title: Prolactin and insulin synergize to regulate the translation modulator PHAS-I via mitogen-activated protein kinase-independent but wortmannin- and rapamycin-sensitive pathway.
abstract: The synergism between insulin and prolactin (PRL) in their effect on protein synthesis in the mammary gland was studied in differentiating mammary epithelial CID-9 cells. Both hormones were needed to induce phosphorylation of PHAS-I which resulted in its dissociation from the eIF-4E translation initiation factor. This step is crucial for the initiation of translation. The induction of PHAS-I phosphorylation was rapid and its rate matched that demonstrated for the JAK2/STAT5a and the binding of STAT5a to its DNA binding motif. However, 120 min was needed for complete phosphorylation of the PHAS-I protein. In the presence of insulin, PRL induced MAP kinase activity, initiated at a comparable rate to that of PHAS-I phosphorylation. However, a line of evidence suggested that although this kinase phosphorylates PHAS-I in vitro, it does not actively participate in its phosphorylation in vivo: (a) the level of insulin needed to enable PRL-induced ERK-1/ERK-2 activation was one order of magnitude higher than that needed for PHAS-I phosphorylation; and (b) PD 098059, a MEK-1 inhibitor, completely inhibited insulin-dependent, PRL-induced ERK-1/ERK-2 activation but had no effect on the PRL-induced PHAS-I phosphorylation. In contrast, wortmannin, a phosphatidylinositol 3-kinase (PI 3'-kinase) inhibitor and the immunosuppressant rapamycin abrogated PHAS-I phosphorylation and caused a reciprocal shift between the fully phosphorylated PHAS-I gamma form and its non-phosphorylated alpha form. Since the partly phosphorylated PHAS-I beta form was not significantly affected by these inhibitors, it is possible that more than a single kinase mediates the synergistic effect of prolactin and insulin on PHAS-I phosphorylation.
authors.affiliation: Institute of Animal Science, The Volcani Center, Bet-Dagan, Israel. barashi@agri.huji.ac.il
authors.forename: I
authors.initials: I
authors.lastname: Barash
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004357:Drug Synergism; D004847:Epithelial Cells; D039642:Eukaryotic Initiation Factors; D005260:Female; D007328:Insulin / Q000494:pharmacology*; D053614:Janus Kinase 2; D007700:Kinetics; D008321:Mammary Glands, Animal; D051379:Mice; D008894:Milk Proteins; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011388:Prolactin / Q000494:pharmacology*; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D050799:STAT5 Transcription Factor; D020123:Sirolimus / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C499527:Eif4ebp1 protein, mouse; D039642:Eukaryotic Initiation Factors; D007328:Insulin; D008894:Milk Proteins; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D050799:STAT5 Transcription Factor; C494093:Stat5a protein, mouse; D015534:Trans-Activators; D011388:Prolactin; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020123:Sirolimus; D000077191:Wortmannin