Mutation of Serine 90 to Glutamic Acid Mimics Phosphorylation of Bovine Prolactin

Abstract

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 50 values of 16.3 and 18.8 pM, respectively) were similar to that of wild-type prolactin (ED 50 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 wildtype bovine prolactin. The biological activity of S90E bovine prolactin (ED 50 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.Prolactin binds and activates receptors on the plasma membrane of target cells (1). The PRL 1 receptor is a member of the cytokine receptor superfamily (2), receptors with extracellular and intracellular domains connected by a single transmembrane domain. Several receptors in this superfamily activate cells by formation of receptor dimers brought about by the binding of two receptors binding to unique surfaces of the ligand (sites 1 and 2) (3). Receptor dimerization promotes the association and/or activation of the Janus family of tyrosine kinases (4) and phosphorylation of specific tyrosines of the receptor and substrate proteins that regulate transcription in the nucleus or the activity of other kinase systems. Substrate proteins appear to be presented to the Janus kinases by binding to the phosphorylated loci of the intracellular domain of the receptor (5).The three-dimensional structure of PRL has not been solved, but it is likely to be a four-helix bundle motif (6) similar to the up-up-down-down structure of hGH, a protein related by sequence able to bind prolactin receptors and induce lactogenic actions. The sequence of several variants of the PRL receptor have been reported; most variation between receptor subtypes is observed in the intracellular domain (7). The three-dimensional structure of hGH bound to the extracellular domain of the hPRL receptor in a 1:1 complex has been reported (8).These data have defined the site 1 surface area and have shown that the four-helix bundle of hGH has undergone little structural change when bound to either the hPRL or hGH receptor. However, there is a major shift of the first ancillary helix of the loop connecti...

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