Identification of Phosphorylation Sites Unique to the B Form of Human Progesterone Receptor

The human progesterone receptor (PR), a member of the steroidlthyroid 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 NH,-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, Ser” and Ser16’. Both sites are located in the amino-terminal region unique to PR-B, and one of these sites (Sersl) is encompassed in a casein kinase