PMC

Src is activated by various growth factor (GF) receptors, but also by E2 stimulation, at least in the presence of GFs. Memo interacts with Src and ERα to promote their binding upon HRG and E2 stimulation. This in turn increases Y418-Src and Y537-ERα phosphorylation as well as the extra-nuclear retention of ERα. Memo is thus a crucial component at the intersection between E2 and HRG (and possibly other GF) signaling, and can thus promote their downstream effect on cell migration and proliferation.

A. Western blot analysis of endogenous ERα and GFP-tagged WT or Y537F ERα. Asterisk indicates an unspecific band. B. Immunofluorescence (IF) analysis showing the cytoplasmic/nuclear localization of ERα-WT-GFP and ERα-Y537F-GFP in NT T47D cells treated with DMSO (vehicle), HRG and/or E2 for 10 minutes. Nuclei were stained with DAPI. 40x magnification, Scale bar: 30 μM. C. Quantification of nuclear ERα-GFP IF intensity as percentage of total ERα IF intensity (n = 6). The data shown in (C) represent means and error bars represent standard deviation (S.D.), P values were determined using one-way ANOVA.

A. Immunofluorescence (IF) analysis showing the cytoplasmic/nuclear localization of ERα and Memo in NT and Sh5 T47D cells treated with 4-OHT, HRG and E2 for 10 minutes. Nuclei were stained with DAPI. 40x magnification, Scale bar: 50 μM. B. Quantification of nuclear ERα IF intensity as percentage of total ERα IF intensity (n = 4). C. Quantification of nuclear Memo IF intensity as percentage of total Memo IF intensity (n = 4). D. Western blot analysis of PY537-ERα, PS118-ERα, and PY418-Src levels in starved T47D cells treated with 2 nM HRG, 10 nM E2, DMSO (Veh), and/or 20 nM 4-OHT for 10 min. E. Quantification of PY537-ERα levels relative to total ERα levels (AU, arbitrary units) (n = 3). F. Quantification of PS118-ERα levels relative to total ERα levels (AU, arbitrary units) (n = 3). G. Quantification of PY418-Src levels relative to β-Actin levels (AU, arbitrary units) (n = 3). The data shown in (B, C, E - F) represent means and error bars represent S.D. Significance levels written in blue show the significance between 4-OHT treatment and the respective treatment without 4-OHT. P values were determined using Student's t-test or one-way ANOVA.

A. Migration assay. Starved T47D cells were seeded into the upper transwell chamber. The lower wells contained phenol-free DMEM supplemented with 0.5% DCC-treated FCS and 2 nM HRG, 10 nM E2, DMSO (Veh), and/or 20 nM 4-OHT. After 24 h the migrated cells were fixed, stained and counted. The migration is expressed relative to respective DMSO treated cells (n = 5). B. Proliferation assay. T47D cells were starved for 5 days in phenol-free DMEM supplemented with 0.5% DCC-treated FCS, and in the presence of 2 nM HRG, 10 nM E2, DMSO (Veh), and/or 20 nM 4-OHT. Viable cells were counted and the proliferation change was assessed relative to respective DMSO treated cells (n = 5). C. Proliferation of LZ (control) and Sh5 (Memo KD) SKBR3 cells was assessed as in (B) in the presence of 2 nM HRG, 10 nM E2, and/or DMSO (Veh). Viable cells were counted and the proliferation change was assessed relative to respective DMSO treated cells (n = 5). D. Western blot analysis of PY418-Src levels in LZ and Sh5 SKBR3 cells treated with 2 nM HRG, 10 nM E2, and/or DMSO (Veh) for 10 min. The data shown in (A - C) represent means and error bars represent S.E.M. The data shown in (D) is representative of 3 independent experiments (n = 3). Significance levels written in blue show the significance between 4-OHT treatment and the respective treatment without 4-OHT. P values were determined using Student's t-test or one-way ANOVA.

A. Western blot analysis of Memo protein levels in T47D NT control and Sh5 Memo KD cells. B. ERα activity upon E2 and or HRG stimulation for 48h. Firefly luciferase activity was normalized to renilla luciferase activity (n = 3). C. Relative mRNA expression of GREB1 in T47D cells cultured in the presence of DMSO (Vehicle, Veh), HRG, E2 and HRG+E2 for 24 h (n = 5). D. Relative mRNA expression of PS2 in T47D cells cultured as in (B) (n = 5). E. Relative mRNA expression of Cyclin D1 in T47D cells cultured as in (B) but for 6h (n = 3). F. ChIP analysis showing the recruitment of ERα to the ERE promoter sequence of Cyclin D1 in T47D NT and Sh5 cells treated with DMSO, HRG, and/or E2 for 30 min (n = 3). Dotted line indicates enrichment level with non-specific IgG. G. Immunofluorescence (IF) analysis showing the cytoplasmic/nuclear localization of ERα and Memo in NT and Sh5 T47D cells treated with DMSO (vehicle), HRG and/or E2 for 10 minutes. Nuclei were stained with DAPI. 40x magnification, Scale bar: 30 μM. H. Quantification of nuclear ERα IF intensity as percentage of total ERα IF intensity (n = 7). I. Quantification of nuclear Memo IF intensity as percentage of total Memo IF intensity (n = 5). The data shown in (B-F) and (H-I) represent means and error bars represent standard deviation (S.D.), P values were determined using Student's t-test or one-way ANOVA.

A. Western blot analysis of ERα phosphorylation status in T47D NT and Sh5 cells treated with DMSO (Veh), HRG, and/or E2 for 10 min. B. Quantification of relative PY537-ERα levels (n = 3) in the presence or absence of 500 nM Src inhibitor-1. C. Quantification of relative PS118-ERα levels (n = 3). D. Western blot analysis of HER2, Src, Akt, and Erk1/2 phosphorylation status in T47D NT and Sh5 cells treated with DMSO, HRG, and/or E2 for 10 min. E. Quantification of relative PY418-Src levels (n = 3). F. Immunoprecipitation (IP) of Src in NT and Sh5 T47D cells treated for 10 min with DMSO (Veh), HRG (H) and/or E2 (E), followed by immunoblotting (IB) for HER2, ERα, Memo and Src. G. Memo and Myc-Memo protein levels in T47D cells transfected with an empty pLHCX vector (Ev), Sh5 KD construct, and Sh5 cells transfected with pLHCX-Myc-Memo (Sh5-Myc-Memo). H. IP of Myc-Memo in T47D Sh5-Myc-Memo cells treated for 10 min with DMSO (Veh), HRG and/or E2, followed by immunoblotting (IB) for HER2, ERα, Src, and Memo. T47D Ev cells were used as a control. I. Proposed model for Src, Memo and ERα interaction (I-L). Under basal conditions, without HRG or E2 stimulation, Memo associates with both Src and ERα. However, Src and ERα do not appear to bind each other directly. J. HRG treatment induces HER2 heterodimerization and phosphorylation as well as recruitment of the Memo-Src-ERα complex to HER2. The HER2 activation promotes phosphorylation and activation of Src as well as Erk1/2 and Akt pathways. This in turn promotes ligand independent activation, likely through its phosphorylation at S167, and a strong Memo-dependent ERα nuclear translocation. K. Upon E2 treatment ERα changes conformation that likely disrupts the ERα-Memo interaction. However, the ERα interaction with the Src-HER2 complex is still Memo dependent. E2 promotes ERα S118 and Y537 phosphorylation, resulting in ERα activation and nuclear translocation. L. Upon combined HRG and E2 treatment, Memo binds to HER2, and the E2 binding to ERα prevents Memo's complex formation with ERα. Nevertheless, Memo is required for this Src-ERα interaction. The HRG activation of HER2 and Src increases the binding of ERα to Src. This in turn increases the phosphorylation of ERα on S118 and especially Y537, resulting in a very tight Src-ERα complex and preventing ERα from entering the nucleus. The data shown in (B, C, E) represent means and error bars represent standard deviation (S.D.). The IPs (F) and (H) are representative of 3 independent experiments. P values were determined using Student's t-test or one-way ANOVA.