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Figure 2 | BMC Biochemistry

Figure 2

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

Figure 2

Identification of ERα phosphorylated at serine residues 47, 282, 294 and 559. 6 × 108 COS-1 cells were cultured in phenol red free DMEM supplemented with 10% charcoal stripped FBS. Cells were transfected with wt ERα expression plasmid as noted in materials and methods. 24 hours post-transfection, cells were phosphate-depleted and medium was exchanged with phosphate free DMEM supplemented with 1% dialyzed FBS. 4 mCi [P32] H3PO4 and 10-8 M estradiol were added to each plate and incubated overnight. Cells were subjected to denaturing lysis, ERα purified by immuno-affinity column, eluted, and fractionated by SDS-PAGE. (A) The band corresponding to the 67 kDa ERα was excised and subjected to tryptic digestion. Tryptic ERα peptides were separated by reverse phase HPLC using a C-18 column and 0-45% acetonitrile gradient over 90 minutes. (B) Fractions were collected, pooled according to HPLC retention times and electrophoresed on 40% acrylamide alkaline peptide gels. Gels were autoradiographed, revealing distinct ERα phosphopeptides. 4 novel phosphopeptides (A, B, C, and D) resulting from tryptic digestion of ERα were identified. Each phosphopeptide was then excised, and subjected to modified manual Edman degradation (MED) and phosphoamino acid analysis as described in Materials and Methods. (C) Phosphoamino acid analysis revealed that the phosphopeptide A, B, C and D contained only phosphoserine. Representative phosphoamino acid analyses autoradiograms are presented with identical results detected for phosphopeptides A, B, C, and D. (D) MED detected 32P release for phosphopeptides A-D. Combined data for phosphoamino acid analysis and MED is presented in Table 1.

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