FIG. 7.

Persistent c-Src activation is required for progestin-induced sustained Erk1/2 activity. (A) T47D-YB cells were pretreated for 30 min with DMSO vehicle control, U0126, the c-Src family inhibitor PP2 or SU6656 (SU), or the PI3-K inhibitor, LY294002 (LY), followed by 18 h of R5020 treatment, and whole-cell lysates were Western blotted for phospho- (p) and total (t) Erk1/2 MAPKs. The specificity and activity of each c-Src inhibitor, and the LY294002 and SB203580 compounds, were confirmed by Western blotting for activated (phospho) forms of c-Src, Akt and p38, respectively (not shown). (B) T47D-YB cells were treated for 18 h with R5020 or vehicle control or 5 min (m) with EGF as a positive control for c-Src activation. Tyrosine-phosphorylated proteins were immunoprecipitated using 4G10 antibody-conjugated agarose beads (p-Tyr) or IP IgG control and Western blotted with c-Src-specific antibodies. Western blotting of c-Src in whole-cell lysates (10% of IP input) indicated no effect of vehicle control or R5020 treatments on total c-Src levels. The results were confirmed in two independent experiments. (C) T47D-YB cells were pretreated (pre) for 30 min with DMSO (lanes 1 and 2, 5 and 6) or PP2 (lanes 3 and 4, 7 and 8) prior to the addition of R5020 (+) or EtOH vehicle control (−) for 10 min or 18 h. In lanes 9 and 10, PP2 was added at the beginning of the 12th hour (post) of R5020 treatment. Cell lysates were Western blotted for phospho- or total Erk1/2 MAPK, cyclin D1, or total PR. (D) Progestin-induced persistent c-Src activity downstream of EGFR signaling to sustained activation Erk1/2 MAPK and cyclin D1 upregulation. (Inset) c-Src functions downstream of EGFR. T47D-YB cells pretreated for 30 min with DMSO, PP2, or SU6656 were treated for 18 h with R5020. EGFR was immunoprecipitated using specific antibodies or control IgG, and immune complexes were subjected to Western blotting to detect active (phospho-Tyr1173) EGFR. The membranes were stripped and reprobed to detect the total EGFR pulled down in each IP reaction. Whole-cell lysates indicate 10% of IP input. (E) T47D-YB cells were plated in soft agar as described in Materials and Methods and treated without (−) or with (+) R5020 in the presence of DMSO, U0126 (U0), PP2, or PD168393 (PD). The bars represent the increase (n-fold) in colony numbers (plus standard deviation) for cell cultures treated with progestin over EtOH DMSO controls, and the asterisk denotes statistical significance (P < 0.01) determined by an unpaired Student's t test. Except where noted, experiments were repeated three times with similar results.