Phosphorylation of calf uterine progesterone receptor by cAMP-dependent protein kinase.
We have examined the potential for using calf uterine progesterone receptor (PR) as a substrate for phosphorylation by cAMP-dependent protein kinase (cAMP-PK), PR was found to interact with anti-PR monoclonal antibody alpha PR6 (Sullivan et al., 1986), which was to immunopurify the receptor. Protein staining of the purified preparation revealed the presence of two major bands corresponding to 114 kDa and 90 kDa peptides; only 114 kDa peptide could be photoaffinity-labeled with R5020. The 90 kDa peptide co-migrated with 90 kDa heat shock protein (hsp-90) precipitated by anti-hsp-90 monoclonal antibody AC88 (Riehl et al., 1985). Incubation of the immunopurified protein-A-Sepharose-adsorbed PR with the catalytic subunit of cAMP-PK in the presence of gamma-[32P]ATP and divalent cations resulted in a Mg++-dependent incorporation of 32P-radioactivity into both the 114 kDa and the hsp-90 peptides. Small 32P-incorporation was also seen in the 114 kDa peptide in the presence of Mn++. A 60 degrees C preincubation of immunopurified PR increased the extent of phosphorylation of the hsp-90 peptide. A pretreatment with alkaline phosphatase reduced the ability of PR to act as a substrate while the steroid occupancy of PR appeared to enhance the phosphorylation of the 114 kDa peptide. The differential cation requirement for the phosphorylation of 114 kDa and hsp-90 peptides and a selective hormone-dependent increase in the phosphorylation of the 114 kDa peptide suggest a possible role of phosphorylation in mediating progesterone action in the calf uterus.
Hurd
C
C
Department of Biological Sciences, Oakland University, Rochester, MI 48309-4401.
Nakao
M
M
Moudgil
V K
VK
D000818:Animals; D000911:Antibodies, Monoclonal; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D005260:Female; D006358:Hot Temperature; D010744:Phosphoric Monoester Hydrolases / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D011980:Receptors, Progesterone / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014599:Uterus / Q000201:enzymology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D011980:Receptors, Progesterone; D011494:Protein Kinases; D010744:Phosphoric Monoester Hydrolases