Phosphorylation of chicken oviduct progesterone receptor by cAMP-dependent protein kinase.
Phosphorylation of immunopurified chicken oviduct progesterone receptor (PR) was studied in intact cells and under cell-free conditions. Cytosol PR was isolated by incubation with anti-PR monoclonal antibody alpha PR22 adsorbed to protein A-Sepharose and suspended in a reaction mixture containing 10 mM Mg2+, 0.1 mM [gamma-32P]ATP, and the catalytic subunit of cAMP-dependent protein kinase (cAMP-PK) from bovine heart. All three major proteins of avian PR (PR-A, 79 kDa; PR-B, 110 kDa; 90 kDa) incorporated 32P-radioactivity on serine residues. The phosphorylation reaction was inhibited by synthetic inhibitors of protein kinases, H-8 and 20-residue peptide IP20. A 40 degrees C preexposure of PR oligomer increased phosphorylation of the 90-kDa protein, known to be a heat-shock protein (hsp-90). The extent of the phosphorylation reaction was temperature-dependent as the 32P-incorporation into PR-A and PR-B increased gradually, showing a maximum at 37 degrees C. Multiple phosphopeptides (4-7) were resolved by two-dimensional electrophoresis chromatography following cleavage of 32P-labeled peptides with trypsin. Both A and B forms of receptor showed similar phosphorylation patterns with B receptor digestion exhibiting two to three additional peptides. Under physiological conditions, preincubation of oviduct mince with forskolin, a regulator of intracellular cAMP levels, caused a greater extent of phosphorylation of PR-A and PR-B proteins. The results of this study demonstrate that chicken oviduct PR is an excellent substrate for the action of cAMP-PK in vitro and that this enzyme may be a physiological regulator of progesterone action in the oviduct.
Nakao
M
M
Department of Biological Sciences, Oakland University, Rochester, Michigan.
Mizutani
T
T
Bhakta
A
A
Ribarac-Stepic
N
N
Moudgil
V K
VK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002413:Cations, Divalent / Q000494:pharmacology; D002645:Chickens; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008970:Molecular Weight; D010057:Oviducts / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011980:Receptors, Progesterone; D000255:Adenosine Triphosphate; D011494:Protein Kinases