EGF stimulates phosphorylation of Ser-167 in vivo. COS-1 cells were transfected with expression vectors for wild-type human ER or for the mutants S104/106/118A-ER and S167A-ER. Cells were incubated with [³²P]orthophosphate, and EGF (100 ng/ml) or vehicle was added for the last 30 min of labelling. The ER was immunoprecipitated, and an aliquot of the immunoprecipitates was resolved on an SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted with monoclonal antibody EVG F9 (1:4,000), and the ER was quantitated (28). The remainder of the immunoprecipitated ER was resolved by SDS-PAGE, transferred to nitrocellulose, and autoradiographed. The ER-containing bands were excised and counted, and counts per minute were normalized for the amount of ER present. The average counts per minute ± the range as for duplicates are shown. The ER was digested with CNBr, and the peptides were resolved by Tricine-SDS-PAGE. The arrows on the autoradiogram of the CNBr digests indicate the 7-kDa peptide band that contained Ser-118 and Ser-167 (a) and the 9-kDa peptide band that contained Ser-104 and Ser-106 (b). Lane 7, ¹⁴C-labelled protein molecular mass standards. The images were obtained as described in the legend to Fig. 1.

pp90^rsk1 and Ser-167 regulate ER-mediated transcription. (A) MCF-7 cells cotransfected with a reporter construct that contained a luciferase gene regulated by two EREs and a plasmid that permitted constitutive expression of β-galactosidase. Additionally, the cells were transfected with expression vectors for either wild-type pp90^rsk1 or the mutant RSK N′ or vector (V) alone. EGF (100 ng/ml) and estradiol (E2) (1 nM), ICI 182,780 (1 μM), or vehicle was added. Luciferase activity was normalized to β-galactosidase activity. The normalized luciferase values were divided by the value obtained in the absence of exogenous agents and multiplied by 100. The means ± standard errors of the means are shown. (B) BHK cells transfected as described for panel A, except that constructs containing a MAPKKc and ER TAF-1 were also cotransfected. E2 or vehicle was added, and the assay and analysis were performed as described for panel A. The means ± standard errors of the means are shown. (C) BHK cells transfected with the reporter and β-galactosidase constructs as described for panel A and the MAPKKc construct as described for panel B. Additionally, constructs that contained either ER TAF-1, ER S118/167A TAF-1, ER S118A TAF-1, or ER S167A TAF-1 were transfected. E2 or vehicle was added, and the assay and analysis were performed as described for panel A. (D) BHK cells were cotransfected with the reporter and β-galactosidase constructs as described for panel A as well as with constructs containing either ER TAF-1 or ER S118/167A TAF-1. Additionally, constructs that contained either MAPKKc or MAPKKd/n were transfected. E2 or vehicle was added, and the assay and analysis were performed as described for panel A. The means ± standard errors of the means are shown. ∗∗, P of <0.001; ∗, P of <0.05.

pp90^rsk1 phosphorylates Ser-167 of the ER in vitro. (A) Extracts made from MCF-7 cells that were serum starved for 24 h and then treated for 10 or 30 min with 100 nM PMA, 100-ng/ml EGF, or vehicle (lanes C). These extracts were used in an in-gel kinase assay containing NER in the gel. Lane 1 contained ¹⁴C-labelled protein molecular weight standards. (B) Western blot of the extracts used in panel A with anti-pp90^rsk1 antibody (1:5,000) (Transduction Laboratories). (C) Serum-starved MCF-7 cells treated for 10 min with EGF, PMA, or vehicle. pp90^rsk1 was immunoprecipitated with an anti-pp90^rsk1 antibody. The immunoprecipitates were used in an in-gel kinase assay with gels containing either NER, S104/106/118A-NER, or S167A-NER. The locations of the molecular weight standards are shown. (D) In vitro kinase reactions performed with either pp90^rskα, a Xenopus homolog of pp90^rsk1, or Erk2 with GST-NER, GST-S104/106/118A-NER, or GST-S167A-NER as substrate. The proteins were resolved on an SDS-PAGE gel and analyzed. The experiment shown represents three independent trials. Panels C and D were obtained by scanning the autoradiograms with a Molecular Dynamics Personal Densitometer with ImageQuant software. The resultant images were cropped in Photoshop and labelled by using Freehand. Stds, standards.

pp90^rsk1 phosphorylates Ser-167 in vivo. (A) COS-1 cells cotransfected with expression vectors for the mutant S104/106/118A-ER and either wild-type pp90^rsk1 or a mutant (RSK N′) or vector control. The cells were incubated with [³²P]orthophosphate, and EGF (100 ng/ml) or vehicle was added for the last hour of labelling. An aliquot of cell extracts was resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-avian pp90^rsk1 antibodies (1:2,000) (10). The mutant ER was immunoprecipitated. The immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, immunoblotted with the monoclonal ER AB 10 (Neo Markers) (1:400), and analyzed, and the ER was quantitated. The relative counts per minute in the ER-containing bands were determined with the Molecular Dynamics PhosphorImager by using ImageQuant software. The data were normalized for the amount of ER present. The means ± standard deviations for triplicate samples are shown. (B) The blot of ER immunoprecipitates shown in panel A stripped and reprobed with anti-avian pp90^rsk1 antibodies (1:2,000). NS, nonspecific band indicating that equal amounts of protein were loaded into each gel well.