Phosphorylation of prolactin by endogenous protein kinases within isolated secretory granules was shown to result in the production of both phosphoserine and phosphothreonine residues. The majority of the radiolabel was determined to be present in the C terminus of the molecule after specific cleavage with glandular kallikrein. Glandular kallikrein cleaves in three places at the C terminus, liberating three small peptides, only one of which contains a phosphorylatable residue. Sequencing of this phosphopeptide showed it to be Arg-Lys. Thus the major site of prolactin phosphorylation was determined to be serine 177. Using a synthetic peptide equivalent to this region of the molecule (Ser-Val), serine 177 was demonstrated to be a substrate for protein kinase A as well as for one of the endogenous granule kinases. Inclusion of the synthetic peptide in an endogenous granule phosphorylation reaction resulted in competition for the kinase and reduced phosphorylation of prolactin. Protein kinase A phosphorylation of purified prolactin resulted in the production of only phosphoserine and primarily the most abundant (monophosphorylated) variant. We conclude that serine 177 is the major in vivo phosphorylation site of rat prolactin and that phosphorylation of this site can be reproduced by protein kinase A in vitro. The minor threonine phosphorylation site was demonstrated by two-dimensional tryptic peptide mapping and mass analysis to be either threonine 58 or 63, both of which are contained within a single peptide.

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