Molecular and Cellular Endocrinology 155 (1999) 37 – 49 Prolactin and insulin synergize to regulate the translation modulator PHAS-I via mitogen-activated protein kinase-independent but wortmannin- and rapamycin-sensitive pathway Itamar Barash * Institute of Animal Science, The Volcani Center, P.O. Box 6, 50250 Bet -Dagan, Israel Received 24 February 1999; accepted 7 May 1999 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  form and its non-phosphory- lated  form. Since the partly phosphorylated PHAS-I  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. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mammary gland; Prolactin; Insulin; Translation; eIF-4E; PHAS-I www.elsevier.com/locate/mce 1. Introduction In a mammary organ culture of transgenic mice, we have recently demonstrated an insulin-dependent pro- lactin (PRL) induction of translation of human serum albumin RNA, directed for expression in the mammary gland by regulatory sequences of the -lactoglobulin milk protein gene (Baruch et al., 1998). This study aims to probe deeper into the molecular mechanisms which mediate the synergism between these two hormones. PRL is a pleiotropic hormone involved in water and salt balance, growth and morphogenesis, metabolism, immune response, steroidegenesis and maternal behav- ior (Bole-Feysot et al., 1998). However, its best-charac- terized role is in the initiation and maintenance of milk protein gene expression in the mammary gland (Von- derhaar and Ziska, 1989). Apparently, PRL transfers its transcriptional activation via the JAK2/STAT5 path- way. The binding of STAT5 to a specific motif, located in the promoters of the casein (Schmitt-Ney et al., 1992), BLG (Streuli et al., 1995) and WAP genes (Li and Rosen, 1994) promotes gene transcription. How- ever, it has been shown in the HC11 mammary cell line that PRL activation of the STAT5/MGF is insulin independent whereas expression of the -casein gene depends on the presence of insulin and hydrocortisone (Welte et al., 1994). * Tel.: +972-8-9484-418; fax: +972-8-9475075. E-mail address: barashi@agri.huji.ac.il (I. Barash) 0303-7207/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII:S0303-7207(99)00116-1