Epidermal Growth Factor Directs Sex-specific Steroid Signaling through Src Activation

Estrogens and androgens exert many biological effects that do not require interactions of their receptors with chromosomal DNA. However, it has been a long-standing question how the sex steroid receptors provoke signal transduction outside the nucleus. Here we have shown that epidermal growth factor (EGF) directs sex-specific steroid signaling through Src activation. We have revealed that estrogen (E2)-induced Src activation takes place in, not only plasma, but also endomembranes. This was found ascribed to the existence of EGF and the occurrence of EGF receptor (EGFR)-involved endocytosis of estrogen receptor together with Src. EGFR, estrogen receptor, and Src were found to form a complex upon E2 stimulation. The cell growth of breast cancer-derived MCF-7 cells was found to remarkably increase through the above EGF-involved estrogen-signaling process. In contrast, the androgen 5α-dihydrotestosterone-induced Src activation occurs only in the plasma membrane free from the interaction of EGFR with androgen receptor, irrespective of EGF. The cell growth occurred only moderately as a result. The spatial difference in Src activation between E2 and 5α-dihydrotestosterone may be responsible for the different extent of observed cell growth. Estrogens and androgens exert many biological effects that do not require interactions of their receptors with chromosomal DNA. However, it has been a long-standing question how the sex steroid receptors provoke signal transduction outside the nucleus. Here we have shown that epidermal growth factor (EGF) directs sex-specific steroid signaling through Src activation. We have revealed that estrogen (E2)-induced Src activation takes place in, not only plasma, but also endomembranes. This was found ascribed to the existence of EGF and the occurrence of EGF receptor (EGFR)-involved endocytosis of estrogen receptor together with Src. EGFR, estrogen receptor, and Src were found to form a complex upon E2 stimulation. The cell growth of breast cancer-derived MCF-7 cells was found to remarkably increase through the above EGF-involved estrogen-signaling process. In contrast, the androgen 5α-dihydrotestosterone-induced Src activation occurs only in the plasma membrane free from the interaction of EGFR with androgen receptor, irrespective of EGF. The cell growth occurred only moderately as a result. The spatial difference in Src activation between E2 and 5α-dihydrotestosterone may be responsible for the different extent of observed cell growth. Besides traditional genomic pathways of sex steroid receptors in the nucleus, the extranuclear non-genomic pathways of these receptors are being revealed to strongly relate to many biological consequences, including vascular protection and cell proliferation (1Losel R. Wehling M. Nat. Rev. Mol. Cell Biol. 2003; 4: 46-56Crossref PubMed Scopus (698) Google Scholar, 2Marquez D.C. Pietras R.J. Oncogene. 2001; 20: 5420-5430Crossref PubMed Scopus (150) Google Scholar, 3Kousteni S. Chen J-R. Bellido T. Han L. Ali A.A. O'Brien C.A. Plotkin L. Fu Q. Mancino A.T. Wen Y. Vertino A.M. Powers C.C. Stewart S.A. Ebert R. Parfitt A.M. Weinstein R.S. Jilka R.L. Manolagas S.C. Science. 2002; 298: 843-846Crossref PubMed Scopus (385) Google Scholar). These non-genomic pathways are rapidly mediated through several critical protein kinases. A non-receptor protein tyrosine kinase, Src, is known to be activated immediately after a steroid stimulation (4Migliaccio A. Castoria G. Di Domenico M. de Falco A. Bilancio A. Lombardi M. Barone M.V. Ametrano D. Zannini M.S. Abbondanza C. Auricchio F. EMBO J. 2000; 19: 5406-5417Crossref PubMed Google Scholar, 5Castoria G. Migliaccio A. Bilancio A. Di Domenico M. de Falco A. Lombardi M. Fiorentino R. Varricchio L. Barone M.V. Auricchio F. EMBO J. 2001; 20: 6050-6059Crossref PubMed Scopus (399) Google Scholar). This activated Src phosphorylates a wide variety of substrate proteins (such as Shc), which finally induce gene transcription (6Migliaccio A. Di Domenico M. Castoria G. de Falco A. Bontempo P. Nola E. Auricchio F. EMBO J. 1996; 15: 1292-1300Crossref PubMed Scopus (861) Google Scholar, 7Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han L. Han K. DiGregorio G.B. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jika R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar). In previous studies, the rapid activation of Src and other kinases by steroids has never been demonstrated in living cells. To answer the question about how the Src activity is nongenomically regulated by steroid receptors in single living cells, we developed a fluorescent indicator for Src kinase activity, and named it Srcus. This indicator can monitor substrate phosphorylation by activated endogenous Src as a fluorescent resonance energy transfer (FRET) 5The abbreviations used are: FRET, fluorescent resonance energy transfer; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; AR, androgen receptor; CFP, cyan fluorescent protein; GFP, green fluorescent protein; YFP, yellow fluorescent protein; PTB, phosphotyrosine binding; DCC, dextran/charcoal; SH2, Src homology 2; ERK, extracellular signal-regulated kinase; TM, transmembrane.5The abbreviations used are: FRET, fluorescent resonance energy transfer; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; AR, androgen receptor; CFP, cyan fluorescent protein; GFP, green fluorescent protein; YFP, yellow fluorescent protein; PTB, phosphotyrosine binding; DCC, dextran/charcoal; SH2, Src homology 2; ERK, extracellular signal-regulated kinase; TM, transmembrane. response in single living cells. Based on the fluorescence imaging with the present fluorescent indicators, we demonstrated that E2-induced Src activation takes place in not only plasma but also endomembranes. This was found ascribed to the existence of epidermal growth factor (EGF) and the occurrence of EGF receptor (EGFR)-involved endocytosis of estrogen receptor (ER) together with Src. EGFR, ER, and Src were found to form a complex upon E2 stimulation. The cell growth of breast cancer-derived MCF-7 cells was found to remarkably increase through the above EGF-involved estrogen-signaling process. In contrast to estrogen-activated Src signaling, the male steroid hormone 5α-dihydrotestosterone (DHT) was found to activate Src only in the plasma membrane irrespective of EGF free from the interaction of EGFR with androgen receptor (AR). The cell growth occurs only moderately as a result. The spatial difference in Src activation between E2 and DHT may be responsible for the different extent of MCF-7 cell growth between E2 and DHT. Materials—CFP mutations were F64L/S65T/Y66W/N146I/M153T/V163A/N212K, and YFP mutations were S65G/V68L/Q69K/S72A/T203Y. 17β-estradiol (E2), EGF, Eagle's minimal essential medium, phenol red-free Eagle's minimal essential medium, and flutamide were purchased from Sigma. Fetal calf serum, Hanks' balanced salt solution, Lipofectamine 2000 reagent, and mammalian expression vector pcDNA3.1(+) were obtained from Invitrogen. PP2 was purchased from Calbiochem. ICI 182,780 was obtained from Wako Pure Chemical Industries (Osaka, Japan). Anti-EGFR (1005G), anti-ERα (F-10), and anti-AR (441) antibodies for immunoprecipitation experiments and anti-Src rabbit polyclonal antibody for immunoblotting experiments were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Src antibody (GD11) for immunoprecipitation experiments was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-GFP antibody was obtained from Clontech (Palo Alto, CA). Anti-EGFR mouse monoclonal antibody for immunoblotting experiments was obtained from BD Transduction Laboratories (San Jose, CA). Other chemicals used were all of analytical reagent grade. Plasmid Construction—To construct the cDNAs encoding the present fluorescent indicators, fragment cDNAs of CFP, YFP with a nuclear export signal, a linker, and substrate domain, phosphorylation recognition domain, phosphotyrosine binding (PTB) domain, and an N-terminal transmembrane domain of Cbp were generated by standard PCR and subcloned into pBluescript (SK+). All cloning enzymes were from Takara Biomedical (Tokyo, Japan) and were used according to the manufacturer's instructions. All PCR fragments were sequenced with ABI310 genetic analyzer. The amino acid sequence containing the substrate and flexible linker is EEIYGEFFGGNNGGNNGGNNGGNNGNNGGNGNG, which is reported to be selectively phosphorylated by Src (8Songyang Z. Carraway K.L. Eck M.J. Harrison S.C. Feldman R.A. Mohammadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantley L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (844) Google Scholar). The phosphorylation recognition domain is an Src homology 2 (SH2) domain of Src-(148-248). The amino acid sequence of the nuclear export signal is LPPLERLTL, which is derived from a human immunodeficiency virus-derived protein, Rev. The PTB domain is derived from Shc-(46-206), and the transmembrane domain is from Cbp-(1-52). In the mutant PTB F198V domain, an amino acid residue at position 198 of Shc-(46-206) is mutated from phenylalanine to valine. All of the constructs were cloned into pcDNA3.1 (+). Imaging of Cells—MCF-7 cells were treated with a medium (phenol red-free Eagle's minimal essential medium) supplemented with dextran/charcoal (DCC)-treated serum, 1% penicillin/streptomycin, 1 mm sodium pyruvate, 0.1 mm non-essential amino acids, and 2 mm l-glutamine for 12 h followed by starvation with a serum-free medium (phenol red-free Eagle's minimal essential medium supplemented with 1% penicillin/streptomycin, 1 mm sodium pyruvate, 0.1 mm non-essential amino acids, and 2 mm l-glutamine for 2 h. After starvation with the serum-free medium, the culture medium was replaced with Hanks' balanced salt solution for imaging. As described previously (9Sato M. Ueda Y. Takagi T. Umezawa Y. Nat. Cell Biol. 2003; 5: 1016-1022Crossref PubMed Scopus (156) Google Scholar), the cells were imaged at room temperature on a Carl Zeiss Axiovert 135 microscope with a cooled CCD camera MicroMAX (Roper Scientific Inc., Tucson, AZ) controlled by MetaFluor (Universal Imaging, West Chester, PA). The exposure time at 440 ± 10 nm of excitation was 100 ms. The fluorescence images were obtained through 480 ± 15- and 535 ± 12.5-nm filters with a 40× oil immersion objective (Carl Zeiss, Jena, Germany). Cell Culture and Transfection—MCF-7 cells were cultured in Eagle's minimal essential medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 1 mm sodium pyruvate, and 0.1 mm non-essential amino acids at 37 °C in 5% CO2.12h after transfection with Lipofectamine 2000 reagent, the MCF-7 cells were placed onto glass-bottom dishes and plastic culture dishes for fluorescence imaging of living cells and Western blotting analysis, respectively. Immunoprecipitation and Immunoblot Analysis—MCF-7 cells were starved with serum-free medium for 2 h following the treatment with medium containing DCC-treated serum for 12 h. After stimulation, the cells were lysed with an ice-cold lysis mm 100 mm 1 mm 10 mm 2 mm sodium 1 mm 10 10 and 10 After the were from the cell with protein The were by onto and with the The obtained signal was an Src by of is shown in phosphorylation of the substrate sequence by activated endogenous Src, the domain with phosphorylated and is between the fluorescent M. T. K. T. Umezawa Y. Nat. 2002; 20: PubMed Scopus Google Scholar, Y. Y. S. S. Nature. PubMed Scopus Google Scholar). of the indicator are observed as a in the fluorescence of We a that and the in MCF-7 cells, which are derived from human breast cells and and G. Migliaccio A. Bilancio A. Di Domenico M. de Falco A. Lombardi M. Fiorentino R. Varricchio L. Barone M.V. Auricchio F. EMBO J. 2001; 20: 6050-6059Crossref PubMed Scopus (399) Google Scholar, A. Di Domenico M. Castoria G. de Falco A. Bontempo P. Nola E. Auricchio F. EMBO J. 1996; 15: 1292-1300Crossref PubMed Scopus (861) Google Scholar). was it was observed in the of MCF-7 cells to a nuclear export signal sequence to the of a steroid stimulation, the cells were by for 12 h with a medium supplemented with DCC-treated serum, as treated in previous on steroid signaling (4Migliaccio A. Castoria G. Di Domenico M. de Falco A. Bilancio A. Lombardi M. Barone M.V. Ametrano D. Zannini M.S. Abbondanza C. Auricchio F. EMBO J. 2000; 19: 5406-5417Crossref PubMed Google Scholar, 5Castoria G. Migliaccio A. Bilancio A. Di Domenico M. de Falco A. Lombardi M. Fiorentino R. Varricchio L. Barone M.V. Auricchio F. EMBO J. 2001; 20: 6050-6059Crossref PubMed Scopus (399) Google Scholar, A. Di Domenico M. Castoria G. de Falco A. Bontempo P. Nola E. Auricchio F. EMBO J. 1996; 15: 1292-1300Crossref PubMed Scopus (861) Google Scholar). stimulation with 17β-estradiol of the fluorescent cell images of the cell that the of a in a We the observed response of was by phosphorylation by Src in MCF-7 cells. the tyrosine in the substrate sequence of was replaced with that in the was observed upon E2 stimulation of MCF-7 cells with a for Src kinase activity, the response of upon E2 stimulation of Src in MCF-7 cells the response of upon E2 stimulation These that and of the substrate phosphorylation of activated endogenous Src. The of 10 nm E2 the response of a and This response after E2 stimulation, as shown in the time of the in the of 10 nm DHT also a response as was observed with E2 stimulation and The response of by E2 DHT was in a and was found to response upon stimulation of the cells with E2 and DHT at 1 not of MCF-7 cells with the ICI 182,780 the E2-induced response of and the flutamide was shown to the response by DHT stimulation These that the present to monitor Src activation by the of E2 through and by that of DHT through in living cells, respectively. EGF for Src but for DCC-treated serum used in the starvation of steroids is known to growth de J. Cell. PubMed Scopus Google Scholar). To the that these may the non-genomic Src we treated the MCF-7 cells with the medium containing DCC-treated serum followed by the starvation of the cells with a serum-free medium for 2 h. To the of serum-free medium on the response of the cells were with E2 and DHT at 1 of which induce the response of Srcus. The E2-induced activation of Src was to the the MCF-7 cells were starved of factor with serum-free medium In contrast, DHT stimulation the response of in the cells treated with the serum-free medium to the extent as treated with the medium containing DCC-treated serum These that androgen Src factor; estrogen was found to activate Src with as growth in of to Src We that of the EGF, is a factor for Src activation and observed cells with E2 and EGF the cells were treated with serum-free medium and with E2 and the different of EGF Src activation was in an EGF the other the cells treated with serum-free medium were with different of EGF activation of Src was observed is that Src is activated by with E2 and EGF, not by stimulation only with to E2-induced Src activation only on EGF other growth we the cells for 12 h in the medium containing DCC-treated serum and 10 antibody that EGF. The cells were with E2 and observed a not response upon E2 stimulation In of the cells for with 100 an EGFR E2-induced response These that E2-induced Src activation in the cells with DCC-treated serum is to EGF and receptor activation. A was to that EGF the activation of in MCF-7 cells. We the cells with serum-free medium and the cells with EGF. To the we the immunoblotting of MCF-7 cell with antibody that the phosphorylated tyrosine of activated and The extent of phosphorylation of is upon EGF stimulation in the cells treated with the serum-free medium Src is not activated by EGF in these cells This that EGF the activation in MCF-7 the EGF stimulation is not to activate Src. of EGF for MCF-7 Cell Src activation has been shown to with cell proliferation (4Migliaccio A. Castoria G. Di Domenico M. de Falco A. Bilancio A. Lombardi M. Barone M.V. Ametrano D. Zannini M.S. Abbondanza C. Auricchio F. EMBO J. 2000; 19: 5406-5417Crossref PubMed Google Scholar). To the of EGF for cell proliferation through Src signaling, we a growth of MCF-7 cells, which are with E2 together with EGF serum-free The EGF E2 stimulation in the serum-free medium on MCF-7 cell growth and with However, the of MCF-7 cells with together with EGF, remarkably MCF-7 cell growth DHT stimulation of the cells in the serum-free medium only moderately MCF-7 cell growth irrespective of EGF and with These that not only E2 but also EGF is for MCF-7 cell growth. The between and in EGF of Src of the of EGFR with but with shown in we found that the Src activation EGF, but the Src activation not require EGF. To the of difference in EGF of Src activation between estrogen and the with the antibody were by anti-ERα After the starvation of the cells with the medium containing DCC-treated serum for 12 the cells were treated with the serum-free medium for 2 h and with E2 DHT at 1 for to the experiments the as a and was found to with EGFR irrespective of E2 and EGF we the immunoblotting of anti-Src with anti-ERα and Src was found to with upon stimulation of the cells with E2 with E2 and EGF EGFR was to of Src the cells were with E2 and EGF These that EGFR, and Src form a complex in an To the activated complex with Src for the Src we be to the activated complex by a PTB domain of to the of and named it This PTB domain is known to with a domain of EGFR EMBO J. 2000; 19: PubMed Google Scholar, G. T. E. L. A. M. J. J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). the activated complex with Src for the Src be phosphorylated by activated Src through complex the mutated that has a mutant PTB F198V domain, which not with EGFR P. P. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google As shown in the MCF-7 cells treated with the serum-free medium were with E2 and EGF, the extent of phosphorylation in was that in This that the of EGF for Src activation is of the binding of the complex with Src. In contrast, interaction with EGFR proteins were with an anti-AR antibody In and were phosphorylated by Src to the extent in the cells These that Src free from the interaction with To the interaction of we and EGFR from MCF-7 cell by anti-ERα and respectively. The cell were with anti-ERα and We that EGFR and of from the the other of EGFR was with the of anti-ERα These that of the is to EGFR and of the EGFR is to in MCF-7 cells. and Src in of the of EGFR with but with the cells were with the of estrogen and the of was to the extent and This that difference between E2 and DHT was observed to the extent of Src activation in the The difference in the of MCF-7 cell growth observed between E2 together with EGF remarkably MCF-7 cell but DHT only moderately MCF-7 cell growth irrespective of EGF Src a in including the plasma membrane and the and T. Y. Y. M. J. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). To in which Src activation is by that with EGFR by that not with EGFR, was to of by a transmembrane domain of Cbp protein M. Y. T. Y. S. K. A. M. Nature. 2000; PubMed Scopus Google Scholar). This membrane a free of the indicator and the of spatial as to phosphorylation by the activated Src This was named transmembrane M. K. and Y. for The MCF-7 cells were treated with the medium containing DCC-treated serum and with E2 DHT. in the plasma membrane and also in the were a and E2 stimulation, the that the of was from to green in plasma and a time of the the response of was immediately observed in the plasma and it a In the E2 the response Src was activated in plasma and upon E2 stimulation. DHT stimulation, the was from to green only in the plasma membrane response of was in the The observed spatial difference in Src activation between and may be to of EGFR in and was found to with EGFR, and not with EGFR EGFR is known to be from the plasma membrane to the by The endocytosis of EGFR is by a mutant (9Sato M. Ueda Y. Takagi T. Umezawa Y. Nat. Cell Biol. 2003; 5: 1016-1022Crossref PubMed Scopus (156) Google Scholar, C. Science. 1996; PubMed Scopus Google Scholar). We in MCF-7 cells and the response of in the which the E2-induced response of in the and However, was observed in the Src activation in the plasma membrane and These that Src is from the plasma membrane to the as the complex by the EGFR In contrast, Src, which not with EGFR, was found not to be to the endomembranes. To in the plasma and Src but in the only Src activation is by the endocytosis of EGFR that with and not with This Src activation in the may the difference in the extent of the observed MCF-7 cell growth between estrogen and androgen In the present we have found that estrogen is not to rapidly activate Src. We have shown that not estrogen but with estrogen and EGF is for the rapid Src activation. In contrast, androgen was found to activate Src the EGF stimulation. In we have shown that not only E2 but also EGF is for MCF-7 cell EGF is not for cell growth. to the present it been that the steroid hormone estrogen rapidly signal as Src by and effects on cells. However, in these the cells have been with estrogen in the of DCC-treated serum that many of as EGF. of the present factor EGF has not been found for the rapid estrogen signaling in previous We have how Src is activated by the with E2 and EGF. was found to to EGFR, and complex to Src in an the other was found to activate Src the interaction of These the at the for the of EGF in Src but is not the for The substrate sequence in is the as a substrate for Src and is phosphorylated by of the other tyrosine kinases (8Songyang Z. Carraway K.L. Eck M.J. Harrison S.C. Feldman R.A. Mohammadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantley L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (844) Google Scholar). In as shown in the E2-induced response of is by PP2 and is with the of Src in MCF-7 cells. However, we the that may activation of other of the Src as PP2 also the activity of other Src kinases. the of by the of estrogen and difference was observed in the extent of Src activation in the The difference in the of MCF-7 cell growth observed between E2 together with EGF remarkably MCF-7 cell but DHT only moderately MCF-7 cell growth irrespective of EGF. we that estrogen and androgen Src, we not how estrogen and androgen the difference in the effects activated Src. the fluorescence imaging with we have shown the difference in the of Src activation between E2 and DHT. E2 was found to activate Src in not only plasma but also endomembranes. We found that Src activation in the plasma and EGF and is regulated by the occurrence of endocytosis of together with Src. In contrast to estrogen-activated Src signaling, the male steroid hormone DHT was found to activate Src only in the plasma membrane irrespective of EGF free from the interaction of EGFR with The spatial difference in Src activation by E2 DHT may be responsible for the different extent of MCF-7 cell growth between E2 and DHT. We have found that EGF a in MCF-7 cell growth. EGFR have previously been reported to growth and an response to E2 stimulation in cells of and P. R. Z. PubMed Scopus Google Scholar). This to a between EGFR and estrogen signaling in other and of the EGFR have not been in to on the interaction of EGFR with for Src activation a at the for the above in of the EGFR In an of E2 binding have been observed in the plasma membrane of MCF-7 cells, and the other the and other D.C. Pietras R.J. Oncogene. 2001; 20: 5420-5430Crossref PubMed Scopus (150) Google Scholar). In of E2 and serum have including the Src activation E2 J. Ali A. J. 1996; Google Scholar, D. E. S. A. 2000; PubMed Scopus Google Scholar). these that the has been to at the plasma have and M. P. A. J. Mol. 2002; PubMed Scopus Google have reported the membrane of through the interaction with and Z. Y. R. R.J. S. A. PubMed Scopus Google have shown the of to the plasma membrane through the with growth factor 1 and L. S. A. 2003; PubMed Scopus Google have described a of of on the L. S. A. 2003; PubMed Scopus Google Scholar). present a that the of with EGFR and how at the plasma membrane and the steroid signal to the Src activation. a of the and as and are known to be in human breast Mol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. PubMed Scopus Google Scholar). We have found that EGFR, ER, and Src form the complex in an and the activation of and EGFR is for Src activation and cell growth of human breast cancer-derived MCF-7 cells. In other the complex an in the in human breast We Y. for