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[Cancer Research 63, 3923-3930, July 15, 2003]
© 2003 American Association for Cancer Research

Biochemistry and Biophysics

Identification and Characterization of Signal Transducer and Activator of Transcription 3 Recruitment Sites within the Epidermal Growth Factor Receptor1

Huang Shao, Haiyun Y. Cheng, Richard G. Cook and David J. Tweardy2

Section of Infectious Diseases, Department of Medicine [H. S., D. J. T.], Department of Pathology [H. Y. C.], and Department of Immunology [R. G. C.], Baylor College of Medicine, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The oncogene signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a wide variety of human cancers, including squamous cell carcinoma of the head and neck. In squamous cell carcinoma of the head and neck, Stat3 activation is mediated by up-regulation of the autocrine ligand-receptor pair, tumor growth factor {alpha} and epidermal growth factor receptor (EGFR), resulting in cell growth and resistance to apoptosis. The initiating molecular event in Stat3 activation is recruitment to specific phosphotyrosine motifs within signaling complexes. Stat3 activation by the EGFR has been mapped to the COOH-terminal region of the EGFR between amino acid residues 1061 and 1123, which contains Y1068 and Y1086. However, it is not known if Stat3 binds directly to the EGFR or if either of these tyrosines is involved in this interaction. In this study, we demonstrated in stably transfected NIH-3T3 cells that activation of Stat3 by EGFR was eliminated by mutation of all five EGFR tyrosines to phenylalanine and that activation was restored with return of two of the mutated tyrosine sites, Y1068 and Y1086, to wild-type. Stat3 was detected in the activated EGFR complex, and its presence within the complex was dependent on Y1068 and/or Y1086. Phosphododecapeptides spanning Y1068 and Y1086 were able to pull down Stat3 with Y1068 being more effective than Y1086 in this regard. Real-time mirror resonance affinity analysis revealed Stat3 bound to phosphododecapeptide Y1068 with a KD of 135 ± 20 nM and to phosphododecapeptide Y1086 with a KD of 243 ± 36 nM (P = 0.044), consistent with the results of the pull-down assays. The lower KD of Y1068 was completely attributable to slower dissociation of Stat3 bound to Y1068 versus Y1086. Each phosphododecapeptide was capable of destabilizing Stat3 homodimers in vitro. When delivered into squamous carcinoma cells, phosphopeptides spanning Y1068 and Y1086 were able to inhibit EGFR-stimulated Stat3 DNA binding activity and cell proliferation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The EGFR3 family consists of four members: EGFR, ErbB2, ErbB3, and ErbB4 (1) . The members of EGFR family, particularly EGFR and ErbB2, are implicated in various forms of human cancers and serve as both prognostic markers and therapeutic targets. EGFR contains an extracellular ligand-binding domain, a single transmembrane region and an intracellular domain harboring intrinsic tyrosine kinase activity (2) . Activation of the receptor tyrosine kinase requires ligand-induced dimerization that allows reciprocal transphosphorylation of residues within the catalytic domain leading to enzymatic activation and autophosphorylation of cytoplasmic tyrosine residues. Five autophosphorylation sites have been identified in EGFR: Y992, Y1068, Y1086, Y1148, and Y1173 (3 , 4) . These phosphorylated tyrosine residues serve as docking sites for signal proteins containing SH2 domains, including phospholipase C-{gamma} (5 , 6) , SHP-1 (7) , Grb-2 (8 , 9) , and Shc (10) .

STAT3 is a SH2 domain-containing transcription factor that is latent in the cytoplasm of cells until stimulation with cytokines and growth factors, including EGF or TGF-{alpha} (11 , 12) . When constitutively activated, Stat3 is oncogenic (13) . Stat3 has been demonstrated to be required for transformation of fibroblasts by v-Src (14 , 15) and for autocrine growth of SCCHN (12) . Constitutive activation of Stat3 has been detected in a wide variety of other cancers, including breast, prostate, renal cell, melanoma, ovarian, lung, leukemia, lymphoma, and multiple myeloma (16) .

Upon ligand-induced kinase activation and protein tyrosine phosphorylation, Stat3 is recruited via its SH2 domain to tyrosine-phosphorylated motifs within receptor complexes and is itself phosphorylated on tyrosine 705 within its COOH terminus. Phosphorylation of Stat3 leads to its dimerization, nuclear translocation, binding to specific DNA sequences, and up-regulation of target gene expression. Activation of Stat3 in SCCHN cells involves activation of EGFR by autocrine production of TGF-{alpha} (12 , 17) . Our understanding of the molecular details of its recruitment and activation by the EGFR are incomplete.

Activation of Stat3 by EGFR requires an intact receptor tyrosine kinase domain. Cell lines deficient in each of the known Jak kinases still demonstrated Stat3 activation in response to EGF (18) , and cells expressing kinase-deficient EGFR mutants were incapable of activating Stat3 after EGF stimulation (19, 20, 21) . Furthermore, EGFR produced in recombinant baculovirus-infected insect cells associated with Stat3 and phosphorylated it on tyrosine 705 in vitro (22) . Using truncated mutants of EGFR stably expressed in NIH-3T3 cells, activation of Stat3 mapped to the region of EGFR between amino acid residues 1061 and 1123 (19) . This region contains two of five tyrosines (Y1068 and Y1086) within the cytoplasmic portion of the EGFR that are autophosphorylated upon ligand binding. However, whether Stat3 binds directly to either of these sites or to another motif within this region or whether it binds indirectly to EGFR through another protein is not known.

In this study, we demonstrated that activation of Stat3 by EGFR was eliminated by mutation of all five EGFR tyrosines to phenylalanine and that activation was restored with return of two of the mutated tyrosine sites, Y1068 and Y1086, to wild-type. Stat3 was detected in the activated EGFR complex, and its recruitment was dependent on Y1068 and/or Y1086. Phosphopeptides spanning Y1068 and Y1086 were able to pull down Stat3 and real-time mirror resonance affinity analysis revealed a KD of 135 ± 20 and 243 ± 36 nM, respectively. Each phosphopeptide destabilized Stat3 homodimers, and when delivered into squamous carcinoma, cell lines inhibited ligand-stimulated Stat3 DNA binding activity and cell proliferation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of EGFR Expression Plasmids and Site-directed Mutagenesis.
Human wild-type EGFR cDNA was kindly provided by Dr. Yoshinori Okabayashi (10) . Mutated EGFR constructions were accomplished by site-directed mutagenesis (Stratagene). The 5F construct (Fig. 1A)Citation was generated by making tyrosine-to-phenylalanine substitutions at amino acids 992, 1068, 1086, 1148, and 1173. The TM construct was made by reverting sites 1068 and 1086 back to tyrosine. The HindIII/XbaI fragment containing wild-type or mutated EGFR cDNA was subcloned into expression vector pcDNA3.1+Zeo. The sequence of each construct was verified by sequencing analysis.



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  		Fig. 1. Phosphorylation and activation of Stat3 in stable cell lines expressing EGFR and mutants. A, schematic representation of the EGFR and the EGFR constructs examined. B, expression of EGFR constructs in NIH-3T3 cell lines determined by immunoblotting. Lysates of equivalent amounts of NIH-3T3 cells before stable transfection (NIH-3T3) and after stable transfection with plasmid vector alone (Vector), or the indicated EGFR constructs were separated by SDS-PAGE, blotted, and probed with EGFR-specific antibody. The location of the EGFR band and molecular weight markers are indicated. C, activation of Stat3 in whole-cell extracts of NIH-3T3 cell lines determined by EMSA. Equivalent numbers of NIH-3T3 cells were incubated with (+) or without (-) TGF-{alpha} for 20 min. Whole-cell extracts were incubated in binding reactions with hSIE duplex oligonucleotide. Reaction samples were separated by electrophoresis and autoradiographed. D, NIH-3T3 cell lysates from an equivalent number of cells were separated by SDS-PAGE, blotted onto membrane, and probed with mAb specific for Stat3 phosphotyrosine 705 (top panel). After stripping, the membrane was incubated with mAb specific for the NH2 terminus of Stat3 (bottom panel). E, supershift analysis of whole-cell extracts of NIH-3T3 cells. Equivalent amounts of whole-cell extract of the indicated NIH-3T3 cells incubated with (+) or without (-) TGF-{alpha} for 20 min were incubated in binding reactions without or with antibodies capable of supershifting Stat1 or Stat3 as indicated. Reaction samples were separated by electrophoresis and autoradiographed. The results shown are representative of two or more experiments.

 
Reagents and Cell Lines.
IL-6 and TGF-{alpha} were purchased from R&D. Polyclonal antibody against EGFR was obtained from Santa Cruz Biotechnology, Inc. mAb against Stat3 and EGFR were purchased from Transduction Laboratories. Protein G and protein A-Sepharose were obtained from Sigma. The cell lines of NIH-3T3, HepG2, and A431 were obtained from the American Type Culture Collection. The SCCHN cell line, UM-SCC-23 (23) , was kindly provided by Dr. Thomas Carey (University of Michigan). Cells were grown in DMEM supplemented with 10% fetal bovine serum. NIH3T3 cells, shown previously to be devoid of endogenous EGFR (24) , were transfected with 5 µg of plasmids using Fugene 6 (Roche). For stable transfections, the clones were selected using Zeocin at 350 µg/ml for 14–20 days. The pooled resistant clones were additionally sorted by EGFR antibody (Upstate) through flow cytometry.

EMSA.
Cells starved in serum-free medium for 4 h were stimulated by cytokine at 37°C for 20 min. Where indicated, cells were transiently transfected with human Stat3-{alpha} cDNA 48 h before ligand stimulation. Whole-cell extracts were prepared, and EMSA was performed on 4.5% native polyacrylamide gels using hSIE as a probe as described previously (25) . Inhibition studies using phosphorylated and nonphosphorylated dodecapeptides were performed by incubating whole-cell extract with peptides at various concentrations at 37°C for 1 h before addition of radiolabeled hSIE as described previously (25) .

Immunoprecipitation and Immunoblotting.
NIH-3T3 cells expressing wild-type and mutated EGFR were starved in serum-free DMEM for 18 h and then stimulated with TGF-{alpha} at 37°C for 20 min. Cells were lysed by ultrasonification in buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Clarified lysate was incubated with anti-EGFR antibody (Santa Cruz Biotechnology) at 4°C for 1 h and mixed with protein G-Sephorose (Sigma) for 1 h. Immunoprecipitates were washed three times with radioimmune precipitation assay buffer (50 mM Tris-HCl, 150 mM NaCl, % Triton X-100, 0.1%SDS, 1 mM EDTA, and 1% sodium deoxycholate). Bound proteins were boiled in SDS-PAGE sample buffer for 5 min and separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes.

Expression and Purification of Stat3 Protein.
Human Stat3-{alpha} and Stat3-ß cDNA were provided by Dr. Rolf Van de Groot (26) . A HindIII/XhoI DNA fragment containing Stat3-{alpha} was cloned into the baculovirus expression vector, pFastBac1 (Invitrogen, Life Technologies, Inc.) with a 6-histidine tag engineered onto the NH2 terminus of human Stat3. The recombinant plasmid was used to transform DH10Bac-competent cells that contain the bacmid with a mini-attTn7 target site and a helper plasmid. Recombinant bacmids were prepared and used to infect Sf9 cells. Sf9 cells (3 x 106 cells/ml) were infected with Stat3 recombinant virus at a multiplicity of infection of 0.05 and harvested after 3 days of culture. Cells (6 x 108) were suspended in 12 ml of precooled lysis buffer [20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 10 mM imidazole] and lysed by ultrasonification on ice. Lysates were centrifuged at 15,000 x g for 30 min at 4°C, and the supernatant was incubated with Ni-NTA-agarose (Qiagen) at 4°C for 1 h. The mixture of lysate and Ni-NTA resin was washed twice with four volumes of wash buffer [20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 20 mM imidazole] to remove unbound proteins. Stat3 was eluted from the Ni-NTA resin with elution buffer [20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 250 mM imidazole]. The purified proteins were dialyzed against 10 mM PBS containing 5 mM NaF and 1 mM Na3VO4 at 4°C and stored at -80°C.

Affinity-binding Assay of EGFR to Stat3.
A431 cells or NIH-3T3 cells expressing EGFR constructs were stimulated with 35 ng/ml TGF-{alpha} for 20 min and then lysed in 0.5 ml of lysis buffer [50 mM Tris-HCl (pH 7.5), 5 mM EGTA, 150 mM NaCl, 1% Triton X-100, 2 mM Na3VO4, 50 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride]. The lysates were clarified by centrifugation at 13,000 x g for 30 min at 4°C and diluted with 0.5 ml of wash buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, and 20 mM imidazole]. His-Stat3 (5 µg) was incubated with the lysates at 4°C for 90 min followed by addition of 50 µl of Ni-NTA resin (Qiagen) and incubation for 2 h. The resin was washed three times with wash buffer. Resin-bound proteins were separated by SDS-PAGE, blotted onto a polyvinylidene difluoride membrane, and developed using anti-EGFR antibody.

Peptide Synthesis.
The peptides listed in Table 1Citation were synthesized in the Baylor College of Medicine Protein Core Facility using a Perkin-Elmer Applied Biosystems Division peptide synthesizer and standard 9-fluorenylmethoxycarbonyl amino acid chemistry. Seventy percent of the peptide reaction mix was biotinylated at the NH2 terminus, whereas the peptide remained on the resin using D-Biotin-LC (AnaSpec, Inc.). All peptides were purified to >90% using reverse-phase high performance liquid chromatography.


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  		Table 1 Tyrosine-phosphorylated and -nonphosphorylated peptides synthesized based upon the EGFR sequence

 
Phosphopeptide Affinity Purification.
NeutrAvidin-agarose (40 µl; Pierce) was incubated with 10 µg of biotinylated peptide in 300 µl of buffer A [20 mM HEPES (pH 7.5), 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 20% glycerol, 0.05% NP40, 1 mM DTT, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 100 mM NaCl] at 4°C for 2 h and washed with buffer A three times. Cells were lysed using buffer A containing 1% NP40 and centrifuged at 13,000 x g for 20 min. The NeutrAvidin-peptide complex was then mixed with 0.5 ml of cell lysate and 0.5 ml of buffer A (without NaCl and NP40) at 4°C for 2 h and washed thoroughly. Bound proteins were separated and immunoblotted as described above.

Resonant Mirror Biosensor Assay.
Kinetics experiments were performed using an IaSys Auto+ resonant mirror biosensor (Affinity Sensor, Paramus, NJ) as described previously (27) . Briefly, 2-welled cuvettes coated on the bottom of each well with biotin were purchased from Affinity Sensor and prepared for immobilization of biotinylated peptides by coating each surface with 0.04 mg/ml NeutrAvidin (Pierce) and washing with PBS-T (20 mM sodium phosphate, 0.05% Tween 20). Biotinylated peptide (5 µg) was added into each well, experimental peptide to one well and control peptide to the other, and change in arc seconds monitored simultaneously in both wells using the biosensor until stable followed by washing with PBS-T. Real-time binding of Stat3 was conducted at 25°C at a stir speed of 70 for 10 min starting at the lowest concentration of Stat3. The wells were washed out with three changes of 60 µl of PBS-T, and dissociation was allowed to proceed for 5 min. Each well bottom was regenerated by washing with 50 µl of 100 mM formic acid for 2 min and equilibrated with PBS-T for the next round of the association assay. Data were collected automatically and analyzed with the FASTplot and GraFit software (28) .

Peptide Delivery and Detection within Cells.
Peptide (10 µg) was mixed with BioPorter (10 µl; Gene Therapy Systems) in 100 µl of PBS [20 mM sodium phosphate, 150 mM NaCl (pH 7.4)] at room temperature for 5 min followed by addition of 900 µl of serum-free DMEM. UM-SCC-23 cells were washed once with serum-free DMEM. The BioPorter/peptide mixtures were transferred directly onto the cells. After 4 h of incubation, the cells were stimulated with TGF-{alpha} and analyzed by EMSA as described above. For cell staining, biotinylated peptides were delivered into HepG2 cells growing on a chamber slide (Nunc) as described above. Cells were washed with PBS and incubated with 4% formaldehyde for 30 min on ice and 0.5% Triton for 5 min at room temperature. After blocking with 1% BSA at room temperature for 4 h, cells were incubated with streptavidin labeled with Texas Red (Molecular Probes) after 4',6-diamidino-2-phenylindole then mounted using the Prolong Antifade Kit (Molecular Probes). Slide images were recorded by digital photography at x200 magnification and imported into Adobe PhotoShop without modification except cropping.

MTT Cell Proliferation Assay.
Cells were plated at 5000 cells/well in triplicate in a 96-well tissue culture plate. Cells were incubated in DMEM containing 10% fetal bovine serum with or without EGFR-based peptides containing a MTS at the COOH-terminal end (29) . Peptides were first solubilized in DMSO before adding to wells. The final volume of cell culture medium in each well was 0.1 ml. MTT solution (0.01 ml of a 5 mg/ml stock) was added into each well, and the plate was returned to the incubator for 4 h. When the MTT formazan appeared as a purple precipitate, 0.1 ml isopropanol/HCl was added and mixed thoroughly by repeated pipetting with a multichannel pipettor. The plate was left in the dark for 1 h, and the absorbance was measured at 570 nm in a microtiter plate reader.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mapping of Stat3 Activation by EGFR within Cells to Y1068 and Y1086.
To determine whether any of the five autophosphorylated tyrosine sites within the cytoplasmic domain of the EGFR contributed to Stat3 activation, we generated an EGFR construct in which each was mutated to phenylalanine (5F; Fig. 1ACitation ). This construct and the wild-type construct were stably transfected into NIH-3T3 cells, which have been shown previously by others not to express detectable endogenous EGFR (24) . After transfection and selection, cells were selected by flow cytometry to generate cell lines that expressed equivalent levels of EGFR protein (Fig. 1BCitation and data not shown). Although NIH-3T3 cells expressing wild-type EGFR demonstrated TGF-{alpha}-induced activation of Stat3 DNA binding activity by EMSA (Fig. 1C)Citation and phosphorylation of Stat3 tyrosine 705 (Fig. 1D)Citation , NIH-3T3 expressing the 5F construct did not, indicating that one or more of these five tyrosines were required for Stat3 activation.

Coffer and Kruijer (19) demonstrated in NIH-3T3 cells stably transfected with full-length and truncated EGFR constructs that, in addition to an active kinase domain, the region of the EGFR between 1061 and 1123 was required for ligand-induced stimulation of Stat3 DNA binding activity. This region contains two of five autophosphorylated tyrosines, Y1068 and Y1086. To determine whether either of these tyrosines was responsible for mediating Stat3 activation, we generated an EGFR construct in which Y1068 and Y1086 were left intact, whereas the other three tyrosines (Y992, Y1148, and Y1173) were mutated to phenylalanine (construct TM; Fig. 1ACitation ). NIH-3T3/TM cells expressed levels of EGFR similar to NIH-3T3/5F (Fig. 1B)Citation . In contrast to NIH-3T3/5F, however, NIH-3T3/TM cells demonstrated activation of Stat3 DNA binding activity (Fig. 1C)Citation and phosphorylation of Stat3 on tyrosine 705 (Fig. 1D)Citation at levels nearly identical to NIH-3T3/WT, indicating that one or both of these tyrosines are involved in recruitment and/or activation of Stat3 by the EGFR. Supershift analysis (Fig. 1E)Citation confirmed that the DNA binding complexes activated by TGF-{alpha} in NIH-3T3 WT and TM cells were composed of both Stat3 and Stat1.

Stat3 Binds to the EGFR Complex.
The initial step in Stat3 activation is generally believed to be recruitment to the receptor complex through its SH2 domain. To assess whether Stat3 binds to the EGFR complex, we immunoprecipitated the EGFR from the squamous cell carcinoma cell line, A431, allowed it to be autophosphorylated in vitro, and incubated it with recombinant Stat3 purified from Sf9 cells using Ni-NTA affinity chromatography (Fig. 2A)Citation . Immunoprecipitates were separated by SDS-PAGE and immunoblotted for Stat3 (Fig. 2B)Citation . Stat3 was readily detected within the activated EGFR complexes. In the reciprocal experiment, EGFR was detected in Stat3 pull-down assays containing lysates of A431 and His-Stat3 bound to Ni-NTA resin but not when A431 lysates were incubated with Ni-NTA resin alone (Fig. 2C)Citation . Stat3 pull-down assays performed using lysates of NIH-3T3 cells transfected with EGFR constructs (wild-type, 5F, or TM) implicated Y1068 and/or Y1086 in the recruitment of Stat3 to the receptor complex (Fig. 2D)Citation .



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  		Fig. 2. Interaction of Stat3 with the EGFR. A, a HindIII/XhoI DNA fragment containing human Stat3-{alpha} was cloned into pFastBac1 with a 6-histidine tag engineered into the NH2 terminus. Recombinant bacmids were prepared and used to infect SF9 cells. The expressed His-tagged Stat3{alpha} was affinity purified by incubation of clarified SF9 cell lysate with Ni-NTA resin at 4°C for 1 h. His-Stat3 protein was eluted with a wash buffer containing the indicated concentrations of imidazole. Cell lysate and eluted samples were separated by SDS-PAGE and immunoblotted using Stat3 mAb (top panel) or the gel stained with Coomassie Blue (bottom panel). The position of His-Stat3-{alpha} is indicated to the right. B, A431 cell lysates were immunoprecipitated with EGFR antibody and protein G-Sepharose 4B, then phosphorylated in vitro by incubation with 1 mM ATP in kinase buffer at room temperature for 10 min. The immunoprecipitates were incubated with His-Stat3-{alpha} (5 µg) at 4°C for 2 h, separated by SDS-PAGE, and immunoblotted with Stat3 mAb. C, A431 cells were stimulated by TGF-{alpha} (35 ng/ml for 20min) and lysed. Equal amounts of clarified lysates were incubated without or with His-Stat3-{alpha} (5 µg) and Ni-NTA resin as indicated. Resin-bound proteins were separated by SDS-PAGE and immunoblotted using EGFR antibody. D, the wild-type (WT) and mutated EGFR constructs (5F and TM) were transiently transfected into NIH-3T3 cells. Forty-eight h later, cells were stimulated with TGF-{alpha} for 20 min then lysed. One aliquot of clarified lysate was incubated with His-Stat3-{alpha} and Ni-NTA resin and the amount of EGFR bound determined by SDS-PAGE and immunoblotting with EGFR antibody (top panel). A second aliquot of clarified lysate was separated by SDS-PAGE and immunoblotted with EGFR antibody to confirm equivalent levels of EGFR expression (bottom panel). The results shown are representative of two or more experiments.

 
Mapping Stat3 Binding to EGFR Tyrosine(Y) 1068 and 1086.
Binding of Stat3 to the EGFR receptor complex may involve the SH2 domain of Stat3 binding to the receptor at Y1068 and/or Y1086 or binding of Stat3 to a protein intermediate that binds to these tyrosines. To establish whether Stat3 is capable of binding directly to EGFR at these tyrosine sites, we synthesized a panel of tyrosine phosphorylated dodecapeptides spanning each of the five autophosphorylated EGFR tyrosine sites (Table 1)Citation . Each peptide was incubated in pull-down assays with purified Stat3 protein (Fig. 3A)Citation . Immunoblotting for Stat3 demonstrated a prominent Stat3 band in pull-down assays using Y1068 phosphododecapeptide and a weaker Stat3 band in pull down assays using Y1086 phosphododecapeptide. None of the other three phosphododecapeptides interacted with purified Stat3 above control level. The ability of both Y1068 and Y1086 dodecapeptides to interact with purified Stat3 depended on whether the tyrosine within the peptide was phosphorylated. The results with Y1068 dodecapeptides were confirmed using lysates of HepG2 (Fig. 3B)Citation and 293T cells as sources of Stat3-{alpha} and Stat3-ß (Fig. 3C)Citation .



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  		Fig. 3. Peptide affinity purification of Stat3. NeutrAvidin-agarose was incubated without (C) or with the indicated peptides that were tyrosine phosphorylated (+P) or nonphosphorylated (-P). The agarose was washed and mixed with purified His-Stat3-{alpha} (3.4 µg; A) or lysates of HepG2 cells (B) or lysates of 293T cells (C) transiently transfected with Stat3-{alpha} (top panel) or Stat3-ß (bottom panel). Bound proteins were separated by SDS-PAGE and immunoblotted using Stat3 mAb. Purified His-Stat3-{alpha} (0.6 µg; Lane S in A) or cell lysate (10 µl; Lane L in B and C) was used as positive controls. The results shown are representative of two or more experiments.

 
Mirror Resonance Biosensor Analysis of Stat3 Binding to Dodecapeptides.
The Stat3 pull-down results suggested that phosphododecapeptide Y1068 had a stronger binding affinity for Stat3 than phosphododecapeptide Y1086. To confirm the pull-down assay results and to characterize the binding of Stat3 to EGFR Y1068 and Y1086 in more detail, we performed real-time affinity measurements using a mirror resonance biosensor. Biotinylated peptides were immobilized onto cuvette wells precoated with NeutrAvidin. The interaction of peptides with Stat3 at different concentrations was measured in real time and analyzed with GraFit software. Mirror resonance analysis (Fig. 4Citation , Table 2Citation ) demonstrated that Stat3 bound to phosphododecapeptide Y1068 with a KD of 135 ± 20 nM and to phosphododecapeptide Y1086 with a KD of 243 ± 36 nM (P = 0.044). The lower KD for Y1068 versus Y1086 is consistent with the results of the Stat3 pull-down assay and is completely attributable to slower dissociation of Stat3 bound to Y1068 versus Y1086. Although it appears from the real-time measurement curves (Fig. 4A)Citation that Stat3 was capable of binding to nonphosphorylated Y1068 and Y1086 dodecapeptides, the level of binding measured was below the level quantifiable by GraFit analysis. No binding of Stat3 to phosphododecapeptides Y992, Y1148, and Y1173 was detected (Fig. 4B)Citation in complete agreement with the peptide pull-down assay results.



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  		Fig. 4. Mirror resonance affinity assay of Stat3 binding to EGFR dodecapeptides. A, His-Stat3-{alpha} was added in the concentrations indicated to two cells of a biotin-coated cuvette pretreated with saturating amounts of NeutrAvidin and the indicated dodecapeptides. In the results shown in the top two panels, one well of the cuvette was pretreated with biotinylated phosphopeptide based on Y1068 (left top panel), whereas the other well of the cuvette was pretreated with biotinylated nonphosphorylated peptide (right top panel). In results shown in the bottom two panels, one well of the cuvette was pretreated with biotinylated phosphopeptide based on Y1086 (left bottom panel), whereas the other well of the cuvette was pretreated with biotinylated nonphosphorylated peptide (right bottom panel). B, the results shown in each of the panels represent one well of a cuvette pretreated with biotinylated phosphopeptide based Y1173 (left top panel), Y992 (right top panel), or Y1148 (bottom panel). The other well of the cuvette in each of these experiments was incubated with biotinylated phosphopeptide based on Y1068 as a positive control (data not shown). Mirror resonance measurements were recorded continuously for 10 min after the addition of Stat3 and for 5 min after removal of Stat3 and analyzed using GraFit software. The results shown are representative of two or more experiments.

 

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  		Table 2 Kinetics of Stat3 binding to phosphododecapeptides determined by mirror resonance biosensor analysis

 
Inhibition of Stat3 DNA Binding, Stat3 Activation, and Cell Proliferation by Y1068 and Y1086 Phosphopeptides.
It has been previously reported (30, 31, 32) that preformed STAT protein dimers can be destabilized and inhibited from binding DNA by incubation with phosphopeptides based on receptor sequences involved in their recruitment and activation. To determine whether this was the case for EGFR phosphododecapeptides Y1068 and Y1086, we preincubated whole-cell extracts of HepG2 stimulated with IL-6 with phosphododecapeptides Y1068 and Y1086 before addition of duplex DNA and EMSA. In a dose-dependent fashion, phosphododecapeptides Y1068 and Y1086 were capable of destabilizing Stat3 homodimers and inhibiting its DNA binding (Fig. 5A)Citation . This ability was dependent on their tyrosine phosphorylation. In contrast, preincubation with phosphododecapeptides Y1148 and Y1173 had no effect on Stat3 DNA binding. Phosphododecapeptide Y992 also was capable of inhibiting Stat3 DNA binding confirming the recent findings of Xia et al. (33) . Because Y992 was unable to bind monomers of Stat3, destabilization of Stat3 homodimers most likely occurred through its interaction at a region of the dimerization interface distinct from that formed by the Stat3 SH2 and the Stat3 tyrosine 705. Alternatively, phosphododecapeptide Y992 may interfere directly with Stat3 binding of DNA.



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  		Fig. 5. Effect of EGFR-derived peptides on Stat3 and cell proliferation. A, whole-cell extracts of IL-6-stimulated HepG2 cells (5 µg) were incubated with the phosphorylated and nonphosphorylated peptides at the indicated concentration for 1 h at 37°C before addition of radiolabeled hSIE followed by EMSA. B, EMSA was performed using whole-cell extracts of SCCHN cells transiently transfected with EGFR and Stat3 after incubation with BioPorter without (Lane C) or with the indicated dodecapeptides (10 µg for 4 h) and stimulated with TGF-{alpha} (35 ng/ml for 20 min). C, EMSA of control whole-cell extract (Lane C in B) was performed after incubation with antibodies capable of supershifting Stat1 or Stat3 as indicated. D, MTT proliferation assay was performed using A431 cells incubated for 1, 2, or 3 days with MTS-tagged Y1068 phosphododecapeptide (150 µM, {bullet}), MTS-tagged Y1068 nonphosphorylated dodecapeptide (150 µM, {blacksquare}), or with vehicle (DMSO) control (x). The results shown are the mean ± SD of triplicate wells and are representative of two or more experiments.

 
One of the long-term goals of these studies is to design peptidomimetics capable of specifically inhibiting Stat3 activation in SCCHN and other cancers by inhibiting the first step in its activation, i.e., recruitment to the active receptor/signaling complex. As proof-of-principle of this approach, we used the BioPorter peptide deliver system to introduce phosphododecapeptides into cells and determined the effect of their intracellular delivery on ligand-stimulated Stat3 activation. Intracellular delivery of phosphorylated versus nonphosphorylated dodecapeptides was equivalent as assessed by fluorescence microscopy (data not shown). However, only delivery of phosphorylated dodecapeptides Y1068 and Y1086 substantially decreased ligand-stimulated DNA binding activity by Stat3 (Fig. 5B)Citation . In contrast, intracellular delivery of nonphosphorylated dodecapeptides Y1068 and Y1086 and of phosphododecapeptide Y992, Y1148, and Y1173 did not affect ligand-stimulated Stat3 DNA binding in these cells. Supershift analysis (Fig. 5C)Citation confirmed the composition of the SIF-A (Stat3 homodimer) and SIF-C (Stat1 homodimer) bands.

To assess the effect of a phosphopeptide capable of blocking Stat3 recruitment to EGFR on EGFR-mediated cell growth, we generated phosphorylated and nonphosphorylated peptides based on Y1068 that contain a MTS as described previously (Ref. 29 ; Table 1Citation ). Peptides containing MTS are capable of entering cells with minimal toxicity. MTS-tagged peptides based on EGFR Y1068 were added to the squamous carcinoma cell line A431. Their uptake and effect on A431 cell growth was assessed over 3 days (Fig. 5D)Citation . Incubation of A431 cells with MTS-tagged phosphopeptide Y1068 dramatically reduced spontaneous growth of A431 cells at 2 and 3 days compared with cells incubated with MTS-tagged nonphosphorylated peptide Y1068 or cells incubated with vehicle control. Intracellular delivery of phosphorylated versus nonphosphorylated peptides was equivalent as assessed by fluorescence microscopy (data not shown). A431 were previously shown to have 20–100-fold up-regulation of EGFR mRNA and to have autocrine production of TGF-{alpha} leading to Stat3 activation (2 , 34 , 35) . Agents that blocked production of TGF-{alpha}, EGFR, or Stat3 or that inhibited activity of the receptor kinase previously were demonstrated to inhibit growth of A431 and other squamous carcinoma cell lines (12 , 17 , 36 , 37) . Thus, these results indicate that in addition to the ability to inhibit ligand-stimulated Stat3 DNA binding activity, intracellular delivery of phosphopeptides spanning Stat3 recruitment sites within the EGFR can inhibit TGF-{alpha}/EGFR-mediated autocrine cell growth.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that activation of Stat3 by EGFR was eliminated by mutation of all five EGFR tyrosines to phenylalanine and that activation was restored with return of two of the mutated tyrosine sites, Y1068 and Y1086, to wild-type. Stat3 was detected in the activated EGFR complex and its recruitment was dependent on Y1068 and/or Y1086. Phosphododecapeptides spanning Y1068 and Y1086 were able to pull down Stat3 with Y1068 being more effective that Y1086 in this regard. Real-time mirror resonance affinity analysis revealed Stat3 bound to phosphododecapeptide Y1068 with a KD of 135 ± 20 nM and to phosphododecapeptide Y1086 with a KD of 243 ± 36 nM consistent with the results of the pull-down assays. The lower KD of Y1068 was completely attributable to slower dissociation of Stat3 bound to Y1068 versus Y1086. Each phosphododecapeptide was capable of destabilizing Stat3 homodimers in vitro. When delivered into squamous cell carcinoma cells, phosphopeptides spanning EGFR Y1068 and Y1086 demonstrated the ability to inhibit ligand-stimulated Stat3 DNA binding activity and TGF-{alpha}/EGFR-mediated autocrine cell growth.

The requirement for COOH-terminal EGFR autophosphorylation sites for Stat3 recruitment and activation is controversial. Initial studies using truncation mutants of EGFR indicated that the COOH-terminal domain of the EGFR was important for Stat3 activation (19 , 38) , especially the region between amino acid residues 1061 and 1123. However, later studies using truncation mutants raised questions about the role of the COOH-terminal domain of EGFR in Stat3 activation (33 , 39) . Our studies strongly support a role for Y1068 and Y1086 in Stat3 recruitment and activation, which is consistent with the initial findings (19 , 38) , as well as some of the results in the later articles (33 , 39) . Our results are consistent with the findings of David et al. (20) using an EGFR mutant truncated at amino acid residue 1000, which demonstrated markedly reduced Stat3 activation compared with wild-type-expressing cells. Their additional finding that an EGFR construct truncated at 973 regained Stat3 activation may have been confounded by the inability of this receptor to internalize after ligand binding. Ours is the first study to map Stat3 activation by the EGFR using Y-to-F mutants, which avoids the confounding effect of truncation mutants on receptor internalization. Our results also confirm Xia et al.’s (33) findings that demonstrated the ability of phosphopeptides Y1068 and Y1086 to destabilize Stat3 homodimers. Their inability to map Stat3 activation using truncation mutants that remove single tyrosine motifs is consistent with our results indicating that both Y1068 and Y1086 can recruit Stat3.

Stat3 has been demonstrated to bind to phosphorylated peptides based on receptor phosphotyrosine motifs that contain the sequence YXXQ (40) and YXXC (32) and phosphorylated peptides based on the Stat3 Y705 sequence YLKT (41) . In each instance, the +3 amino acid is a polar residue. It is interesting to note that the sequence context for Y1068 (YINQ) and Y1086 (YHNQ) each conform to the consensus sequence YXXQ first identified within the IL-6 receptor (42) . Upon examination of the sequence context for EGFR Y992 (YRAL) and Y1173 (YQQD), one would have predicted that phosphopeptides based on these sequences would not bind Stat3, which is consistent with the results obtained. From the sequence context of EGFR Y1148 (YLNT), however, one might have predicted the result opposite to the one we obtained. Not only is there a threonine (T) at the +3 position, but there also is a leucine (L) at the +1 position. In alanine mutagenesis studies, leucine at the +1 position has been shown to be important for the ability of short peptides based on the Stat3 Y705 region (YLKT) to destabilize Stat3 DNA binding activity (41) . However, Stat3 binding to EGFR Y1148 phosphododecapeptide was not observed in either pull-down studies or mirror resonance studies, underscoring the fact that our understanding of the structural requirements of Stat3-phosphotyrosine binding is incomplete.

The KD we obtained for Stat3 binding to EGFR-derived phosphopeptides was similar to that obtained in affinity studies of Stat1 and Stat6 performed using fluorescence polarization (31) and surface plasmon resonance (30) . In those studies, Stat1 bound to IFN-{gamma} receptor-derived phosphopeptide with an apparent KD of 50–137 nM, whereas Stat6 bound to IL-4 receptor-derived phosphopeptides with an apparent KD of 300 nM.

Grb2 SH2 binding previously was mapped to two tyrosine sites within the EGFR identical to those we identified as binding Stat3, Y1068, and Y1086 (9) . The KD measured by mirror resonance analysis was 30 nM for binding of Grb2 to a phosphopeptide containing Y1068 (14 residues) and 60 nM for binding of Grb2 to a phosphopeptide containing Y1086 (16 residues). The 2-fold greater affinity of binding of Grb2 for Y1068 versus Y1086 is identical in magnitude to the difference in binding between these two sites that we observed for Stat3. The basis for the lower KD of Grb2 binding to Y1068 versus Y1086, however, was not explored.

Identification of specific motifs required for Stat3 recruitment and activation may be exploited to target its activation in cancers such as SCCHN where it contributes to cell growth and resistance to apoptosis. Our studies show that when introduced into SCCHN cells using a lipid-based system, phosphopeptides Y1068 and Y1086 interfere with ligand-induced Stat3 signal transduction. In addition, when introduced into A431 cells using a MTS sequence, phosphopeptide Y1068 inhibited autocrine cell growth. Turkson et al. (41) demonstrated that introduction of a tyrosine-phosphorylated hexapeptide based on Stat3 Y705 (PYLKTK) linked to a MTS-blocked Stat3 activation and suppressed v-Src transformation of NIH-3T3 cells. Our findings extend the spectrum of peptides that can block Stat3 activation and its growth-promoting and oncogenic effects when introduced into transformed cells.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported, in part, by NIH R01 Grant CA86430. Back

2 To whom requests for reprints should be addressed, at Section of Infectious Diseases, Baylor College of Medicine, One Baylor Plaza, BCM 286, Room 1319, Houston, TX 77030. Phone: (713) 798-8918; Fax: (713) 798-8299; E-mail: dtweardy{at}bcm.tmc.edu Back

3 The abbreviations used are: EGFR, epidermal growth factor receptor; hSIE, high-affinity sis-inducible element; SH2, Src homology; Stat, signal transducer and activator of transcription; EGF, epidermal growth factor; TGF-{alpha}, tumor growth factor {alpha}; SCCHN, squamous cell carcinoma of the head and neck; IL, interleukin; mAb, monoclonal antibody; EMSA, electrophoretic mobility shift assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTS, membrane translocation sequence. Back

Received 10/15/02. Accepted 5/12/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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