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Figure 3 | BMC Biochemistry

Figure 3

From: Identification of four novel phosphorylation sites in estrogen receptor α: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

Figure 3

Mutation of serine residues to alanine eliminates specific phosphorylation of peptides. To confirm the identity of phosphorylated serine residues within peptides A, B, C, and D, serine to alanine mutations were introduced into wt ERα (S47A, S282A, S294A, or S559A). 12 plates of COS-1 cells (4 × 107 /plate) were transfected with 500 ng/plate of wt ERα, S47A, S282A, S294A, or S559A expression plasmids. 18 hours post-transfection, cells were phosphate-depleted, labeled with 4 mCi [32P]H3PO4 and incubated with 10-8 M estradiol overnight. ERα was immunopurified and tryptic peptides were separated by HPLC using a C-18 reversed phase column. Fractions were collected and electrophoresed on a 40% alkaline polyacrylamide gel followed by autoradiography. (A) Peptide map of wt ERα displaying 4 novel phosphopeptides A-D. (B) S294A resulted in loss of peptide C. C) S559A resulted in loss of peptide B. (D) S47A resulted in a modest decrease in peptide D compared to wt ERα. E) S47A/S104A/S106A/S118A resulted in loss of peptide D. (F) Mutation of S282 to alanine reduces ERα protein following 24 h incubation with estradiol. 106 COS-1 monkey embryonic kidney cells which had been cultured in phenol-red free DMEM supplemented with 10% fetal bovine serum were transfected with 2.5 μg of wt ERα or S282A expression plasmid. 24 hours after transfection, cells were incubated with vehicle (veh) or estradiol (10-8M) for an additional 24 hours. Cell lysates were collected and ERα protein levels determined, using α-tubulin as a loading control.

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