Identification of the Major Site of Rat Prolactin Phosphorylation as Serine 177

Abstract

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...

Administration of unmodified prolactin (U-PRL) and a molecular mimic of phosphorylated prolactin (PP-PRL) during rat pregnancy provides evidence that the U-PRL:PP-PRL ratio is crucial to the normal development of pup tissues

“…We have previously produced such a mimic by substitution of the serine, which is phosphorylated in PRL secretory granules (Wang et al 1996), by an aspartate residue to produce S179D PRL (Chen et al 1998). This molecular mimic of phosphorylated PRL, referred to as pseudophosphorylated PRL (PP-PRL), cannot be dephosphorylated, as it contains no phosphate group.…”

“…The possible posttranslational modifications are many, including glycosylation (Lewis et al 1984), phosphorylation (Oetting et al 1986, Brooks et al 1990, Aramburo et al 1992 and proteolytic cleavage (Oetting & Walker 1985, Sinha et al 1985, Clapp 1987. This laboratory has focused its attention on the biology of phosphorylated PRL , 1993a,b, Greenan et al 1989, Wang & Walker 1993, Wang et al 1996, Chen et al 1998), a form of posttranslationally modified PRL that occurs in all species thus far examined (Oetting et al 1986, Brooks et al 1990, Aramburo et al 1992, Chen et al 1998.…”

A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone

“…S179D PRL, a mimic of phosphorylated human PRL (Wang et al 1996, Chen et al 1998, Tuazon et al 2002, was originally developed as a PRL receptor antagonist since we had shown that naturally phosphorylated PRL had this activity (Krown et al 1992, Wang & Walker 1993. It was anticipated therefore that S179D PRL would impair the growth of tissues utilizing PRL as a growth factor by blocking the effects of both pituitary and autocrine PRL.…”

The Effect of Estrogen on Phosphorylation of Prolactin in the Mouse Pituitary Gland

“…Phosphate analysis of phosphorylated PRL ( P P R L ) h a s s h o w n t h a t t h e r a t i o o f n o nphosphorylated PRL to PPRL is species-specific [5,6]. Analysis of phosphorylation sites has demonstrated one at serine 179 th (177 th in rat) in human PRL [7]. A minor phosphorylation site has also been identified at threonine 58 rd or 63 rd in rat PRL [7].…”