Identification of phosphorylation sites unique to the B form of human progesterone receptor. In vitro phosphorylation by casein kinase II.
The human progesterone receptor (PR), a member of the steroid/thyroid receptor superfamily of ligand-activated transcription factors, is expressed in most tissues as two forms that exhibit differential transcriptional activation potentials, full-length PR-B and NH2-terminally truncated PR-A. In human breast cancer cells (T47D) both forms of PR are constitutively phosphorylated but phosphorylation is increased in response to hormone treatment, suggesting that this modification has a role in regulating the activation state of the receptor. To more directly define the functional role of phosphorylation in the action of A and B receptors requires knowledge of the phosphorylated amino acid residues and the protein kinase(s) involved. Toward this end we have developed a strategy that combines isolation of PR phosphotryptic peptides by reverse phase high performance liquid chromatography, secondary analytical protease digestion, manual Edman degradation, and release of 32P that resulted in identification of two major phosphorylation sites, Ser81 and Ser162. Both sites are located in the amino-terminal region unique to PR-B, and one of these sites (Ser81) is encompassed in a casein kinase II (CKII) consensus sequence. Although human PR contains 11 potential CKII consensus sequences, CKII in vitro phosphorylated purified PR-B only at Ser81 suggesting that this may be an authentic site for CKII in vivo.
Zhang
Y
Y
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Beck
C A
CA
Poletti
A
A
Edwards
D P
DP
Weigel
N L
NL
D000595:Amino Acid Sequence; D047390:Casein Kinase II; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014357:Trypsin; D014407:Tumor Cells, Cultured
D010748:Phosphopeptides; D011980:Receptors, Progesterone; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D014357:Trypsin