title: Threonine 391 phosphorylation of the human prolactin receptor mediates a novel interaction with 14-3-3 proteins.
abstract: The prolactin receptor (PrlR) is a member of the cytokine receptor superfamily that lacks an intrinsic kinase domain and relies on the cytoplasmic Jak tyrosine kinases to transduce signals. Prolactin-induced Jak2 activation and consequent tyrosine phosphorylation of the receptor and downstream signaling molecules have been studied, but phosphorylation of the PrlR on serine or threonine residues has not been reported. Here we describe a novel interaction between the PrlR and the phosphoserine/phosphothreonine-binding 14-3-3 proteins. This association is mediated by the KCST391WP motif, which occurs in the major functional isoform of the human receptor and is conserved among a wide variety of species. Mutagenesis of threonine 391 to alanine significantly impaired 14-3-3 binding to the PrlR in both glutathione S-transferase pulldown and coimmunoprecipitation assays. In breast carcinoma and mouse mammary epithelial cell lines, the endogenous receptor was found to associate with glutathione S-transferase-14-3-3 proteins independent of prolactin stimulation. A phospho-specific peptide antibody was generated and used to demonstrate phosphorylation of Thr391 in vivo. Phosphorylation of this site was found to be sensitive to okadaic acid, a specific inhibitor of serine/threonine protein phosphatases. Interestingly, the T391A PrlR mutant exhibited increased basal and prolactin-induced tyrosine phosphorylation compared with the wild-type receptor. This was accompanied by a ligand-induced increase in protein kinase B and Erk activation but not that of Stat5a. Phosphorylation of the receptor on Thr391 may therefore provide a new mechanism by which prolactin signaling is attenuated.
authors.affiliation: Walter and Eliza Hall Institute of Medical Research and Bone Marrow Research Laboratories, 1G Royal Parade, Victoria 3050, Australia.
authors.forename: Monilola A
authors.initials: MA
authors.lastname: Olayioye
authors.forename: Mark A
authors.initials: MA
authors.lastname: Guthridge
authors.forename: Frank C
authors.initials: FC
authors.lastname: Stomski
authors.forename: Angel F
authors.initials: AF
authors.lastname: Lopez
authors.forename: Jane E
authors.initials: JE
authors.lastname: Visvader
authors.forename: Geoffrey J
authors.initials: GJ
authors.lastname: Lindeman
mesh_terms: D048948:14-3-3 Proteins; D016475:3T3 Cells; D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008024:Ligands; D020935:MAP Kinase Signaling System; D051379:Mice; D008894:Milk Proteins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019319:Okadaic Acid / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D010769:Phosphothreonine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011388:Prolactin / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011981:Receptors, Prolactin / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry*; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D014443:Tyrosine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D004268:DNA-Binding Proteins; D008024:Ligands; D008894:Milk Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D011981:Receptors, Prolactin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494093:Stat5a protein, mouse; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019319:Okadaic Acid; D013912:Threonine; D014443:Tyrosine; D011388:Prolactin; D014446:Tyrosine 3-Monooxygenase; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases