PROLACTIN INDUCES PHOSPHORYLATION OF TYR694 OF STAT5 (MGF), A PREREQUISITE FOR DNA-BINDING AND INDUCTION OF TRANSCRIPTION

Mammary gland factor (MGF) is a transcription factor discovered initia lly in the mammary epithelial cells of lactating animals. It confers t he lactogenic hormone response to the milk protein genes. We reported recently the isolation of the cDNA encoding MGF, MGF is a novel member of the cytokine-regulated transcription factor gene family. Members o f this gene family mediate interferon alpha/beta and interferon gamma induction of gene transcription, as well as the response to epidermal growth factor and interleukin-6, and have been named signal transducer s and activators of transcription (Stat). The name Stat5 has been assi gned to MGF. We studied the mechanisms involved in the prolactin activ ation of Stat5 in COS cells co-transfected with cDNA encoding Stat5 an d the prolactin receptor. Prolactin treatment of the transfected cells caused activation of Stat5 within 5-10 min. This activation does not require ongoing protein synthesis. Tyrosine kinase inhibitors prevent Stat5 activation in transfected COS cells. Treatment of recombinant St at5 with a tyrosine-specific protein phosphatase in vitro abolishes it s DNA binding activity. Prolactin stimulation of transfected cells ind uces Stat5 phosphorylation on tyrosine. Phosphorylation of iii vitro t ranscribed and translated Stat5 with the Jak2 tyrosine kinase, but not with fyn, lyn or lck, confers DNA binding activity, The prolactin res ponse of the beta-casein milk protein gene promoter can be observed in COS cells transfected with cDNA vectors encoding Stat5 and the long f orm of the prolactin receptor. The short form of the prolactin recepto r is unable to promote Stat5 phosphorylation and confer transcriptiona l induction in COS cells, Phosphorylation of the tyrosine residue at p osition 694 in the Stat5 sequence is essential for prolactin regulatio n. Replacement of Tyr694 by a phenylalanine residue prevents tyrosine phosphorylation, induction of DNA binding and transactivation by prola ctin. Signal transduction via the prolactin receptor shares common fea tures with other members of the cytokine/hematopoietin receptor gene f amily, i.e. the rapid activation of a factor through tyrosine phosphor ylation which serves as a second messenger and an activator of transcr iption.