Figure 1. Enhancement of ERα activity by Constitutively Active MEKK1 in Endometrial Cancer Cells A, Ishikawa cells were transfected with 0.5 μg EREe1bCAT and 0.5 μg pLENβgal with or without 0.1 μg pLEN-hERα and treated with or without 10⁻¹⁰m 17β-estradiol (E₂) as described in Materials and Methods. CAT activity was determined and normalized to β-gal activity. B, Cells were transfected with the same constructs as in panel A but together with 0.2 μg of SRαMEKK1(CT), SRαHAMEKK2(CT), SRαRAF(B×B) or parental vector SRα3. After treatment with indicated concentrations of 17β-estradiol, CAT activity was determined, and normalized activity is expressed as fold activation relative to SRα3 activity in the presence of 10⁻¹⁰m 17β-estradiol. C, Cells were transfected with the same constructs as in panel A and 0.2 μg of SRαMEKK1(CT) or SRα3. Transfected cells were treated with or without 10⁻⁹m 17β-estradiol and 10⁻⁷m ICI 182,780 (ICI) as indicated. CAT activity is expressed as in panel B. D, Cells were transfected with 0.5 μg EREe1bCAT or EREtkCAT, 0.5μ g pLENβgal, 0.1 μg pLEN-hERα, and 0.2 μg of SRα3 or SRαMEKK1(CT). Transfected cells were treated with 10⁻⁸m 17β-estradiol, and CAT activity is expressed as in panel B. E, Immunodetection of human ERα from lysates of MCF-7 (positive control) or Ishikawa cells transfected with SRα3, SRαMEKK1(CT), SRαHAMEKK2(CT), or SRαRAF(B×B) and with or without pLEN-hERα. Transfected cells were treated with (+E₂) or without (−E₂) 10⁻⁸m 17β-estradiol. Duplicated samples from the transfected cells were analyzed and shown. From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society

Figure 2. Correlation of MEKK1-Induced ERα Activation with MEKK1-Induced Activation of Endogenous AP1 A, Ishikawa cells were transfected with 0.5 μg EREe1bCAT, 0.5μ g pLENβgal, 0.1 μg pLEN-hERα, and the indicated amounts of SRαMEKK1(CT), SRαHAMEKK2(CT), SRαRAF(B×B), or SRα3. Transfected cells were treated with 10⁻⁸m 17β-estradiol. B, Ishikawa cells were transfected with 0.2 μg AP-1-CAT, 0.5 μg pLENβgal, and indicated amounts of SRαMEKK1(CT), SRαHAMEKK2(CT), SRαRAF(B×B), or SRα3; CAT activity was determined, and normalized activity is expressed as fold activation relative to SRα3 activity. The total amount of DNA used in transfections was equalized with SRα3. From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society

Figure 3. ERα Activation by Full-Length MEKK1 and Active Rac1 A, Ishikawa cells were transfected with 0.5 μg EREe1bCAT, 0.5 μg pLENβgal, 0.1 μg pLEN-hERα, and indicated amounts of SRα3 or SRα₅1p1 which encodes full-length MEKK1. Transfected cells were treated with (+E₂) or without (−E₂) 10⁻⁸m 17β-estradiol. B, Ishikawa cells were transfected with 0.5 μg EREe1bCAT, 0.5 μg pLENβgal, 0.1 μg pLEN-hERα, 0.2 μg Rac1L61 or SRα3, and indicated amounts of dominant negative MEKK1 (DN-MEKK1) or dominant negative MEKK2 (DN-MEKK2). Transfected cells were treated with 10⁻⁸m 17β-estradiol; CAT activity was determined and normalized activity is shown. From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society

Figure 4. Blockage of MEKK1-Mediated ERα Activation by Dominant Negative JNKK1 but Not by a MEK Inhibitor A, Ishikawa cells were transfected with 0.5 μg EREe1bCAT, 0.5 μg pLENβgal, 0.1 μg pLEN-hERα, and, as indicated, with or without 0.2 μg SRαMEKK1(CT) or SRαHAJNKK1(AL), which encodes dominant negative JNKK1 (DN-JNKK). The total amount of DNA used in transfections was equalized with SRα3. Transfected cells were treated with 10⁻⁸m 17β-estradiol and CAT activity was determined. B, Cells were transfected with 0.5 μg EREe1bCAT, 0.5μ g pLENβgal, 0.1 μg pLEN-hERα, and either 0.2 μg SRα3 or 0.2 μg SRαMEKK1(CT). Transfected cells were treated with or without 10⁻⁸m 17β-estradiol (E₂) and indicated concentrations of PD98059. PD98059 was dissolved in dimethylsulfoxide (DMSO), and the absolute amount of DMSO added to all samples was balanced. CAT activity was determined. C, Cells were transfected with 0.2 μg SRα3 or SRαRAF(B×B) and with or without 0.5 μg HA-ERK2. Transfected cells were treated with indicated concentrations of PD98059. HA-ERK2 was immunoprecipitated from cell lysates using anti-HA antibody, and in vitro immunocomplex kinase assays were performed using MBP (3 μg) as substrate (top panel). The level of HA-ERK2 expression was determined by Western blotting using the same anti-HA antibody (bottom panel). P-MBP, Phosphorylated MBP. Duplicated samples were analyzed and shown for both assays. From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society

Figure 11. The Effect of Active MEKK1 on Both the Agonistic and Antagonistic Activity of 4-Hydroxytamoxifen Ishikawa cells were transfected with 0.5 μg EREe1bCAT, 0.5 μg pLENβgal, 0.1 μg pLEN-hERα, and 0.2 μg of SRαMEKK1(CT) or SRα3. Transfected cells were treated with either vehicle (none), 10⁻⁸m 17β-estradiol (E₂), 10⁻⁶m 4-hydroxytamoxifen (4-HT), 10⁻⁸m 17β-estradiol plus 10⁻⁶m 4-hydroxytamoxifen (E₂+ 4-HT), or 10⁻⁸m 17β-estradiol plus 10⁻⁷m ICI 182,780 (E₂ + ICI) as indicated. CAT activity was determined. From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society

Figure 10. Activation of ERα by MEKK1 in Endometrioid Ovarian Cancer Cells A, MDAH-2774 endometrioid ovarian cancer cells were transfected with 0.5 μg EREe1bCAT, 0.5 μg pLENβgal, 0.1 μg pLEN-hERα, and 0.15μ g of either SRαMEKK1(CT), SRαHAMEKK2(CT), SRαRAF(B×B), or SRα3. Transfected cells were treated with vesicle (−hormone), 10⁻⁹m 17β-estradiol (E₂) or 10⁻⁹m 17β-estradiol plus 10⁻⁷m ICI 182,780 (E₂ + ICI) as indicated. B, OV1063 serous ovarian cancer cells were transfected with 0.5 μg EREe1bCAT, 0.5 μg pLENβgal, 0.1 μg pLEN-hERα, and indicated amounts of SRαMEKK1(CT), SRαHAMEKK2(CT), SRαRAF(B×B), or SRα3. Transfected cells were treated with 10⁻⁹m 17β-estradiol; cells were harvested and assayed for CAT activity. From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society

Figure 9. Phosphorylation of Purified ERα Protein by p38 A, Ishikawa cells were transfected with 0.2 μg of either SRαMEKK1(CT) or SRα3 and where indicated, 0.5 μg Flag-p38. Flag-p38 was immunoprecipitated with anti-Flag antibody, and in vitro immunocomplex kinase assays were performed using recombinant human ERα protein (0.5 μg) as substrates (top panel). The level of Flag-p38 expression was determined by Western blotting using the same anti-Flag antibody (bottom panel). P-ER, Phosphorylated ER. B, Cells were transfected with 0.5 μg Flag-p38 and 0.2 μg of either SRαMEKK1(CT) or SRα3. Flag-p38 was immunoprecipitated, and in vitro immunocomplex kinase assays were performed using either recombinant ERα protein (0.5 μg) or GST-ATF2 (3 μg) as substrates (top panel). The level of Flag-p38 expression was determined by Western blotting using the same anti-Flag antibody (bottom panel). P-GST-ATF2, Phosphorylated GST-ATF2. C, Cells were transfected with or without 0.5 μg HA-JNK1 and 0.2 μg of either SRαMEKK1(CT) or SRα3. HA-JNK1 was immunoprecipitated with anti-HA antibody, and in vitro immunocomplex kinase assays were performed using either recombinant human ERα protein (0.5 μg), GST-c-Jun (3 μg), or both as substrates (top panel). The level of HA-JNK1 expression was determined by Western blotting using the same anti-HA antibody (bottom panel). P-GST-c-Jun, phosphorylated GST-c-Jun; duplicated samples were analyzed and shown for both assays in all panels. From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society

Figure 8. Effect of MEKK1 on the Activity of ERα Phosphorylation Site Mutants Ishikawa cells were transfected with 0.5 μg EREe1bCAT, 0.5 μg pLENβgal, 0.2 μg of SRαMEKK1(CT) or SRα3, and 0.1 μg pLEN vector encoding either wild-type or mutant ERα. Transfected cells were treated with or without 10⁻⁸m 17β-estradiol and CAT activity was determined. Solid bars, Cells were transfected with SRα3 and treated with 10⁻⁸m 17β-estradiol (SRα3 + E₂); hatched bars, cells were transfected with SRαMEKK1(CT) and treated with 10⁻⁸m 17β-estradiol (MEKK1 + E₂); double hatched bars, cells were transfected with SRα3 and treated with ethanol (SRα3 − E₂). From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society

Figure 7. Attenuation of MEKK1- and Estrogen-Induced ERα Activation by a p38 Inhibitor, SB203580 A, Effect of SB203580 on MEKK1-induced ERα activation. Ishikawa cells were transfected with 0.5 μg EREe1bCAT, 0.5 μg pLENβgal, 0.1 μg pLEN-hERα, and 0.2 μg of either SRα3 or SRαMEKK1(CT). Transfected cells were treated with or without 10⁻⁸m 17β-estradiol (E₂) and indicated concentrations of SB203580. SB203580 was dissolved in DMSO, and the absolute amount of DMSO added to all samples was balanced. CAT activity was determined. B, Effect of SB203580 on estrogen-induced ERα activation in the absence of ectopic MEKK1 expression. As in panel A except that cells were not transfected with SRαMEKK1(CT). C, Inhibition of p38 activity by SB203580. Cells were transfected with 0.2μ g of either SRα3 or SRαMEKK1(CT) and with or without 0.5 μg Flag-p38. Transfected cells were treated with indicated concentrations of SB203580. Flag-p38 was immunoprecipitated with anti-Flag antibody, and in vitro immunocomplex kinase assays were performed using GST-ATF2 (3 μg) as substrates (top panel). The level of Flag-p38 expression was determined by Western blotting using the same anti-Flag antibody (bottom panel). Duplicated samples were analyzed and shown for both assays. P-GST-ATF2, Phosphorylated GST-ATF2. D, Effect of p38 inhibitor on MEKK1-induced activation of endogenous AP1. Cells were transfected with 0.2 μg AP-1-CAT, 0.5 μg pLENβgal, and 0.2 μg of SRαMEKK1(CT) or SRα3. Transfected cells were treated with indicated concentration of SB203580, harvested, and assayed for CAT activity. From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society

Figure 6. Inhibition of MEKK1- and Estrogen-Induced ERα Activation by Dominant Negative JNK1 A, Inhibition of MEKK1-induced ERα activation by dominant negative JNK1. Ishikawa cells were transfected with 0.5 μg EREe1bCAT, 0.5 μg pLENβgal, 0.1 μg pLEN-hERα, 0.2 μg of either SRα3 or SRα3MEKK1(CT), and indicated amounts of SRαHAJNK1(APF), which encodes a dominant negative JNK1 (DN-JNK1). Transfected cells were treated with or without 10⁻⁸m 17β-estradiol (E₂). B, Inhibition of estrogen-induced ERα activation by DN-JNK1 in the absence of ectopic MEKK1 expression. As in panel A except that cells were not transfected with SRα3MEKK1(CT) DNA. C, Inhibition of MEKK1-induced c-Jun activation by DN-JNK1. Cells were transfected with 0.2 μg 3×17 mer-Gal-CAT, 0.5μ g pLENβgal, 0 .1 μg of pGal-Jun(1–223) or pGal-Jun(1–223)(Ala^63/73), 0.1 μg of SRα3 or SRαMEKK1(CT), and indicated amounts of SRαHAJNK1(APF). D, Lack of inhibition of RAF-induced activation of endogenous AP1 by DN-JNK1. The cells were transfected with 0.2 μg AP-1-CAT, 0.5 μg pLENβgal, 0.2μ g of SRα3 or SRαRAF(B×B), and indicated amounts of SRαHAJNK1(APF). CAT activity was assayed and normalized with β-gal activity in all panels. From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society

Figure 5. Activation of JNK and p38, but Not ERK, by MEKK1 in Ishikawa Cells A, Ishikawa cells were transfected with 0.5 μg HA-JNK1 and 0.2μ g of either SRαMEKK1(CT) or SRα3. HA-JNK1 was immunoprecipitated from the cell lysates using anti-HA antibody, and in vitro kinase assays were performed using GST-c-Jun (3 μg) as substrate (top panel). The level of HA-JNK1 expression was determined by Western blotting using the same anti-HA antibody (bottom panel). P-GST-c-Jun, Phosphorylated GST-c-Jun. B, Cells were transfected with 0.5 μg HA-ERK2 and 0.2 μg of either SRαMEKK1(CT) or SRα3. HA-ERK2 was immunoprecipitated from the cell lysates using anti-HA antibody, and in vitro immunocomplex kinase assays were performed using MBP (3 μg) as substrate (top panel). The level of HA-ERK2 expression was determined by Western blotting using the same anti-HA antibody (bottom panel). P-MBP, Phosphorylated MBP. C, Cells were transfected with 0.2 μg of either SRαMEKK1(CT) or SRα3 and with or without 0.5 μg Flag-p38. Flag-p38 was immunoprecipitated from the cell lysates using anti-Flag antibody, and in vitro immunocomplex kinase assays were performed with GST-ATF2 (3 μg) as substrate (top panel). The level of Flag-p38 expression was determined by Western blotting using the same anti-Flag antibody (bottom panel). P-GST-ATF2, Phosphor-ylated GST-ATF2; duplicated samples were analyzed and shown for both Western blotting and kinase assays in all panels. From: MEKK1 Activation of Human Estrogen Receptor α and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells Mol Endocrinol. 2000;14(11):1882-1896. doi:10.1210/mend.14.11.0554 Mol Endocrinol | Copyright © 2000 by The Endocrine Society