Phosphorylated prolactin has been identified and isolated from bovine pituitaries. The biological activity of this phosphoprotein is severely reduced in comparison with nonphosphorylated prolactin. The sites of phosphorylation are serines 26, 34, and 90, and the stoichiometry is 1:1:10, respectively. In this report, the phosphoserine residues have been individually replaced with glutamic acid in recombinant methionyl bovine prolactins in order to mimic phosphorylation at each site. Substitution of glutamic acid for serine at positions 26, 34, and 90 reduced protein helical contents by 10, 6, and 14%, respectively. UV absorbances for S26E and S34E bovine prolactins were blue-shifted, similar to the biological isolates of phosphorylated bovine prolactin, but the biological activities of the S26E and S34E mutants (ED values of 16.3 and 18.8 pM, respectively) were similar to that of wild-type prolactin (ED value of 18.6 pM) in the Nb2 rat lymphoma assay. S90E bovine prolactin had the greatest reduction in helical content but showed similar UV and fluorescent spectra to the wild-type bovine prolactin. The biological activity of S90E bovine prolactin (ED value of 672 pM) was reduced to an activity similar to that of phosphorylated bovine prolactin. The data indicate that the phosphorylation of serine 90 is responsible for the reduction in biological activity.

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