitate complexes isolated from cells before and after EGF treatment. An SRM method that tracked the top six EGFR-associated proteins (according to spectral counts associated with WT EGFR; ) was formulated based on transitions summarized in supplemental Table 2
. The fragmentation patterns obtained during discovery analyses with the LTQ-Orbitrap were a useful starting point toward the development of the SRM transitions. However, the resonance excitation-based fragmentations produced in the linear ion trap were not identical with the true CIDs undertaken in Q2 of the triple quadrupole TSQ instrument that was used for SRM. The intensity of b ions was generally greater in the linear ion trap, which also had a low molecular weight cutoff (i.e.
⅓ rule) that made low m
ions more predominant in the triple quadrupole TSQ instrument. In addition, the LC gradients were of different durations (120 min with the Orbitrap and 40 min with the TSQ instrument). The SRM method was applied in three independent biological experiments resulting in the quantification the EGFR and the six receptor-associated proteins ( and ). The EGFR peptide YSFGATCVK (residues 285–293; described above), which is common to the WT and variant proteins and not subject to phosphorylation, was quantified and used to normalize for the amount of EGFR in each sample.
Fig. 5. SRM-based measurement of EGFR-associated proteins. EGFR was recovered by anti-FLAG IP from HEK cells stably expressing WT EGFR or one of the variants Y998F or S991A. The immune complexes were converted to tryptic peptides, which were subjected to SRM (more ...)
SRM-based measurement of six EGFR-associated proteins
The interactions of all six proteins were verified by the SRM analysis (). The largest EGF-stimulated -fold increases in receptor binding were observed for CBL followed by GRB2. CBL association with WT EGFR increased more than 7-fold following EGF treatment but was less than 5-fold with S991A and was ~4-fold with the Y998F variant. The drop in CBL association associated with the Y998F mutation was significantly different from WT (p
= 0.01; ). EGF-stimulated receptor association of GRB2 was roughly 3-fold, which was a statistically significant increase over that observed without EGF treatment, and the measurements for GRB2 were not different between WT and the variant receptors (). The associations of VAV2 and SOS1 were measured to be essentially constitutive and unaffected by the phosphorylation site mutations because no statistically significant differences were noted following EGF treatments or between the WT and the variant receptors (). The binding of SHC1 to WT EGFR was stimulated more than 2-fold by EGF, but for the receptor variants, EGF-stimulated SHC1 association was associated with too much variation to be considered significant. The capture of ubiquitin with WT and Y998F was increased more than 3-fold by EGF but to a lesser extent with the S991A variant. Furthermore with EGF-treated cells the amount of receptor-associated ubiquitin was significantly reduced in S991A compared with WT (). shows the peptides, SRM transitions, and coefficients of variance associated with the data depicted in . A more complete listing of these measurements including SRM intensities for each of the three repeated experiments is provided in supplemental Table 4
The observed changes in receptor-associated CBL and ubiquitin were supported by Western blot analysis (). The variant receptors were not generally impaired for phosphorylation at the CBL binding site Ser(P)1069
, but the Y998F and S991A protein complexes captured by anti-FLAG IP were observed to contain less CBL and ubiquitin than did WT (). The co-immunoprecipitation of ubiquitin may reflect the ubiquitination of any of the proteins in the receptor (anti-FLAG) immunoprecipitates. However, prominent anti-ubiquitin immunostaining was concentrated at or slightly above the migration of the receptors, suggesting that ubiquitination of the receptors, as expected (19
), may account for a portion of the ubiquitin measured by MS.
Fig. 6. Western blot analysis of EGFR and associated proteins. FLAG-tagged wild type receptor (EGFR) and the variants Y998F and S991A, as indicated, were collected by anti-FLAG IP from total cell lysates prepared from cells treated without or with EGF (15 min, (more ...)
The level of endogenous EGFR in HEK293 cells is very minor (22
), and association with other ErbB family receptors was not detected. This indicated that dimerization of FLAG-tagged ectopic receptors with endogenous ErbB family members to which associated proteins might bind was not a factor in these experiments.
Among the peptides characterized by LC-MS/MS analysis of EGFR immunoprecipitates were phosphopeptides. Following EGF stimulation, the total number of spectral counts for EGFR-derived phosphorylated peptides was 69 for WT EGFR, 72 for the Y998F variant, and 73 for S991A. Thus, by this estimation the mutants missing one or another kinase domain proximal phosphorylation site were not generally impaired for phosphorylation. As shown in , there is evidence for phosphorylation at 18 sites on the receptor, including three Thr(P), six Tyr(P), and nine Ser(P). Additional information related to these assignments including protein search algorithm scores and MS/MS spectra are included in supplemental Table 3
and supplemental Fig. 2
. The low number of observed phosphopeptides was not surprising because no phosphopeptide enrichment was used in this analysis. For several sites, the number of observed spectra was too low to warrant further discussion.
EGFR phosphorylation site analysis suggests increased levels of EGF-induced phosphorylation compared with WT at Ser1039 and Thr1041 in the Y998F and S991A variants
Surprisingly in the region spanning EGFR residues 1000–1052 there was evidence for increased phosphorylation with the Y998F and S991A variants (see bold region in ). There were, respectively, 20 and 23 phosphorylated peptides identified by spectral counting for these variants but only six in the case of WT (). Most notable were phosphorylations at positions Thr1041
and especially Ser1039
for which spectral counts were higher in the two receptor variants than in WT (). Both of these phosphorylations were observed previously in EGF-stimulated HeLa cells (10
). These sites reside in the same tryptic peptide spanning residues 1032–1052. This peptide was also detected doubly phosphorylated at Ser(P)1039
, but the ion current intensities were relatively low (data not shown).
The monophosphorylated isobaric isomers containing either Ser(P)1039 or Thr(P)1041 were analyzed by LC-MS/MS, which confirmed their identities. For example, their y12 fragment ions were clearly distinctive (, A and B). The two species did not co-elute during reverse phase chromatography, and this facilitated their resolution and ion current measurement by high resolution LC-MS (C). To facilitate the quantification of these species by SRM, the measurement of transitions involving distinctive y ions was attempted but proved inefficient. Therefore, an optimized SRM method was developed that included neutral loss of phosphoric acid (i.e. doubly charged parent − 98/2) and y5-associated transitions, which were common to the two phosphoisomers and efficiently detected (, A, B, and D). Note that different elution gradients were used for the LC-MS and SRM analyses shown in , C and D, respectively, so the retention times were not identical. The Orbitrap MS extracted ion current chromatograph and triple quadrupole SRM traces were similar (, compare C and D), and several repeats of the LC-MS/MS experiment indicated that the Ser(P)1039 peptide consistently eluted immediately before the Thr(P)1041 isomer.
Fig. 7. Detection and quantification of phosphorylation at EGFR Ser1039 and Thr1041 by LC-MS/MS and SRM. A and B, trypsin-digested immuno (anti-FLAG) purified EGFR proteins were analyzed by high resolution LC-MS/MS. MS/MS spectra of the EGFR peptide TPLLSSLpS (more ...)
Having established the SRM method, it was applied to quantify phosphorylation at Ser1039
and address three questions: first, to determine whether these modifications were affected by EGF ligand; second, to determine whether they were elevated with the Y998F and S991A variant receptors compared with WT as implied by the semiquantitative spectral counting analysis shown in ; and third, to determine whether these modifications require p38 kinase. Stress (e.g.
TNFα or UV irradiation)-induced p38 kinase-dependent phosphorylation within the Ser/Thr-rich segment of the EGFR spanning residues 1026–1046, but not at Ser1039
specifically, was shown by Zwang and Yarden (17
). , E
, present a summary of three independent repeats of an experiment that addressed these questions. Stimulation of cells with EGF caused an ~3-fold elevation in the abundance of the Ser(P)1039
peptide (3.4 ± 1.3 S.D.) (E
), whereas both the Y998F and S991A variants showed a roughly 10-fold elevation in the abundance of the Ser(P)1039
peptide after EGF treatment. The pronounced EGF-stimulated increase in Ser(P)1039
was largely eliminated by treatment of cells with SB-202190, which is a potent inhibitor of MAP kinases p38α/β (23
), indicating that these phosphorylation sites are regulated by p38 kinase (E
). A similar phenomenon was observed for Thr(P)1041
wherein EGF treatment stimulated Tyr(P)1041
more than 2-fold in WT (2.7 ± 1.1 S.D.) but to an even greater extent with the variants (F
). The Thr1041
modifications were also sensitive to the p38 inhibitor (F