Intragranular prolactin phosphorylation and kallikrein cleavage are regulated by zinc and other divalent cations

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.