Phosphorylation of MNAR promotes estrogen activation of phosphatidylinositol 3-kinase

Abstract

Estrogen actions are mediated by a complex interface of direct control of gene expression (the so-called "genomic action") and by regulation of cell signaling/phosphorylation cascades, referred to as the "nongenomic," or extranuclear, action. We have previously described the identification of MNAR (modulator of nongenomic action of estrogen receptor) as a novel scaffold protein that regulates estrogen receptor alpha (ERalpha) activation of cSrc. In this study, we have investigated the role of MNAR in 17beta-estradiol (E2)-induced activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Consistent with our previous results, a direct correlation was established between MNAR expression levels and E2-induced activation of PI3 and Akt kinases. Endogenous MNAR, ERalpha, cSrc, and p85, the regulatory subunit of PI3 kinase, interacted in MCF7 cells treated with E2. The interaction between p85 and MNAR required activation of cSrc and MNAR phosphorylation on Tyr 920. Consequently, the mutation of this tyrosine to alanine (Y920A) abrogated the interaction between MNAR and p85 and the E2-induced activation of the PI3K/Akt pathway, which was required for the E2-induced protection of MCF7 cells from apoptosis. Nonetheless, the Y920A mutant potentiated the E2-induced activation of the Src/MAPK pathway and MCF7 cell proliferation, as observed with the wild-type MNAR. These results provide new and important insights into the molecular mechanisms of E2-induced regulation of cell proliferation and apoptosis.

FIG. 1.

MNAR affects E2-induced activation of PI3 kinase and Akt. (A) MNAR expression analysis. MCF7 cells were transfected with MNAR-specific siRNA or a plasmid for MNAR overexpression. Forty-eight hours after transfection, whole-cell extracts were evaluated using Western blot analysis with anti-MNAR antibody. (B) MCF7 cells were transfected with the indicated plasmid; 48 h after transfection, cells were treated with dimethyl sulfoxide or E2 (10 nM) for 20 min in the absence or presence of 1 μM ICI-182,780 or 10 μM LY294002. Cells were harvested, and PI3K activity in material immunoprecipitated using p85 antiserum was evaluated using a PI3K activity kit according to the manufacturer's protocol (Echelon Inc.). Bars indicate means ± standard errors of the means for triplicate determinations. (C) MCF7 cells were treated as described above, and the levels of total and phosphorylated Akt in cell extracts were evaluated using Western blot analysis. (D) MCF7 cells were transfected with pcDNA vector or an MNAR expression plasmid. Forty-eight hours after transfection, cells were treated with dimethyl sulfoxide or treated with E2 (10 nM) for 20 min. Akt activity in material immunoprecipitated using Akt antiserum was evaluated. GSK3 was used as a substrate. The results are representative of experiments performed at least in triplicate.

FIG. 2.

Endogenous cSrc, ERα, MNAR, and p85 interact in MCF7 cells treated with E2. Quiescent MCF7 cells were treated with dimethyl sulfoxide or 10 nM E2 for 20 min. Total cell extracts were used for immunoprecipitation (IP) with anti-ERα (A), anti-p85 (B), or anti-cSrc (C) antibodies. Immunoprecipitates were then probed with antibodies against the indicated proteins. (D) cSrc activation is required for MNAR interaction with p85. MCF7 cells were treated with 10 nM E2 alone or in combination with PP2 (10 μM) or ICI (10 μM). MNAR was immunoprecipitated using anti-MNAR antibody, and the obtained material was probed with p85 and MNAR antiserum. The results are representative of experiments performed at least in triplicate.

FIG. 3.

Cell treatment with E2 leads to MNAR phosphorylation by cSrc on tyrosine 920. (A) Treatment of MCF7 cells with E2 leads to MNAR phosphorylation. MCF7 cells were treated with 10 nM E2 for 20 min, and cell lysates were used for immunoprecipitation (IP) with MNAR antiserum. Precipitated material was probed using Western blot analysis with antiphosphotyrosine and anti-MNAR antibodies. (B) MNAR phosphorylation on Y920 requires activation of cSrc and ERα. MCF7 cells were treated for 20 min with dimethyl sulfoxide or E2 (10 nM) alone or in combination with the indicated inhibitor and then probed with anti-phospho-Y920 MNAR or anti-MNAR antibodies. Material immunoprecipitated with anti-MNAR antibody was probed with anti-phospho-Y920 MNAR antibody in the presence of competing (phosphorylated) or control (nonphosphorylated) peptide, both at 50 μM. (C) Tyrosine 920 is required for MNAR interaction with p85. MCF7 cells transfected with an empty vector or plasmids for expression of wild-type FLAG-tagged MNAR or the Y920A FLAG-tagged MNAR mutant were treated with 10 nM E2 for 20 min. Cell lysates were used for immunoprecipitation with anti-FLAG antibody. Precipitated material was probed with antibodies against the indicated proteins. The results are representative of experiments performed at least in triplicate.

FIG. 4.

Mutation of MNAR tyrosine 920 to alanine abrogates E2-induced activation of PI3 kinase. (A) COS7 cells were transfected with wild-type FLAG-MNAR or the Y920A FLAG-tagged MNAR mutant. Cell extracts were evaluated using Western blot analysis with anti-MNAR antibody 48 h after transfection. (B) COS7 cells were transfected with empty vector or plasmids for expression of the wild-type MNAR or the MNAR Y920A mutant. Transfected cells were treated with 10 nM E2 (red bars) or vehicle (black bars) for 20 min, and the relative PI3K activities were determined. Means ± standard errors of the means for two experiments are presented. (C) MCF7 cells were transfected with 50 or 500 ng of MNAR expression plasmid or 500 ng of empty vector prior to treatment with 10 nM E2. Levels of phosphorylated Akt (S473), total Akt, and MNAR were determined using Western blot analysis. The results are representative of experiments performed at least in triplicate. DMSO, dimethyl sulfoxide.

FIG. 5.

MNAR Y920A mutant stimulates E2-induced activation of the Src/MAPK pathway. (A) MNAR expression in COS7 cells. COS7 cells were left untransfected or transfected with an empty vector or vectors for expression of the FLAG-tagged wild type or the Y920A MNAR mutant. Cell extract was used for Western blot analysis with MNAR antibody. (B) MNAR Y920A mutant stimulates E2-induced activation of cSrc in COS7 cells. Transfected cells were treated with 10 nM E2 (red bars) or dimethyl sulfoxide (black bars) for 20 min, and cSrc kinase activity was evaluated. Bars indicate means ± standard errors of the means for triplicate determinations. (C) MNAR Y920A mutant stimulates E2-induced activation of Erk 1/2 kinases in MCF7 cells. MCF7 cells were transfected with 500 ng of MNAR-expressing or control (pcDNA) plasmid. Transfected cells were treated with 10 nM E2 or vehicle for 20 min. Levels of phosphorylated Erk 1/2 and total Erk 1/2 kinases and total MNAR were evaluated using Western blot analysis. (D) Gels presented in panel C were scanned, and the relative band densities were evaluated. Presented graphs show the normalized pErk activation levels (the ratios of the phosphorylated- to total-Erk levels) and the MNAR expression levels. The results are representative of experiments performed in triplicate. NT, not transfected; ERa, ERα; INT, intensity.

FIG. 6.

MNAR is an important regulator of MCF7 cell proliferation and survival. (A) MNAR expression in MCF7 cells. MCF7 cells were transfected with specific or unspecific siRNA, empty vector, or vectors for expression of the wild type or the Y920A MNAR mutant. Cell extract was used for Western blot analysis with MNAR antibody. (B) MNAR potentiates E2-induced proliferation of MCF7 cells. MCF7 cells were transfected with the indicated plasmid or siRNA. Cells were treated with 10 nM E2 for 24 h and then with BrdU at 10 μM. BrdU incorporation was determined as described in Materials and Methods. (C) MNAR plays an important role in E2-induced protection from apoptosis. MCF7 cells were transfected with specific or unspecific siRNA. Cells were treated with E2 (10 nM) and TNF-α (100 ng/ml). Forty-eight hours after the treatment, cell lysates were evaluated using Western blot analysis with antiserum against MNAR, cleaved PARP, and beta-actin. (D) Normalized PARP cleavage levels (%) (ratios of cleaved- to total-PARP levels). (E) Transfection with the MNAR Y920A mutant abrogates E2-induced protection of MCF7 cells from apoptosis. MCF7 cells were transfected with a GFP-expressing plasmid and empty-vector (black), wild-type MNAR (red), or MNAR Y920A (green) expression plasmids. Cells were incubated with vehicle (open bars) or 10 nM E2 (filled bars) alone or in combination with TNF-α (100 ng/ml). Forty-eight hours after treatment, percentages of apoptotic cells were determined microscopically by calculating TUNEL-positive staining or condensed chromatin in GFP-expressing cells. Averages for at least eight fields are shown. The results are representative of experiments performed in triplicate. Bars indicate means ± standard errors of the means. DMSO, dimethyl sulfoxide.

FIG. 7.

MNAR coordinates E2-induced activation of important cell signaling pathways. Interactions with ERα and MNAR lead to activation of cSrc, MNAR phosphorylation on tyrosine 920, interaction with p85, and activation of PI3K. While E2-induced activation of the Src/MAP kinase pathway stimulates MCF7 cell proliferation, activation of the PI3K/Akt pathways by E2 controls cell resistance to apoptosis.