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 ), 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. For many years prolactin (PRL)1 was considered an unmodified polypeptide hormone. It is now clear, however, that posttranslational processing of PRL causes it to be phosphorylated (e.g. Refs. 1 and 2), glycosylated (e.g. Ref.3), and variously proteolytically cleaved (e.g. Refs. 4 -6). The phosphorylation of PRL within pituitary cells has been demonstrated to occur in vivo in the rat (1), chicken (7), and cow (2). Phosphate analysis of purified preparations of PRLs from different species showed them to be variously phosphorylated with molar ratios of hormone to phosphate of 1.0:0.2 for ovine and rat and 1.0:0.7 for turkey (7).Functional studies from this laboratory have determined that monophosphorylated PRL is an antagonist to non-phosphorylated PRL in two cell systems where non-phosphorylated PRL promotes cell proliferation (8, 9). It is therefore important to establish the sites of PRL phosphorylation so that these may be reproduced in vitro for further analysis of this antagonism which operates through a single receptor (9, 10).In our earlier studies, we used a variety of purified protein kinases in an attempt to identify potential phosphorylation sites (1). This approach, however, while illustrating that PRL is a very readily phosphorylated molecule, did not narrow the search because such a variety of protein kinases with very different consensus recognition sequences were found capable of phosphorylating PRL. For PRL from other species, only protein kinase A (PKA) has been tried and shown to successfully phosphorylate ovine, chicken, and turkey PRL (7).In this article we present evidence that PRL is phosphorylated on both a serine and threonine residue and that on...

Previous studies have demonstrated that naturally phosphorylated PRL antagonizes the growth-promoting effects of unmodified PRL in two different PRL-responsive cell lines. In this study our aim was to produce a molecular mimic of phosphorylated PRL by substituting a fairly bulky, negatively charged amino acid (glutamate or aspartate) for the normally phosphorylated serine [serine 179 in human PRL (hPRL)]. In addition, because of the marked effect of phosphorylation on biological activity, we investigated the importance of the unmodified serine in the growth-promoting activity of PRL. hPRL complementary DNA was obtained from the American Type Culture Collection and subcloned into pT7-SCII after site-directed mutagenesis using the deoxyuridine approach. Proteins were expressed in Escherichia coli BL21 (DE3) and were primarily found in inclusion bodies. Agonist and antagonist activities of each serine 179 mutant were assessed using the Nb2 bioassay. Compared with standard hPRL, the recombinant wild-type was more active in the Nb2 assay, attesting to both the absence, or low level, of endotoxin contamination in preparations from these cells and the appropriate folding of the molecule. The aspartate and glutamate mutants had no intrinsic agonist activity, but both antagonized the growth-promoting activity of wild-type PRL, with the aspartate mutant proving to be a very effective antagonist. Two hundred picograms per ml of the aspartate mutant negated 75% of the growth response to 400 pg/ml wild-type PRL. When serine 179 was mutated to alanine or valine, mutant PRLs with 0% and 14% of the biological activity of wild-type PRL, respectively, were produced. These results demonstrate 1) that molecular mimicry of the phosphorylated hormone does produce a PRL antagonist, and 2) that the serine at position 179 is crucial to the growth-promoting activity of PRL. The aspartate mutant can now be used to study many aspects of the physiology of PRL.

Estrogen and progesterone replacement in ovariectomized rats in an often-used experimental system for determination of the specific effects of these hormones. In this study, two different delivery systems and two different dosage levels of estrogen, progesterone or a combination of the two have been used. Estrogen and progesterone in the circulation have been measured in response to each treatment. It is reported that estrogen treatment (237.2 +/- 49.2 pg/mL) results in physiologically significant levels of circulating progesterone (11.1 +/- 1.3 ng/mL). Also, co-administration of progesterone (23.7 +/- 2.0 ng/mL) with estrogen decreases the level of estrogen over that seen with estrogen alone (96.7 +/- 19.2 pg/mL with progesterone vs 237.2 +/- 49.2 pg/mL without progesterone). Thus, contrary to expectations, estrogen replacement therapy is not specific to estrogen and some of the antagonistic effects of progesterone are the result of a decrease in circulating estrogen, and not a specific effect on a target tissue. Whereas the mechanism of these effects has not been determined, obvious artifactitous phenomena have been excluded as being their cause. These results could have a major impact on the interpretation of past and future experiments of this kind.

Rat prolactin (PRL) secretory granules contain enzymes for proteolytic cleavage and serial phosphorylation, but hormone cleavage products and phosphorylated PRL are not detected until just prior to exocytosis. Similarly, although PRL is stored in granules, in part, as high-mol-wt oligomers, PRL is primarily monomeric in the circulation. PRL secretory granules contain zinc, calcium, and magnesium, which inhibit depolymerization and dissolution of granules. Divalent cations also protect cysteine free thiol residues in the carboxy-terminal region of the intragranular hormone. The present studies examined the effect of removal and replacement of divalent cations on kallikrein cleavage and phosphorylation of secretory granule PRL.Kallikrein cleavage was assessed utilizing two experimental protocols. First, granules were treated with or without 3 mM EDTA, free hormone thiols were alkylated, the PRL was cleaved by kallikrein, and the small kallikrein-cleavage peptides were assessed by reversephase HPLC. No differences in hormone cleavage owing to removal of divalent cations were observed at this concentration of EDTA. Second, divalent cations in granules were reduced/removed by 10 mM EDTA/ 3 mM o-phenanthroline (OP), followed by addition of either 5 mM zinc, magnesium, calcium, or additional EDTA. Kallikrein cleavage was then initiated. In this instance, the extent of proteolysis was analyzed by two-dimensional polyacrylamide gel electrophoresis (PAGE) of the larger remnant PRL pieces. After treatment with 10 mM EDTA/3 mM OP, results indicated that cleavage between R174 and R175 (site 1) was unaffected by added cations or additional EDTA. Recovery of site 2 cleaved PRL (L1-K185) and site 3 cleaved PRL (L1-R188) was∼40% reduced by zinc, but unaffected by calcium or magnesium. Additional EDTA resulted in increased recovery of site 2 cleaved PRL, but no change in site 3 recovery, suggesting the presence of tightly bound intragranular zinc around site 2, even after the initial EDTA/OP treatment.Phosphorylation of PRL at S177 was studied using the same protocols. Phosphorylation was increased by added EDTA, even at 3 mM, and decreased by divalent cations, with no marked specificity for zinc observed. An additional experiment studied phosphorylation without exposure to kallikrein. Comparisons between the plus and minus kallikrein experiments showed kallikrein to have no apparent preference for unmodified or phosphorylated PRL.From the kallikrein cleavage and phosphorylation studies and modeling of PRL, we suggest D181 as a likely site for intragranular zinc coordination. When C189 and C197 are present as free thiols in intragranular PRL, these may also contribute to binding. Zinc coordination in this region of the molecule apparently regulates proteolytic processing by kallikrein, as well as contributing to the stability of the hormone storage forms.