Protein kinase activity of purified rat liver glucocorticoid receptor

Abstract

Molybdate-stabilized nonactivated rat liver glucocorticoid receptor (GR) was purified to near homogeneity using a biospecific affinity adsorbent, Bio Gel A 0.5m and DEAE-Sephacel. The purified GR sedimented in the 9–10S region in 5–20% sucrose gradients containing 0.10M KCl and 20mM Na 2MoO 4. SDS-polyacrylamide gel electrophoresis revealed a major single band with an apparent molecular weight of 90,000 ± 2,000. Affinity labeling of GR with [ 3H]-dexamethasone mesylate showed association of the radioactivity with a peptide of 90,000 molecular weight. Purified receptor preparation was dialyzed to remove molybdate and was incubated with different protein substrates in the presence of 50μM [λ- 32P]-ATP and divalent cations. Radioactive phosphate from [λ- 32P]-ATP was seen to be incorporated into calf thymus histones, turkey gizzard myosin light chain kinase and rabbit skeletal muscle kinase in the presence of Mg 2+ and Ca 2+ ions. Addition of steroid ligand exogenously to the reaction mixture appeared to increase the extent of protein phosphorylation. No autophosphorylation of GR was evident under the above conditions. The data suggest that purified rat liver GR displays protein kinase activity.