Requirement of Tyr-992 and Tyr-1173 in Phosphorylation of the Epidermal Growth Factor Receptor by Ionizing Radiation and Modulation by SHP2*

The epidermal growth factor receptor (EGFR) is activated by ionizing radiation (IR) in many human carcinomas, mediating a cytoprotective response and subsequent radioresistance. The underlying molecular mechanisms remain to be understood, and we propose here a specific role for the Tyr-992 residue of EGFR and examine its regulation by the phosphatase, SHP2. The -fold increase in phosphorylation of Tyr-992 in response to IR is twice that seen with ligand (EGF) binding. Mutation of Tyr-992 blocked completely IR-induced EGFR phosphorylation and reduced activation of the downstream signaling molecule, phospholipase Cγ. IR has previously been demonstrated to inhibit activity of protein-tyrosine phosphatases. Following protein-tyrosine phosphatase inhibition by sodium vanadate both EGFR expressing Chinese hamster ovary (CHO) and A431 exhibited up to an 8-fold increase in the basal level of Tyr-992 phosphorylation, significantly higher than that seen with Tyr-1173, Tyr-1068, and total EGFR Tyr. CHO cells expressing a SHP2 mutant also demonstrated up to an 8-fold increase in the basal level of Tyr-992 phosphorylation. In this study we show the unique association of SHP2 with EGFR in response to IR, with up to a 2.5-fold increase in the direct association of endogenous SHP2 with EGFR-wt in response to 2 gray of IR in both CHO and A431 cells. Mutation of Tyr-992 abolished this response. In conclusion we have identified several differentially activated Tyr residues, one of which is not only more sensitive to activation by IR, translating into differential activation of downstream signaling, but uniquely modulated by the phosphatase SHP2.

The epidermal growth factor receptor (EGFR) 1 is overexpressed in a variety of human tumors where it has been linked to radiation resistance and poor prognosis (reviewed by Ref. 1).
We, and others have shown EGFR to be activated by ionizing radiation (IR) resulting in cytoprotective downstream signaling primarily through the mitogen-activated protein kinase (MAPK) cascade, thus enhancing sensitivities of cancer cells to the toxic effects of IR (2). Overexpression of a dominant-negative EGFR mutant, EGFR-CD533, disrupts the cytoprotective response by preventing radiation-induced activation of the receptor and its downstream effectors, conferring radiosensitization of tumor cells (3). Although EGFR has been established as an important molecular target and prognostic marker in cancer therapy, the initial molecular events involved in EGFR activation by radiation are not well understood.
The general phosphorylation of EGFR Tyr residues by radiation is a well established phenomenon (2,3), but there are currently no reports showing the effects of radiation on any specific EGFR Tyr phosphorylation sites. Because the individual Tyr sites demonstrate differing affinities for the many adaptor molecules involved in downstream signaling from EGFR we are particularly interested in which of these sites respond specifically to IR rather than the ligand EGF. Thus, we have addressed the molecular mechanisms of EGFR activation by examining IR-and EGF-induced phosphorylation of specific EGFR Tyr residues in A431 squamous carcinoma cells and EGFR expressing CHO cells.
The EGFR autophosphorylation sites Tyr-992 and Tyr-1173 have been shown to play a critical role in the activation of the MAPK cascade following EGF stimulation. Tyr-992 is a highaffinity binding site for phospholipase C␥ (PLC␥), and is required for PLC␥ activation by EGF (4,5). We have also shown radiation-induced changes in free cytosolic (Ca ϩ2 ) to be dependent on PLC␥ (2,6,7). PLC␥ activates the Ras/MAPK cascade through inositol 1,4,5-triphosphate production and oscillations in cytosolic (Ca 2ϩ ) (1,7,8). Tyr-1173 serves as a major binding site for Shc, an adaptor protein involved in signaling between EGFR and Ras (9,10). In addition, Tyr-992 and Tyr-1173 are minor binding sites for Shc and PLC␥, respectively (10,11). Thus, these two Tyr residues are potentially involved in MAPK activation. In this study we have tested the role of the EGFR in radiation-induced PLC␥ and Shc phosphorylation using the specific EGFR kinase inhibitor, AG1478. We also evaluated the role of Tyr-992 and Tyr-1173 in these responses, using EGFR constructs specifically mutated (Tyr-Phe) in these residues.
Tyr-992 and Tyr-1173 have also been demonstrated to be binding sites for phosphatases (12,13), which play an important role in Tyr dephosphorylation and hence modulation of EGFR activity. In studies on the radiation-induced radicals on phosphatases, we previously measured radiation-induced reactive oxygen/nitrogen species (ROS/RNS) at the single cell level by fluorescence microscopy (14 -16). ROS/RNS generation oc-curred within seconds of radiation exposure (1-10 Gy) and persisted for 2-5 min after irradiation. ROS species, such as H 2 O 2 , have been shown to induce phosphorylation of the EGFR (4,5) and because H 2 O 2 also causes inhibition of protein-tyrosine phosphatase (PTP) activity (17)(18)(19)(20), it has been suggested that ROS/RNS-induced EGFR activation may, in part, be because of PTP inhibition (16,17). It is therefore possible that IR may also activate the EGFR by a similar mechanism of secondary ROS/RNS generation. Preliminary evidence for this includes the demonstration that radiation S-nitrosylates the active site Cys in the PTPs, SHP-1, and SHP-2 (16). Sequence specificity in recognition of the EGFR has been shown for PTP1B and SHP2 at Tyr-992 (13,21,22), and SHP-1 has been demonstrated to bind to the EGFR at Tyr-1173 (23). The modulation of EGFR phosphorylation by both of these phosphatases, however, has only been demonstrated in response to ligand binding. In this study we examined the modulation of EGFR by phosphatases, including the Tyr-992-specific phosphatase, SHP2. We also studied the interaction of SHP2 and EGFR in response to IR in both A431 and CHO cells expressing either wild type EGFR or its Tyr-992 mutation.  (24). These same constructs were subcloned into adenovirus (25).

Reagents-Unless
Cell Irradiation and EGF Treatment-Culture of A431 cells has been previously described (2). A431 cells were plated at 4.25 ϫ 10 5 cells in 6-cm dishes, incubated for 2 days at 37°C, and serum-starved for 16 -18 h (0.5% fetal bovine serum). Cells were then treated at 70 -80% confluence with 2 Gy ( 60 Co gamma rays) or 10 ng/ml EGF, as previously described (2). Chinese hamster ovary (CHO) cells were maintained in RPMI supplemented with 5% fetal bovine serum. They were plated at 6 ϫ 10 5 cells/6-cm dish and transfected with the appropriate DNA described below. Following a 24-h recovery period, cells were starved for 16 -18 h prior to IR or EGF treatment.
Transfection-CHO cells were plated at 6 ϫ 10 5 cells/6-cm dish, and transfected 24 h later with 1 g/6-cm dish of DNA (EGFR-wt, -Y992F, or -Y1173F mutants) using Lipofectamine PLUS (according to manufacturer instructions). For SHP2 experiments cells were transfected (as described previously) with 0.5 g of EGFR or Y992F mutant EGFR plus either carrier DNA, wt-SHP2 or c/s SHP2. Cells were lysed 24 and 48 h following transfection for Western blot analyses.
Adenoviral Infection-A431 cells were plated at 4.25 ϫ 10 5 cells/6-cm dish and cultured at 37°C for 2 days. Cells were exposed for 3 h to adenovirus containing lacZ, wild type SHP2, or a dominant negative c/s SHP2. The three viruses were tested at multiplicity of infections of 5, 10, and 50. Cells were lysed for Western blot analyses 24 and 48 h following infection.
Cell Lysis and Western Blotting-Cells were rinsed with ice-cold phosphate-buffered saline and snap-frozen on dry ice at the appropriate times after treatment. Cells were scraped into a lysis buffer (25 mM Tris, pH 7.4, 50 mM ␤-glycerophosphate, 1.5 mM EGTA, 0.5 mM EDTA, 5% glycerol, 1% Triton-X) containing protease inhibitors (1 mM sodium pyrophosphate, 1 mM sodium vanadate, 1 mM benzamidine, 10 g/ml leupeptin, 10 g/ml aprotinin, and 100 g/ml phenylmethylsulfonyl fluoride) and passed 5 times through a 20-gauge needle and syringe. Samples were centrifuged at 11,000 ϫ g for 15 min at 4°C, and supernatant protein concentrations were determined by the Bradford assay. For whole cell lysates, 5ϫ loading buffer (50 mM NaPO 4 , 5% SDS, 0.25% bromphenol blue, 12.5% 2-mercaptoethanol, and 10% glycerol) was added to lysates to achieve 1ϫ concentration; samples were then boiled for 5 min prior to Western blotting. Equal amounts of protein were fractionated on SDS, 6% polyacrylamide gels and protein was transferred electrophoretically onto nitrocellulose membranes. Membranes were probed with the appropriate primary and secondary antibodies. Blots were analyzed by chemiluminescence detection and densitometry.
Immunoprecipitation-Cells were lysed on ice immediately following treatment. 4 g of anti-EGFR (Ab-15), ERBB2 (Ab-11), or 2 g of anti-SHP2 antibody was added to 400 g of lysate in a total volume of 500 l. After mixing for 2 h at 4°C, 30 l of protein A/G-agarose beads were added to each lysate and mixed for 1 h at 4°C. Beads were rinsed three times with lysis buffer (plus inhibitors). 30 l of 2ϫ loading buffer was added to each sample before boiling for 10 min.

Ionizing Radiation and EGF Differentially Phosphorylate Specific Tyr Residues within EGFR-We have previously
shown that radiation activates the EGFR by general phosphorylation of certain Tyr residues, and that inhibition of EGFR phospho-Tyr results in radiosensitization (2,3). The purpose of this study was to examine specific EGFR Tyr residues with reported links to MAPK cascade activation, and their potential roles in radiation-induced EGFR activation and downstream signaling. To focus on individual phosphorylation sites, phospho-specific antibodies were used against various Tyr residues within the cytoplasmic domain of the EGFR known to be in involved the binding of adapter proteins involved in downstream signaling.
Examination of the phosphorylation pattern of individual Tyr residues identified three distinct categories. The first consisted of residues unaffected by either IR or EGF. These residues, e.g. Tyr-1068 and Tyr-1086, exhibited a relatively high basal level of phosphorylation that remained unchanged after exposure to 2 Gy of IR or 10 ng/ml EGF ( Fig. 1A; Table I).
The second category consisted of Tyr residues more highly phosphorylated by EGF than IR, paralleling the Tyr phosphorylation response of the EGFR as a whole. This included Tyr-845, Tyr-1045, and Tyr-1173. Tyr-845 demonstrated a 11.6-fold increase in phosphorylation compared with the untreated control in response to EGF but only a 6.3-fold increase in response to IR. This results in a 2 Gy/EGF (10 ng/ml) ratio of 0.7 Ϯ 0.1 (Table I). Similarly, phosphorylation of Tyr-1045 was increased 10.7-fold by EGF but only 5.2-fold by IR (Fig. 1B), resulting in a 2 Gy/EGF ratio of 0.5 Ϯ 0.2. Tyr-1173 exhibited a higher basal level of phosphorylation relative to the other Tyr residues, and this was increased 5.7-fold by EGF and 2.7-fold by IR, resulting in a 2 Gy/EGF ratio of 0.6 Ϯ 0.09 (see Table I).
Finally we identified one residue, Tyr-992, which was more highly phosphorylated by IR than EGF. IR induced a 10.9-fold increase in Tyr-992 phosphorylation, whereas EGF only induced a 5.8-fold increase. The consequent 2 Gy/EGF ratio was 2.1 Ϯ 0.3 ( Fig. 1C and Table I).
In time course experiments total EGFR Tyr phosphorylation (Tyr(P)) was increased by up to 3-fold over that of an untreated control by 2 Gy IR in A431 human carcinoma cells (Fig. 2). The peak in phosphorylation occurred at 2 min following irradiation and decreased over the subsequent 8 min. IR induced an almost 6-fold increase in Tyr-992 phosphorylation over that seen in the untreated control and similar to total Tyr(P) of EGFR, phosphorylation peaked at 2 min following IR exposure and decreased over the next 8 min. Exposure to concentrations of EGF (10 ng/ml) that provide comparable changes in EGFR phosphorylation increased total Tyr(P) by up to 6-fold peaking at 5 min, but Tyr(P)-992 was only increased 3-fold (Fig. 2). The kinetics of ligand-induced phosphorylation of Tyr-992 were similar to total Tyr(P), peaking at 5 min and decreasing over the next 8 min. Treatment times for subsequent experiments were chosen to reflect the times of maximal receptor phosphorylation of 2 min for IR and 5 min for EGF (Fig. 2).
Tyr-992 and Tyr-1173 Are Required for Radiation-induced Activation of EGFR-We demonstrated a significant radiationinduced phosphorylation of Tyr-992 and Tyr-1173 in A431 cells. The functional importance of these two residues was examined in CHO cells, which do not express EGFR-wt, and are also easily transfected. CHO cells expressing EGFR-wt were compared with cells expressing either EGFR-Y992F or -Y1173F mutants. The pattern of total phosphorylation induced in cells expressing EGFR-wt was similar to that observed in A431 cells.
Total Tyr phosphorylation was increased 3.1-fold in response to IR and 6.1-fold in response to EGF (Fig. 3A).
Expression of either mutant abolished the radiation-induced increase in total Tyr phosphorylation, whereas the EGF-induced increase was only reduced by 50% (Fig. 3A). As expected, no significant signal relative to control levels was observed when the antibody against Tyr-992 (Fig. 3B) or Tyr-1173 ( Fig.  3C) was used in combination with cells expressing the corresponding mutants, verifying the specificity of both mutant and antibody. Although CHO cells expressing EGFR-wt showed both radiation-and EGF-induced increases in Tyr-992 phosphorylation, the effect of radiation on Tyr-992 relative to Tyr-1173 was not as pronounced in CHO cells as in A431 cells.
EGFR Dependence of Radiation-induced PLC␥ and Shc Phosphorylation in A431 Cells-Because PLC␥ and Shc have been shown to bind to Tyr-992 and Tyr-1173 with subsequent activation of the MAPK cascade (4, 5, 7-10), we tested whether the radiation-induced phosphorylation of these effectors was dependent upon EGFR. In addition, phosphorylation of Src was examined, because Src-mediated phosphorylation of the EGFR at Tyr-845 has been shown to modulate receptor function (26). Previous studies have shown that critical phosphorylation sites for activation of PLC␥1 and Shc are Tyr-783 and Tyr-317, respectively (27,28); thus, the phosphorylation of these particular sites was examined.
Using the same time and dose strategies as before, a mean 6.2-fold induction of PLC␥ phosphorylation was observed following radiation treatment (Fig. 4). Increases in phosphorylation of Shc family members were observed following irradiation; the mean -fold changes (n ϭ 3) were 2.3, 3.0, and 3.5 for the 66-, 52-, and 46-kDa isoforms, respectively. For both PLC␥ and Shc, EGF at 10 ng/ml induced a significantly greater increase in phosphorylation than IR (p Ͻ 0.05). Pretreatment of cells with 500 nM AG1478, a specific inhibitor of EGFR Tyr phosphorylation (29), abolished the radiation-and EGF-induced activation of both PLC␥ and Shc in A431 cells. Levels of total PLC␥ and Shc were not significantly altered by AG1478. Phosphorylation of Src at Tyr-416, a measure of Src activation, was not modulated by radiation, EGF, or AG1478. Thus, EGFR activation is essential for phosphorylation of PLC␥ and Shc, but not Src, in response to both IR and EGF in A431 cells.
Mutation of either Tyr-992 or Tyr-1173 reduced both IR-and EGF-induced Tyr phosphorylation of PLC␥. Total PLC␥ was unaffected (Fig. 4B). This data indicates that Tyr-992 and Tyr-1173 are important for EGFR activation of PLC␥.
FIG. 1. Ionizing radiation and EGF differentially phosphorylate specific tyrosine residues within EGFR (EGFR). A431 human carcinoma cells were exposed to 2 Gy IR or EGF (10 ng/ml), snap-frozen at 2 min (IR) or 5 min (EGF), and whole cell lysates were generated. EGFR was immunoprecipitated and Western blot used to detect phosphorylation at Tyr-1068 and -1086 (A), Tyr-845, -1045, and -1173 (B), and Tyr-992 (C). Blots were stripped and re-probed for total EGFR to verify equal loading (bottom panels for each Tyr); all bands shown are 170 kDa.

Inhibition of Protein-tyrosine Phosphatases Enhances the Basal Tyr Phosphorylation of EGFR in Both A431 and EGFRexpressing CHO Cells with Tyr-992 Being the Most Signifi-
cantly Affected-We demonstrated previously that radiationinduced EGFR phosphorylation is tightly regulated, peaking at 2 min following IR and recovering to basal levels over the next 10 -30 min (Fig. 2). Phosphatases catalyze the dephosphorylation of Tyr-phosphorylated proteins either potentiating or antagonizing downstream cellular signaling. PTPs have been previously implicated in the dephosphorylation of EGFR (21,23,30,31) with sequence specificity being evident for PTP1B and SHP2 at Tyr-992 (12,13,21,22), and SHP-1 at Tyr-1173 (23). One mechanism by which IR may promote sustained EGFR phosphorylation is by the disruption of dephosphorylation via inhibition of phosphatases. Previous studies demonstrated that therapeutic doses of IR result in the transient S-nitrosylation of the active site Cys of SHP1 and SHP2 with consequent inhibition of their catalytic activities (16). 2 To test this hypothesis we used sodium vanadate to inhibit general phosphatase activity in both EGFR expressing CHO and A431 cells. CHO cells cultured in the presence of 100 M sodium vanadate demonstrated enhanced and sustained EGFR Tyr phosphorylation. Total EGFR Tyr phosphorylation was increased 6-fold in sodium vanadate-treated CHO cells over that seen in the untreated control (Fig. 5A).
The vanadate-stimulated phosphorylation kinetics of Tyr-1068, Tyr-1173, and Tyr-992, one residue from each of the three categories described earlier were also examined in CHO cells. The phosphorylation profiles of Tyr-1068 and Tyr-1173 matched that seen for total EGFR Tyr phosphorylation. Basal Tyr phosphorylation increased up to 10 min following introduc-

FIG. 4. EGFR dependence of radiation-induced PLC␥ and Shc phosphorylation.
A, cells were pretreated with 500 M AG1478 for 1 h prior to IR or EGF treatment. Whole cell lysates were subjected to Western blot analyses using phospho-specific antibodies against PLC␥1 (Tyr-783), Shc (Tyr-317), or Src (Tyr-416). Blots were then stripped and reprobed for total PLC␥1, Shc, or Src. A representative of three independent experiments is shown. B, CHO cells transfected with EGFR-wt, EGFR-Y992F, or EGFR-Y1173F were exposed to 10 ng/ml EGF for 5 min or 2 Gy IR and harvested 2 min later. Cell lysates were subjected to Western blot and analyzed using a phospho-specific PLC␥ antibody. Total PLC␥ and EGFR are shown as transfection and loading controls (con). Representative blots from two independent experiments are shown. tion of sodium vanadate and was sustained for the 30-min duration of the study (Fig. 5A). The magnitude of this increase in basal phosphorylation, however, was significantly greater for Tyr-992. In post-hoc pairwise comparisons of the areas under each curve (Turkey method to adjust for multiple testing), Tyr-992 phosphorylation was shown to be significantly more sensitive to inhibition of phosphatases by sodium vanadate than either Tyr-1068 or Tyr-1173 (p ϭ 0.0073, and 0.01 respectively). Total Tyr, Tyr-1173, and Tyr-1068 were not sig-nificantly different to each other (p values ranging from 0.72 to 0.98).
Similarly in A431 cells Tyr-992 was significantly more sensitive to phosphatase inhibition than Tyr-1173, Tyr-1068, or total receptor Tyr (Fig. 5B, p ϭ 0.0013, 0.0022, and 0.0008, respectively). In the presence of 100 M sodium vanadate basal Tyr-992 phosphorylation was increased up to 8.6-fold, whereas both Tyr-1173 and total EGFR Tyr phosphorylation were only increased up to 3-fold. Tyr-1068 remained unchanged.  5. Tyr-992 of EGFR is significantly more sensitive to phosphatase inhibition than any of the other tyrosine residues. EGFR expressing CHO (A) and A431 (B) cells were starved for 16 h prior to sodium vanadate treatment. Cells were exposed to 100 M sodium vanadate for 5, 10, 20, and 30 min prior to lysis and Western blot. EGFR phosphorylation was determined using phospho-specific antibodies for Tyr-992, -1173, -1068, and total Tyr. Results are shown as scaled mean -fold change in phosphorylation as compared with an untreated control ϮS.E. (n ϭ 3). To remove the variability because of replication, the -fold change values were scaled. Within a given replication the area under each curve was determined. The maximum of these values within each replication was determined. Each -fold value was divided by the maximum area within replication.

Inhibition of SHP2 Enhances Basal Phosphorylation of EGFR with Tyr-992 Being Significantly More Sensitive Than
Total Tyr and Tyr-1173-SHP2 specifically has been shown to bind and dephosphorylate Tyr-992 in response to EGF (13), discriminating between EGFR and the other Tyr kinase receptors (31). Because IR in the therapeutic dose range has been shown to nitrosylate SHP2 with a consequent reduction in its phosphatase activity (16), 2 we examined the role of SHP2 in regulation of EGFR phosphorylation. To do this we utilized a dominant negative SHP2 construct (c/s SHP2) mutated in its phosphatase active site.
CHO cells co-transfected with EGFR-wt and dominant negative SHP2 demonstrated enhanced phosphorylation of EGFR. Total Tyr phosphorylation was increased up to 2-fold over that seen in the control, whereas phosphorylation of Tyr-1173 was enhanced up to 2.5-fold (Fig. 6A). Phosphorylation of Tyr-992 was much more significantly affected, with an up to an 8-fold increase in phosphorylation over that of the mock-transfected control (p Ͻ 0.0001). In contrast overexpression of wt-SHP2 induced a 3-fold decrease in EGFR Tyr-992 phosphorylation. Phosphorylation of Tyr-1173 and EGFR were not significantly affected by overexpression of wt-SHP2. This data suggests that Tyr-992 is the most sensitive of the Tyr residues within EGFR to changes in phosphatase activity.
Similarly in A431 cells (Fig. 6B) Tyr-992 was significantly affected by knock-out of SHP2. Cells infected with a dominant negative SHP2 adenovirus exhibited an average 2.6-fold increase in Tyr-992 phosphorylation compared with a LacZ-infected control (p ϭ 0.014). The phosphorylation of Tyr-1068 and -1173 remained unchanged. Unlike the CHO cells the wild type SHP2 expressing virus did not have a significant effect on the basal phosphorylation of Tyr-992. This may in part be explained by the endogenous expression of SHP2 in these cells. These cells may already express saturating levels of wt-SHP2 in which case they cannot respond further to the additional wt-SHP2 to which we are subjecting them. Previous studies also noted a lack of effect with the wt-SHP2 on global tyrosine phosphorylation of EGFR (12), reporting it to be because of the relatively few phospho-Tyr targets. The effect of the wt-SHP could therefore be masked by phosphorylation of Tyr residues unaffected by SHP2.
IR Enhances SHP2 Association with EGFR, an Interaction That Is Dependent on Tyr-992-To examine the physical interaction between SHP2 and EGFR we exposed EGFR expressing CHO cells to 2 Gy of IR and immunoprecipitated EGFR and SHP2. We then determined the presence of associated endogenous SHP2 and EGFR, respectively, by Western blot. We found SHP2 association with EGFR to increase significantly in response to 2 Gy. Up to a 2.6-fold increase over that seen in the untreated control was observed at 5 min following IR (Fig. 7A; p ϭ 0.01). This peak correlated with the previously observed decrease in EGFR Tyr phosphorylation supporting the hypothesis that SHP2 was involved in regulation of EGFR phosphorylation following IR. At 60 min following exposure to IR, SHP2 association had decreased to levels not significantly different to those seen in the untreated controls (p ϭ 0.34).
In contrast, CHO cells expressing the Tyr-992 mutant of EGFR did not demonstrate an IR-induced increase in SHP2 association with EGFR (Fig. 7A), p Ͼ 0.9999 at 5 min following irradiation. This data suggests that Tyr-992 is critical for IRinduced association of SHP2 and EGFR.
Similarly in A431 cells 2 Gy of IR induced a significant increase in SHP2 association with EGFR (Fig. 6B). As with CHO cells, SHP2 association with EGFR peaked at 10 min following IR with a 1.8-fold increase over the untreated control (p ϭ 0.0001).

DISCUSSION
Earlier work established a fundamental role for EGFR in tumor cell resistance to IR via cytoprotective cell signaling (1). EGFR is phosphorylated in response to IR with consequent stimulation of signaling via the pro-proliferative and anti-apoptotic MAPK/PI3K and AKT pathways (1,32,33). Although several Tyr residues have been identified within the kinase domain of EGFR, to date no differentiation has been made between ligand-induced phosphorylation and that induced by IR. Because there have been various studies outlining the specificity of these Tyr residues for the different adaptor molecules and consequently downstream signaling from EGFR we quantified the phosphorylation of EGFR at the individual Tyr residues in response to EGF and IR.
In the present study we clearly defined three groups of Tyr residues within the kinase domain of EGFR. The first, which included Tyr-1068 and -1086, appeared completely unaffected by either radiation or EGF, possibly reflecting high constitutive phosphorylation levels. Previous work demonstrated Tyr-1068 to directly bind Grb2, an exchange factor involved in Ras stimulation, in response to EGF binding. A similar finding was reported with Tyr-1086 but to a lesser extent (10). The second group, including Tyr-845, Tyr-1045, and Tyr-1173, although responsive to both EGF and radiation, exhibited a greater response to EGF than to radiation. Tyr-845 has been shown to be phosphorylated by Src, and this phosphorylation is involved in the regulation of EGF-stimulated DNA synthesis (26). Phosphorylation of Tyr-1045 creates a major docking site for c-Cbl, which binds to the activated EGFR (34). This leads to the assembly of the ubiquitination machinery on the receptor, en- abling receptor ubiquitination and consecutive degradation. Thus, exposure of cells to EGF and IR may also promote EGFR degradation as a negative feedback mechanism. Although Tyr-845 and Tyr-1045 were phosphorylated in response to both EGF and radiation they have not been directly associated with MAPK activation. Tyr-1173, however, has been shown to be a functional binding site for PLC␥ and Shc (4, 5, 9 -11), both of which could potentially mediate the activation of MAPK by EGF and IR (2).
The final group consists of one residue, Tyr-992, which was more highly phosphorylated in response to IR than EGF. Tyr-992 has also been shown to bind PLC␥ and is believed to be the major site involved in activation of PLC␥ (4,5). This is consistent with the finding that mutation of residue Tyr-992 com-pletely abolished radiation-induced EGFR phosphorylation, whereas it only blocked EGF-induced phosphorylation by 50%. These studies implicate Tyr-992 as an important effector in the radiation-induced activation of EGFR and downstream pathways. Tyr-1173 also contributes to the general induction of EGFR phosphorylation and mutation of Tyr-1173 also eliminated the radiation-induced increase in Tyr phosphorylation, indicating an important role for this molecule in radiationinduced signaling from EGFR. In A431 cells, however, this residue was more significantly affected by EGF than IR, suggesting that in the presence of the other ERBB molecules it is not as IR-specific as Tyr-992. We have shown previously that EGFR in tumor cells exists in both homo-and heterodimers with itself and ERBB2 or ERBB4 (35). Therefore the magnitude of EGFR phosphorylation in response to IR may well rely on heterodimerization with ERBB2 and/or ERBB4.
Another interesting observation is the increase in basal phosphorylation of EGFR when Tyr-992 or -1173 are mutated. This is not surprising when one considers the specificity of these two sites for binding of PTPs. Studies of RTKs, including EGFR, demonstrate that the overall phosphorylation state in unstimulated cells is a net result of basal RTK and PTP activities (35,36). Because the catalytic activity of PTPs can be up to 1000-fold greater than that of kinases (37), perturbation of PTP activity may have a significantly more profound effect on signal propagation than that of kinases. It has also been shown, at least in response to ROS, that activation of EGFR is coupled to the inhibition of PTP via hydrogen peroxide production with a consequent amplification of ligand-induced activation (17)(18)(19)(20).
In accordance with the minimal reaction network described by Reynolds et al. (37) inhibition of endogenous PTP activity by sodium vanadate increased the basal phosphorylation of EGFR in both EGFR expressing CHO and A431 cells. In cells overexpressing EGFR, constitutive basal phosphorylation of EGFR is commonly observed, and is reported to be because of autocrine stimulation by ligands such as transforming growth factor-␣. The consequent survival signaling supports a cellular growth advantage in EGFR overexpressing tumor cells. We have clearly demonstrated in this study that this phosphorylation is particularly sensitive to changes in phosphatase activity.
The unique finding in our study was the much more dramatic effect that PTP inhibition had on Tyr-992 compared with the other residues. This was evident in both EGFR expressing CHO cells and A431 cells in which this residue is critical for IR-induced phosphorylation. Consequently we focused our attention on the Tyr-992-specific PTP, SHP2, and found inhibition of endogenous SHP2 activity to enhance EGFR phosphorylation. Again Tyr-992 was significantly more sensitive than either Tyr-1173 or total receptor Tyr phosphorylation. This data suggests that not only is Tyr-992 specifically involved in IR-induced signaling from EGFR but it is uniquely modulated by PTPs, particularly SHP2. Although we corroborated the finding that Tyr-992 is essential for SHP2 action (13), previous data all pertains to ligand-induced EGFR stimulation. In our study we demonstrated a unique association between SHP2 and EGFR Tyr-992 in response to therapeutically applied doses of IR with the peak association occurring at a time when we observe the most rapid dephosphorylation of EGFR. This association, however, is maintained for up to 20 min following dephosphorylation of the receptor. This may reflect a dual role for SHP2 as both a phosphatase and an adaptor molecule. In addition to a phosphatase domain, responsible for recognition and dephosphorylation of the phosphorylated Tyr residue, SHP2 also has both C-and N-terminal SH2 domains (39,40). Both of these sites face away from the active phosphatase domain and are potentially available to interact with other FIG. 7. Endogenous SHP2 associates with EGFR in response to ionizing radiation, an association dependent on Tyr-992. A, wild type or Y992F mutant EGFR expressing CHO cells were starved for 16 h prior to exposure to 2 Gy IR and lysed at 2, 5, 10, 30, and 60 min post-irradiation. EGFR and SHP2 were independently immunoprecipitated and subsequent Western blots probed for SHP2 and EGFR, respectively. Quantification data are shown as -fold change in SHP2 association as compared with an untreated control. Results are shown as mean -fold change in SHP2 association ϮS.E. (n ϭ 3). p values were determined using an unpaired t test. B, A431 cells were starved for 16 h prior to exposure to 2 Gy IR and lysed at 2, 5, 10, 30, and 60 min post-irradiation. EGFR was immunoprecipitated from cell lysates and subsequent Western blots probed for SHP2. Quantification data are shown as mean -fold change in SHP2 association ϮS.E. (n ϭ 3). p values were determined using an unpaired t test.
phosphopeptides. The N-terminal domain seems to be particularly important in modulation of SHP2 phosphatase activity by directly blocking the PTP active site. Catalytic activity increases dramatically upon occupancy of the N-terminal SH2 domain because of release of the PTP domain. Because the same is true in reverse, we hypothesize that upon binding of the PTP domain to phosphorylated Tyr-992 the N-terminal SH2 domain is released to bind to other signaling adaptor molecules. This is supported by a study of SHP2 mutations in Noonan syndrome (41). Mutations within the N-terminal SH2 domain or the phosphatase domain at the interface between the two sites not only enhances phosphatase activity but also prolongs binding to the adaptor molecule GAB2. Unfortunately trapping mutants of SHP2 have only been developed to identify substrates of the phosphatase domain and not the SH2 domain (12). The role of SHP2 as an adaptor molecule in IR-induced signaling through EGFR therefore requires further study.
We have previously shown that therapeutic doses of radiation transiently stimulate a constitutive, Ca 2ϩ -dependent NOS-1 activity inhibitable pharmacologically or by expression of a dominant negative mutant of NOS-1 (15). These studies further demonstrate that the transient stimulation of MAPK activity commonly observed in epithelial cells following radiation exposure requires NOS activity (15). We speculated that one possible mechanism involves the S-nitrosylation of active site Cys and resulting inhibition of protein-tyrosine phosphatases. Blocking the Tyr phosphatases responsible for either inhibiting Tyr kinase receptors that activate MAPK or Tyr phosphatases that inhibit MAPK downstream (e.g. MKP1) would result in a net MAPK activation. Preliminary evidence for this proposal came from the demonstration that SHP2 (and SHP1) when transiently overexpressed in CHO cells are S-nitrosylated in their active site Cys in response to therapeutic doses of IR (16), with consequent inhibition of their phosphatase activities. 2 These data in addition to the demonstrated interaction of SHP2 with EGFR suggest a novel mechanism by which IR may potentiate its own resistance. IR induces S-nitrosylation and subsequent loss of activity of SHP2, a specific modulator of EGFR Tyr-992 phosphorylation, with consequent up-regulation of downstream survival signaling enabling cells to resist the cytotoxic effects of IR. The effect of phosphatase inhibition on cell survival following IR will likely yield novel information regarding the molecular mechanisms of IRinduced receptor activation.
In conclusion we have uncovered a much more complex mechanism of EGFR activation by IR than previously described. We have identified several differentially activated Tyr residues, one of which is not only more sensitive to activation by IR but uniquely modulated by the phosphatase SHP2. Stabilization of this very specific interaction could have important therapeutic implications in terms of radiosensitization of tumors through specifically targeted drugs.