Figure 8
Protein Kinase CK2 phosphorylates of S282 and S559. (A) 400 ng baculovirus expressed ERα was incubated in CK2 kinase buffer supplemented with 10 mM ATP, in the presence or absence of 200 ng recombinant catalytic α subunit of CK2. Reactions were stopped with Laemmli buffer, subjected to Western blot analysis, and probed with α-pS282, α-pS559, α-pS118 or αER. These studies show that the CK2α catalytic subunit specifically phosphorylates ERα at S282 and S559. Western blot for phosphorylation of S118, a site that exhibits strong phosphorylation in baculovirus expressed ERα, is shown for comparison to demonstrate absence of nonspecific phosphorylation by CK2 on other ERα phosphorylation sites. (B) 106 MCF7 breast cancer cells were pretreated with DMAT (4 uM) for 90 minutes, followed by 30 minutes with estradiol (10-8 M) or vehicle. Immunoprecipitation of S282 or S559 was performed using phosphoantibodies and Western blot for total ERα. DMAT inhibited phosphorylation at both sites, indicating that CK2 phosphorylates these sites in vivo.