Phosphotryptic peptide analysis of human progesterone receptor. New phosphorylated sites formed in nuclei after hormone treatment.
Human progesterone receptors (PR) exist as two independent naturally occurring steroid-binding forms of approximately 120 kDa (B-receptors) and 94 kDa (A-receptors). Both are phosphorylated in hormone-untreated T47Dco breast cancer cells. Hormone treatment leads to receptor transformation and an increased phosphorylation state: the 32P-labeling intensity is 3-5 times higher after progestin treatment and 8-10 times higher after RU 486 treatment. Only serine residues are phosphorylated. To determine whether there are unique phosphorylation sites in transformed nuclear PR, we analyzed the phosphopeptides of untransformed and transformed A- and B-receptors by tryptic cleavage and reverse-phase high pressure liquid chromatography. Untransformed A- and B-receptors share at least five common phosphopeptides, and a sixth is unique to B. Following transformation by either R5020 or RU 486, A-receptors generate at least six and B-receptors seven phosphopeptides. Compared with untransformed PR, there are at least two different phosphopeptides in transformed nuclear PR. Cyanogen bromide cleavage of transformed nuclear A-receptors, which lack the proximal 165 amino-terminal residues of the 933 amino acid B-receptors, produces two large fragments of approximately 43 and 19 kDa. These fragments contain all of the 32P label and comprise amino acids 165-595. Cleavage of transformed B-receptors also produces peptides of 43 and 19 kDa plus an additional 36-kDa fragment corresponding to residues 1-165. No 32P-labeled low molecular mass peptides are detected. Thus, all the hormone-dependent phosphoserine residues produced in nuclei are located in the first 595 amino acids of human PR, representing the amino terminus and 28 residues of the DNA-binding domain.
Sheridan
P L
PL
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Evans
R M
RM
Horwitz
K B
KB
D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D006801:Humans; D015735:Mifepristone; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D011372:Progestins / Q000494:pharmacology; D011980:Receptors, Progesterone / Q000032:analysis*; D014357:Trypsin
D004963:Estrenes; D010446:Peptide Fragments; D010750:Phosphoproteins; D011372:Progestins; D011980:Receptors, Progesterone; D015735:Mifepristone; D003513:Cycloheximide; D014357:Trypsin; D003488:Cyanogen Bromide