title: Biological activity of phosphorylated and dephosphorylated bovine prolactin.
abstract: Phosphorylation is a mechanism by which cells regulate structure and function of proteins. We have previously demonstrated in vivo synthesis and secretion of phosphorylated bovine prolactin (bPRL) from the pituitary, and have isolated and partially characterized the phosphorylated bPRL. In order to investigate the structure/function role of phosphorylation on the biological activity of bPRL, we compared the activities of nonphosphorylated and phosphorylated bPRL isolated from pituitaries, with bPRL provided by the NIDDK (NIDDK-bPRL) to stimulate Nb2 cell proliferation. Nonphosphorylated bPRL has activity similar to, although slightly lower than that of NIDDK bPRL (ED50 = 7.03 pM and 22.8 pM, respectively). The activity of phosphorylated bPRL is significantly reduced (ED50 = 1066 pM). Using Nb2 lymphoma cell homogenate, NIDDK and nonphosphorylated bPRLs are equally effective in competitive receptor binding assays (Kd = 0.252 and 0.269 nM, respectively). Phosphorylated bPRL does not compete for the PRL receptor at concentrations up to 65 nM. Following enzymatic removal of the phosphate group using alkaline phosphatase, there is an increase in the biological activity of phosphorylated bPRL (ED50 = 73.3 pM) while the activity of nonphosphorylated BPRL remained unchanged following enzyme treatment (21.4 pM). We conclude that (1) structural changes induced by phosphorylation of bPRL are responsible for loss of bioactivity, (2) dephosphorylation of phosphorylated bPRL restores biological activity, and (3) the reduction in biological activity of phosphorylated bPRL is mediated by a decrease in receptor binding.
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D008223:Lymphoma / Q000378:metabolism / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011388:Prolactin / Q000378:metabolism* / Q000494:pharmacology; D011981:Receptors, Prolactin / Q000378:metabolism; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: D010750:Phosphoproteins; D011981:Receptors, Prolactin; D011388:Prolactin; D000469:Alkaline Phosphatase