Binding of zinc finger protein ZPR1 to the epidermal growth factor receptor

Science

Volumn 272, Issue 5269, 1996, Pages 1797-1802

  Galcheva Gargova, Zoya ^a   Konstantinov, Konstantin N ^b   Wu, I Huan ^a   Klier, F George ^b   Barrett, Tamera ^a   Davis, Roger J ^a  

^a UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL   (United States)

^b SCRIPPS RESEARCH INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EPIDERMAL GROWTH FACTOR; EPIDERMAL GROWTH FACTOR RECEPTOR; ZINC FINGER PROTEIN;

ANIMAL CELL; ARTICLE; BINDING SITE; CONTROLLED STUDY; HORMONE RECEPTOR INTERACTION; LIGAND BINDING; NONHUMAN; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; PROTEIN PROTEIN INTERACTION; RECEPTOR BINDING;

ANIMALIA; MAMMALIA;

EID: 0030037017     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5269.1797     Document Type: Article

References (42)

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17. 32P]cytidine triphosphate by random priming (Amersham International PLC).
18. 32P]cytidine triphosphate by random priming (Amersham International PLC).
19. We dialyzed against water 6 mg of purified glutathione S-transferase (GST)-Zpr1 and lyophilized it. The protein was solubilized by incubation for 30 min at 65°C in concentrated nitric acid (0 5 ml). The amount of zinc was determined by inductively coupled plasma emission spectroscopy.
20. 291 with Ser. We constructed ZPR1 expression vectors by subcloning the ZPR1 cDNA in the Hind Ill and Eco Rl sites of pCDNA3 (Invitrogen) Bacterial ZPR1 expression vectors were constructed by subcloning PCR fragments of the ZPR1 cDNA in the Eco Rl and Xho l sites of pGEX-5X (Pharmacia LKB Biotechnology). The wild-type human EGFR and a mutant receptor truncated at residue 958 have been described [R. J. Davis, J Biol. Chem. 263, 9462 (1988); A J. Ekstrand, N. Sugawa, C. D. James, V. P. Collins, Proc. Natl. Acad. Sci. U.S.A. 89, 4309 (1992)]. The EGFR mutant truncated at residue 908 was prepared by restriction digestion with Bgl Il. The sequences of all plasmids were confirmed by automated sequencing with an Applied Biosystems model 373A machine.
21. 291 with Ser. We constructed ZPR1 expression vectors by subcloning the ZPR1 cDNA in the Hind Ill and Eco Rl sites of pCDNA3 (Invitrogen) Bacterial ZPR1 expression vectors were constructed by subcloning PCR fragments of the ZPR1 cDNA in the Eco Rl and Xho l sites of pGEX-5X (Pharmacia LKB Biotechnology). The wild-type human EGFR and a mutant receptor truncated at residue 958 have been described [R. J. Davis, J Biol. Chem. 263, 9462 (1988); A J. Ekstrand, N. Sugawa, C. D. James, V. P. Collins, Proc. Natl. Acad. Sci. U.S.A. 89, 4309 (1992)]. The EGFR mutant truncated at residue 908 was prepared by restriction digestion with Bgl Il. The sequences of all plasmids were confirmed by automated sequencing with an Applied Biosystems model 373A machine.
22. 291 with Ser. We constructed ZPR1 expression vectors by subcloning the ZPR1 cDNA in the Hind Ill and Eco Rl sites of pCDNA3 (Invitrogen) Bacterial ZPR1 expression vectors were constructed by subcloning PCR fragments of the ZPR1 cDNA in the Eco Rl and Xho l sites of pGEX-5X (Pharmacia LKB Biotechnology). The wild-type human EGFR and a mutant receptor truncated at residue 958 have been described [R. J. Davis, J Biol. Chem. 263, 9462 (1988); A J. Ekstrand, N. Sugawa, C. D. James, V. P. Collins, Proc. Natl. Acad. Sci. U.S.A. 89, 4309 (1992)]. The EGFR mutant truncated at residue 908 was prepared by restriction digestion with Bgl Il. The sequences of all plasmids were confirmed by automated sequencing with an Applied Biosystems model 373A machine.
23. A smaller amount of binding was observed in experiments with ZPR1 proteins containing the B domain. This may be caused by a function of the B domain to negatively regulate EGFR binding. Alternatively, the presence of the B domain may induce partially improper folding of bacterially expressed ZPR1 protein.
24. 721]EGFR have been described [J. L. Countaway, A. C Nairn, R J Davis, J Biol Chem. 267, 1129 (1992)]. These cells were maintained in Ham's F-12 medium supplemented with 5% fetal bovine serum (Gibco-BRL) Transfection studies were performed with the lipofectamine reagent (Gibco-BRL) and purified plasmid DNA (8). The cells were cultured in 100-mm dishes and solubilized with 1 ml of lysis buffer [20 mM tris (pH 7.4), 2 mM EDTA, 2 mM sodium pyrophosphate, 25 mM sodium β-glycerophosphate, 1 mM sodium orthovanadate, 25 mM NaCl, 0 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ ml of leupeptin, and 10 μg/ml of aprotinin]. Some extracts were prepared with the use of lysis buffer without the Ser-Thr phosphatase inhibitors β-glycerophosphate and pyrophosphate or the Tyr phosphatase inhibitor orthovanadate. We performed calpain cleavage of the EGFRs by harvesting cells in lysis buffer without EDTA, PMSF, leupeptin, and aprotinin. The extracts were clarified by centrifugation at 100,000g for 20 min at 4°C
25. Z. Galcheva-Gargova, unpublished data.
26. M. Gregoriou, A. C. Willis, M. A. Pearson, C. Crawford, Eur. J. Biochem. 223, 455 (1994).
27. S. R. Hubbard, L Wei, L Ellis, W A Hendrickson, Nature 372, 746 (1994).
28. G. Pelicci et al., Cell 70, 93 (1992).
29. EGF causes increased phosphorylation and reduced electrophoretic mobility of the EGFR during SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Contributions to the gel shift arise from increased Ser, Thr, and Tyr phosphorylation. The EGFR gel shift was observed in EGF-treated cells overexpressing ZPR1 (Fig. 5A). As ZPR1 inhibits EGFR tyrosine phosphorylation (Fig. 5B), the gel shift observed indicates that ZPR1 does not inhibit the EGF-stimulated Ser and Thr phosphorylation of the EGFR This increased Ser and Thr phosphorylation is caused, in part, by activation of cytoplasmic protein kinases [J. L Countaway, I. C. Northwood, R. J. Davis, J. Biol Chem. 264, 10828 (1989)] Thus, the reduced EGF-stimulated tyrosine phosphorylation observed in cells overexpressing ZPR1 (Fig. 5B) is not sufficient to completely block signaling by the EGFR. Indeed, control experiments demonstrated that EGF can activate the MAP kinase ERK2 in cells overexpressing ZPR1.
30. K. N. Konstantinov and Z. Galcheva-Gargova, unpublished data.
31. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu, F, Phe; G, Gly; H, His, I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, GIn; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
32. Proteins were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (immobilon-P; Millipore). The blots were probed with the monoclonal antibody to phosphotyrosine PY20 (ICN Biomedicals), the anti-phosphotyrosine horseradish peroxidase conjugate RC20 (Transduction Labs), a monoclonal antibody to HA(BabCo), the monoclonal antibody to Flag M2 (IBl-Kodak), the monoclonal antibody to the EGFR 20.3.6, a rabbit polyclonal antibody to the PDGF receptor β (Upstate Biotechnology), the monoclonal antibody to the IGF-1 receptor αlR3 (Oncogene Science), the monoclonal antibody to the insulin receptor CT-1, the rabbit polyclonal antibody to TrkA 203, a rabbit polyclonal antibody to SHC (Transduction Labs), and a rabbit polyclonal antibody to ZPR1 that was prepared with the synthetic peptide NDMKTEGYEAGLAPQ (17) as an antigen (Research Genetics). Immune complexes were detected by enhanced chemiluminescence (Amersham)
33. 5 cells in lysis buffer (10). The binding reaction was incubated at 22°C for 1 hour. The agarose beads were washed extensively with lysis buffer, and bound EGFRs were detected by protein immunoblot analysis The binding of TrkA was examined with the use of extracts of Sf9 cells infected with a TrkA baculovirus. The binding of the insulin receptor, IGF-1 receptor, PDGF receptor β, MAP kinases, and MAP kinase kinases was examined with extracts prepared from transfected COS cells. The expression vectors were pCMV5-PDGF-Rβ, pCMV5-INS-R, pCMV5-IGF1 -R, pCMV-Flag-p38 [J. Raingeaud et al., J. Biol. Chem. 270, 7420 (1995)], pCDNA3-Flag-JNK1 [B Dérijard et al , Cell 76, 1025 (1994)], pCMV-HA-ERK2, pCMV-HA-MKK1, and pRSV-Flag-MKK3 and pCDNA3-Flag-MKK4 [B Dérijard et al.. Science 267, 682 (1995)]
34. 5 cells in lysis buffer (10). The binding reaction was incubated at 22°C for 1 hour. The agarose beads were washed extensively with lysis buffer, and bound EGFRs were detected by protein immunoblot analysis The binding of TrkA was examined with the use of extracts of Sf9 cells infected with a TrkA baculovirus. The binding of the insulin receptor, IGF-1 receptor, PDGF receptor β, MAP kinases, and MAP kinase kinases was examined with extracts prepared from transfected COS cells. The expression vectors were pCMV5-PDGF-Rβ, pCMV5-INS-R, pCMV5-IGF1 -R, pCMV-Flag-p38 [J. Raingeaud et al., J. Biol. Chem. 270, 7420 (1995)], pCDNA3-Flag-JNK1 [B Dérijard et al , Cell 76, 1025 (1994)], pCMV-HA-ERK2, pCMV-HA-MKK1, and pRSV-Flag-MKK3 and pCDNA3-Flag-MKK4 [B Dérijard et al.. Science 267, 682 (1995)]
35. 5 cells in lysis buffer (10). The binding reaction was incubated at 22°C for 1 hour. The agarose beads were washed extensively with lysis buffer, and bound EGFRs were detected by protein immunoblot analysis The binding of TrkA was examined with the use of extracts of Sf9 cells infected with a TrkA baculovirus. The binding of the insulin receptor, IGF-1 receptor, PDGF receptor β, MAP kinases, and MAP kinase kinases was examined with extracts prepared from transfected COS cells. The expression vectors were pCMV5-PDGF-Rβ, pCMV5-INS-R, pCMV5-IGF1 -R, pCMV-Flag-p38 [J. Raingeaud et al., J. Biol. Chem. 270, 7420 (1995)], pCDNA3-Flag-JNK1 [B Dérijard et al , Cell 76, 1025 (1994)], pCMV-HA-ERK2, pCMV-HA-MKK1, and pRSV-Flag-MKK3 and pCDNA3-Flag-MKK4 [B Dérijard et al.. Science 267, 682 (1995)]
36. 5 cells in lysis buffer (10). The binding reaction was incubated at 22°C for 1 hour. The agarose beads were washed extensively with lysis buffer, and bound EGFRs were detected by protein immunoblot analysis The binding of TrkA was examined with the use of extracts of Sf9 cells infected with a TrkA baculovirus. The binding of the insulin receptor, IGF-1 receptor, PDGF receptor β, MAP kinases, and MAP kinase kinases was examined with extracts prepared from transfected COS cells. The expression vectors were pCMV5-PDGF-Rβ, pCMV5-INS-R, pCMV5-IGF1 -R, pCMV-Flag-p38 [J. Raingeaud et al., J. Biol. Chem. 270, 7420 (1995)], pCDNA3-Flag-JNK1 [B Dérijard et al , Cell 76, 1025 (1994)], pCMV-HA-ERK2, pCMV-HA-MKK1, and pRSV-Flag-MKK3 and pCDNA3-Flag-MKK4 [B Dérijard et al.. Science 267, 682 (1995)]
37. A-431 cells cultured in 100-mm dishes were serum-starved (12 hours) and treated without and with 100 nMEGF for 5 min at 37°C (10). Soluble extracts were prepared with 1 ml of lysis buffer (10). ZPR1 was immunoprecipitated with a rabbit polyclonal antibody that was prepared with the antigen GST-ZPR1 (residues 292 to 416). Control experiments were done with preimmune serum. The immunoprecipitates were washed three times with 20 mM tris (pH 7.4), 2 mM EDTA, 137 mM NaCl, 2 mM sodium pyrophosphate, 25 mM sodium β-glycerophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 0 5% deoxycholate, 0.1% SDS, 10% glycerol, 1 mMPMSF, 10 μg/ml of leupeptin, and 10 μg/ml of aprotinin The ZPR1 immunoprecipitates were examined by protein immunoblot analysis with the monoclonal EGFR antibody 20.3.6 (18).
38. 32P]phosphate into the receptors was determined after immunoprecipitation by SDS-PAGE and Phosphorlmager analysis
39. A-431 cells were cultured on glass cover slips (22-mm squares; Coming). The cells were rinsed briefly with phosphate-buffered saline (PBS) and fixed at -20°C with methanol (5 min) and acetone (2 min) DNA was detected with the monoclonal antibody 1 D12 [B L Kotzin et al., J Immunol. 133, 2554 (1984)], EGFRs were detected with the monoclonal antibody 528 [T. Kawamoto et al., Proc. Natl. Acad. Sci. U.S.A. 80, 1337 (1983)] (American Type Culture Collection), the Golgi region was detected with a human antibody to a Golgi-associated antigen [M. J. Fritzler, K. J. Griffith, E. K. L. Chan, Mol Biol. Cell 5, 1043 (1994)], and ZPR1 was detected with a rabbit polyclonal antibody to the peptide NDMK-TEGYEAGLAPQ (17, 18) The incubation with the primary antibodies (1 hour) was done at 25°C. The cover slips were washed three times with PBS and incubated (1 hour) with species-specific secondary antibodies coupled to fluorescein or rhodamine (Caltag Laboratories). The cover slips were washed three times with PBS and mounted on slides with Vectashield media (Vector Laboratories). Control experiments with preimmune immunoglobulin and competition analysis with antigen demonstrated the specificity of the ZPR1 immunofluorescence. Microscopy was done with an MRC-600 confocal laser scanning microscope with an argon-krypton mixed gas laser (Bio-Rad) fitted to a Zeiss Axiovert epifluorescence microscope with an oil immersion objective lens (1,4 numerical aperture; 63X) Images were collected from a single focal plane (approximately 0.4 μm) with the use of Kalman averaging of 30 scans (Bio-Rad COMOS program). The rhodamine and fluorescein images were collected simultaneously, digitized, and subsequently merged. Differential interference contrast (DIC) images were collected after fluorescence imaging. The images were recorded on Kodak Ektar 25 film.
40. A-431 cells were cultured on glass cover slips (22-mm squares; Coming). The cells were rinsed briefly with phosphate-buffered saline (PBS) and fixed at -20°C with methanol (5 min) and acetone (2 min) DNA was detected with the monoclonal antibody 1 D12 [B L Kotzin et al., J Immunol. 133, 2554 (1984)], EGFRs were detected with the monoclonal antibody 528 [T. Kawamoto et al., Proc. Natl. Acad. Sci. U.S.A. 80, 1337 (1983)] (American Type Culture Collection), the Golgi region was detected with a human antibody to a Golgi-associated antigen [M. J. Fritzler, K. J. Griffith, E. K. L. Chan, Mol Biol. Cell 5, 1043 (1994)], and ZPR1 was detected with a rabbit polyclonal antibody to the peptide NDMK-TEGYEAGLAPQ (17, 18) The incubation with the primary antibodies (1 hour) was done at 25°C. The cover slips were washed three times with PBS and incubated (1 hour) with species-specific secondary antibodies coupled to fluorescein or rhodamine (Caltag Laboratories). The cover slips were washed three times with PBS and mounted on slides with Vectashield media (Vector Laboratories). Control experiments with preimmune immunoglobulin and competition analysis with antigen demonstrated the specificity of the ZPR1 immunofluorescence. Microscopy was done with an MRC-600 confocal laser scanning microscope with an argon-krypton mixed gas laser (Bio-Rad) fitted to a Zeiss Axiovert epifluorescence microscope with an oil immersion objective lens (1,4 numerical aperture; 63X) Images were collected from a single focal plane (approximately 0.4 μm) with the use of Kalman averaging of 30 scans (Bio-Rad COMOS program). The rhodamine and fluorescein images were collected simultaneously, digitized, and subsequently merged. Differential interference contrast (DIC) images were collected after fluorescence imaging. The images were recorded on Kodak Ektar 25 film.
41. A-431 cells were cultured on glass cover slips (22-mm squares; Coming). The cells were rinsed briefly with phosphate-buffered saline (PBS) and fixed at -20°C with methanol (5 min) and acetone (2 min) DNA was detected with the monoclonal antibody 1 D12 [B L Kotzin et al., J Immunol. 133, 2554 (1984)], EGFRs were detected with the monoclonal antibody 528 [T. Kawamoto et al., Proc. Natl. Acad. Sci. U.S.A. 80, 1337 (1983)] (American Type Culture Collection), the Golgi region was detected with a human antibody to a Golgi-associated antigen [M. J. Fritzler, K. J. Griffith, E. K. L. Chan, Mol Biol. Cell 5, 1043 (1994)], and ZPR1 was detected with a rabbit polyclonal antibody to the peptide NDMK-TEGYEAGLAPQ (17, 18) The incubation with the primary antibodies (1 hour) was done at 25°C. The cover slips were washed three times with PBS and incubated (1 hour) with species-specific secondary antibodies coupled to fluorescein or rhodamine (Caltag Laboratories). The cover slips were washed three times with PBS and mounted on slides with Vectashield media (Vector Laboratories). Control experiments with preimmune immunoglobulin and competition analysis with antigen demonstrated the specificity of the ZPR1 immunofluorescence. Microscopy was done with an MRC-600 confocal laser scanning microscope with an argon-krypton mixed gas laser (Bio-Rad) fitted to a Zeiss Axiovert epifluorescence microscope with an oil immersion objective lens (1,4 numerical aperture; 63X) Images were collected from a single focal plane (approximately 0.4 μm) with the use of Kalman averaging of 30 scans (Bio-Rad COMOS program). The rhodamine and fluorescein images were collected simultaneously, digitized, and subsequently merged. Differential interference contrast (DIC) images were collected after fluorescence imaging. The images were recorded on Kodak Ektar 25 film.
42. We thank the following for reagents: A. Ross for the TrkA baculovirus, A. Kazlauskis for pCMV5-PDGF-Rβ, R L Lewis for pCMV5-INS-R, and pCMVS-IGF1-R, M. Weber for pCMV-HA-ERK2, N. Ahn for pCMV-HA-MKK1, M. Hayman for the monoclonal antibody to the EGFR 20.3 6, K. M. Pollard for the fibrillanan antibody 72B9, K. Siddle for the monoclonal antibody to the insulin receptor CT-1, A. Ross for the rabbit polyclonal antibody to TrkA 203, R. L Rubin for monoclonal antibody 1.D12, and E. K. L Chan for human antibody to a Golgi-associated antigen. We also thank W. Royer for assistance with computer graphics, T. Gilbert for assistance with spectroscopic analysis; and M. Roberts for secretarial assistance. Confocal microscopy was supported by the Lucille P. Markey Charitable Trust. R J.D is an Investigator of the Howard Hughes Medical Institute. Supported by grant R01-CA58396 from the National Cancer Institute.