Fig. 2. Effect of androgen and 17β-oestradiol on Erk-2, Raf-1 and Src activities in LNCaP cells. (A) (Left) Cells were treated for 5 min with 10 nM R1881 alone or in the presence of 10 µM Casodex or 10 µM ICI 182,780, or with 10 µM Casodex alone or 10 µM ICI 182,780 alone. (Right) Cells were treated for 5 min with 10 nM oestradiol alone or in the presence of 10 µM Casodex or 10 µM ICI 182,780. Cell lysates were immunoprecipitated with anti-Erk-2 antibodies. Erk-2 kinase activity was assayed using MBP as a substrate The arrows indicate the MBP position. (B) Cells were left untreated or treated for 2 min with either 10 nM oestradiol or 10 nM R1881 alone or in the presence of 10 µM Casodex or ICI 182,780. Cell lysates were immunoprecipitated with anti-Raf antibodies and Raf-1 kinase activity was assayed in the immunoprecipitates using histone H1 as substrate. (C and D) Cells were left untreated or treated for 2 min with the indicated compounds. Cell lysates were immunoprecipitated with anti-Src antibodies and immunoprecipitates assayed for Src kinase activity using acidified enolase as substrate. The arrows indicate the enolase position (en). (E) Cells were left unstimulated or stimulated for 5 min with 10 nM R1881 (left) or 10 nM oestradiol (right) in the absence or presence of 50 µM PD98059 or 10 µM PP1. Control cells were treated with inhibitors alone (not shown).

Fig. 4. Effects of androgen and oestradiol in T47D and MCF-7 cells. (Left) Quiescent T47D cells were left unstimulated or stimulated with the indicated compounds. The cells were then analysed for growth rate (A), which was expressed as increase in cell number and BrdU incorporation (B), which was calculated as in Figure 1C. (B1) Quiescent T47D cells, either uninjected or injected with a SrcK^–-expressing plasmid, were stimulated with R1881 (10 nM). BrdU was included in the cell medium and DNA synthesis analysed. It was calculated as in Figure 1C. Basal incorporation of BrdU (<5%) was evaluated and subtracted. Data derived from at least 150 scored cells were pooled. The means ± SEM are shown. Lysates from quiescent T47D cells were immunoprecipitated with anti-Src antibody. (C) Src kinase activity was assayed using enolase as substrate. (D) Immunocomplexes were probed with either anti-Src (upper), anti-AR (middle) or anti-ERα (lower) antibodies. (Right) Quiescent MCF-7 cells untreated or treated with the indicated compounds were analysed for growth rate (A) and BrdU incorporation (B). (B1) MCF-7 cells, either uninjected or injected with SrcK^–-expressing plasmid, were stimulated with R1881 (10 nM). BrdU was included in the cell medium and DNA synthesis analysed. Basal incorporation of BrdU (<3%) was evaluated and subtracted. Data derived from at least 150 scored cells were pooled. The means ± SEM are shown. Lysates from quiescent MCF-7 cells were immunoprecipitated with anti-Src antibody. (C) Src kinase activity was assayed. (D) Immunocomplexes were probed with either anti-Src (upper), anti-AR (middle) or anti-ERα (lower) antibodies.

Fig. 6. Androgen- and oestradiol-induced association of ER–AR–Src in transiently transfected Cos cells. (A) Cos cells were transfected with void pSG5 or pSG5-AR vectors, then treated for 2 min with 10 nM R1881 alone or in the presence of 10 µM Casodex or 10 µM ICI 182,780. Cell lysates were immunoprecipitated with anti-Src antibody and immunoprecipitates subjected to SDS–PAGE. (B) Cos cells were transfected with pSG5 or pSG5-ERβ and treated for 2 min with 10 nM oestradiol alone or in the presence of 10 µM ICI 182,780 or 10 µM Casodex. Lysates were immunoprecipitated with anti-ERβ antibody and immunoprecipitates subjected to SDS–PAGE. (C) Cos cells were transfected with void pSG5 vector or with both pSG5-AR vector and pSG5-ERβ. They were treated for 2 min with 10 nM oestradiol alone or in the presence of 10 µM ICI 182,780 or 10 µM Casodex, or with 10 nM R1881 alone or in the presence of 10 µM ICI 182,780 or 10 µM Casodex. (D) Cos cells were transfected with pSG5 or with both pSG5-hAR and pSG5-HEG0 encoding human ERα. They were treated for 2 min with 10 nM oestradiol alone or in the presence of 10 µM ICI 182,780 or 10 µM Casodex, or with 10 nM R1881 alone or in the presence of 10 µM ICI 182,780 or 10 µM Casodex. Cell lysates were immunoprecipitated with anti-Src antibody and immunoprecipitates subjected to SDS–PAGE. (C and D) Proteins transferred onto nitrocellulose filters were probed with anti-hAR, anti-ERβ, anti-Src or H222 anti-ERα antibodies. The arrows indicate the expected position of the indicated proteins.

Fig. 8. Steroid-stimulated in vitro association of hAR with Src via its SH3 domain. (A) (Left) GST–Ag, GST–Src, GST–ΔSH2 and GST–ΔSH3 (ΔSH3 is Src with the SH3 domain deleted) were incubated in the presence of 10 nM R1881 with [³⁵S]AR synthesized in reticulocyte lysate. (Right) GST–Ag, GST–Src, GST–SH2 and GST–SH3 were incubated with [³⁵S]AR in the presence of 10 nM R1881. (B) GST–Src or GST–SH3 was incubated with [³⁵S]AR in the absence or presence of R1881. Proteins were eluted with SDS, subjected to SDS–PAGE and revealed by fluorography. (C) Different ³⁵S-labelled AR deletion mutants were incubated with GST–SH3 in the presence of 10 nM R1881. Equivalent aliquots from samples incubated with GST–SH3 (loaded), break-through (BT) and SDS-eluted (El.) proteins were subjected to SDS–PAGE and revealed by fluorography. The bars below the figure are a diagrammatic representation of AR deletion mutants used in the experiment. Mutants 1–3 include amino acids 1–370, 1–422 and 1–564 of hAR, respectively. W indicates the wild-type receptor. The dashed bar corresponds to the 372–379 proline stretch of AR.

Fig. 1. Effect of androgen and oestradiol on LNCaP cell growth and S-phase entry under different conditions. (A) Quiescent LNCaP cells were left untreated (filled circles) or treated for the indicated times with either 10 nM oestradiol in the absence (open circles) or presence of 10 µM ICI 182,780 (upward filled triangles) or with 10 nM R1881 in the absence (filled squares) or presence (downward filled triangles) of 10 µM Casodex. Control cells were also treated with 10 µM ICI 182,780 (open squares) or 10 µM Casodex (crosses) alone. (B) Cells were left untreated (filled circles) or treated for the indicated times with either 10 nM oestradiol in the absence (open circles) or presence (downward filled triangles) of 10 µM Casodex or with 10 nM R1881 in the absence (filled squares) or presence (upward filled triangles) of 10 µM ICI 182,780. (C) Quiescent LNCaP cells were left unstimulated or stimulated with the indicated compounds for 24 h. BrdU was added and the cells were fixed and permeabilized. DNA synthesis was followed as described in Materials and methods. Several coverslips were analysed and BrdU incorporation calculated according to the formula: percentage BrdU-positive cells = (BrdU-positive cells/total cells) × 100. Results from different experiments were pooled. The means ± SEM are shown. (D) Quiescent LNCaP cells were stimulated for 24 h with either 10 nM oestradiol (right) or 10 nM R1881 (left) in the absence or presence of the indicated inhibitors. PD98059 was utilized at 50 µM and PP1 at 10 µM. BrdU was included in the cell medium and DNA synthesis analysed as described in Materials and methods. Data from different coverslips were pooled and residual BrdU incorporation was expressed as a percentage of steroid-stimulated incorporating cells, which was 28 and 25% for oestradiol- and androgen-stimulated cells, respectively. The basal BrdU incorporation (<5%) was evaluated and subtracted in each case. (E) Quiescent LNCaP cells were either not injected or injected with the indicated constructs (either SrcK^– or A221 MEK-1). Either 10 nM oestradiol (left) or 10 nM R1881 (right) was added to the cells for 24 h. BrdU incorporation into DNA was analysed as in (D). Steroid-stimulated BrdU incorporation was 26 and 27% for oestradiol- and androgen-stimulated cells, respectively. The basal BrdU incorporation (<4%) was evaluated and subtracted. For each plasmid, data are derived from at least 150 scored cells. The means ± SEM are shown. A construct expressing the pSG5 empty plasmid was also microinjected into nuclei of quiescent LNCaP cells together with plasmid pEGFP, as an injection marker. The cells injected with pSG5 were left unstimulated or stimulated with either oestradiol or R1881. A total of 23 and 24% of the GFP-expressing LNCaP cells entered S-phase upon stimulation with oestradiol and R1881, respectively. BrdU incorporation of unstimulated cells was <3.5%.

Fig. 3. Androgen and oestradiol trigger ERβ–AR–Src association in LNCaP cells. Quiescent LNCaP cells were left untreated or treated for 2 min with either 10 nM oestradiol alone or in the presence of 10 µM Casodex or 10 µM ICI 182,780, or with 10 nM R1881 in the absence or presence of 10 µM ICI 182,780 or 10 µM Casodex. (A) Cell lysates were immunoprecipitated with anti-Src antibodies. Parallel samples were precipitated with control antibodies. Immunoprecipitated proteins were resolved by SDS–PAGE, transferred onto nitrocellulose filters, then probed with anti-Src, anti-hAR or anti-hERβ antibodies. (B) Cell lysates were immunoprecipitated with anti-hAR or control antibodies. Immunoprecipitates were resolved by SDS–PAGE, transferred to nitrocellulose filters and probed with anti-hAR, anti-Src or anti-ERβ antibodies. The arrows indicate the expected position of the indicated proteins.

Fig. 5. Effect of androgen and oestradiol on Src activity in Cos cells transiently transfected with ER and AR cDNAs. (A) Cos cells were transfected with pSG5-ERβ vector encoding human ER (β form) and treated for 2 min with 10 nM oestradiol alone or in the presence of 10 µM ICI 182,780 or 10 µM Casodex. (B) Cells were transfected with pSG5-AR vector encoding human AR and treated for 2 min with 10 nM R1881 alone or in the presence of 10 µM Casodex or 10 µM ICI 182,780. (C) Cells were co-transfected with pSG5-ERβ and pSG5-AR. They were either untreated or treated for 2 min with 10 nM oestradiol alone or in the presence of 10 µM Casodex or 10 µM ICI 182,780, or with 10 nM R1881 alone or in the presence of 10 µM Casodex or 10 µM ICI 182,780. (D) Cells were co-transfected with pSG5-ERα and pSG5-AR. They were treated for 2 min either with 10 nM oestradiol alone or in the presence of 10 µM ICI 182,780 or 10 µM Casodex, or with 10 nM R1881 alone or in the presence of 10 µM ICI 182,780 or 10 µM Casodex. Lysates were immunoprecipitated with anti-Src antibody and immunoprecipitates assayed for Src kinase activity using acidified enolase as substrate. The arrows (en) indicate the enolase position.

Fig. 7. Role of the Src SH2 domain in hER–Src association and signalling activation by steroids. (A) (Left) GST–agarose (GST-Ag), GST–Src and GST–SH2 were incubated in the presence of 10 nM oestradiol with either HEG0 or HEG537F labelled with [³⁵S]methionine during their synthesis in reticulocyte lysate. (Middle) GST–Ag, GST–SH2 and GST–SH3 were incubated with [³⁵S]HEG0 in the presence of 10 nM oestradiol. (Right) GST–Ag, GST–Src and GST–SH2 were incubated with [³⁵S]hERβ in the presence of 10 nM oestradiol. Proteins were eluted with SDS, subjected to SDS–PAGE and revealed by autoradiography. (B) (Left) GST–Ag and GST–HEG14 (HEG14 is the C-terminal half of HEG0) were incubated in the presence of 10 nM oestradiol alone or together with either [³⁵S]Src or [³⁵S]Src lacking the SH2 domain (ΔSH2). (Right) GST–HEG14 was incubated in the absence or presence of 10 nM oestradiol with either Src or ΔSH2. Proteins were eluted with SDS, subjected to SDS–PAGE and revealed by fluorography. (C) Cos cells, transfected with either empty pSG5 vector or vector pSG5-HEG0 or pSG5-HEG537F, were treated with 10 nM oestradiol for 2 min. Cell lysates were immunoprecipitated with anti-Src antibody. The immunoprecipitates were blotted either with H222 anti-ER (lower) or anti-Src (upper) antibody. (D) Cells transfected as in (C) were incubated in the absence or presence of 10 nM oestradiol for 2 min. Immunoprecipitates by anti-Src antibody were assayed for Src activity using enolase (en) as substrate. (E) Cos cells were transfected with the empty pSG5 vector or PSG5-PR-B together with either PSG5-HEG0 or pSG5-HEG537F. Cells were stimulated for 2 min with 10 nM R5020 progestin. Src activity of the immunoprecipitates from cell lysates was assayed. (F) Quiescent LNCaP cells were injected with either GFP- or GFP-SH2-expressing plasmids. Cells were then left unstimulated or stimulated with either 10 nM oestradiol or R1881. BrdU was included in the cell medium and after 24 h DNA synthesis was analysed. It was calculated as in Figure 1C. Data from several coverslips were pooled and evaluated statistically. The means ± SEM are also shown.

Fig. 9. Model of Src activation by androgen or oestradiol. The kinase domain of Src is held in a restrained, inactive conformation by intramolecular interactions: binding of SH2 with PTyr527 and binding of SH3 with the SH2 kinase linker. Each steroid (A or E₂) in the presence of both AR and ERα is able to enhance kinase activity by inducing binding of a proline-rich sequence of AR to SH3 of Src and phosphotyrosine 537 of ERα to SH2, thus changing the inactive conformation of Src (left) to a ‘fully’ active conformation (right). In the presence of either AR or ER alone, androgen or oestradiol leads to a binary AR/Src or ER/Src complex. In this complex, Src might assume an intermediary conformation corresponding to ‘partial’ activation. ERβ behaves like ERα.