Lactogenic Hormone Activation of Stat5 and Transcription of the β-Casein Gene in Mammary Epithelial Cells Is Independent of p42 ERK2 Mitogen-activated Protein Kinase Activity

doi:10.1074/jbc.271.50.31863

Lactogenic Hormone Activation of Stat5 and Transcription of the β-Casein Gene in Mammary Epithelial Cells Is Independent of p42 ERK2 Mitogen-activated Protein Kinase Activity*

From the Friedrich Miescher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland, the
^∥ Institute for Experimental Cancer Research, Tumor Biology Center, D-79106 Freiburg, Germany, and
^″ Laboratory of Biochemistry and Metabolism, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

^‴ To who correspondence and reprint requests should be addressed. Tel.: 41 61 697 8107; Fax: 41 61 697 8102; E-mail: hynes{at}fmi.ch

↵^¶ Present address: Ciba-Geigy Ltd., K-125.3.04, CH-4002 Basel, Switzerland.

Abstract

HC11 mammary epithelial cells have been used to characterize molecular events involved in the regulation of milk protein gene expression. Treatment of HC11 cells with the lactogenic hormones prolactin, insulin, and glucocorticoids results in transcription of the β-casein gene. Prolactin induces a signaling event which involves tyrosine phosphorylation of the mammary gland factor, Stat5, a member of the family of signal transducers and activators of transcription (Stat). Here we show that HC11 cells express two Stat5 proteins, Stat5a and Stat5b. Phosphopeptide and phosphoamino acid analysis of Stat5a and Stat5b immunoprecipitated from phosphate-labeled HC11 cells revealed that both proteins were constitutively phosphorylated on serine. Lactogenic hormone treatment resulted in the appearance of a tyrosine-phosphorylated peptide in both Stat5 proteins. Consistent with this observation, a Western blot analysis of Stat5a and Stat5b showed that lactogenic hormones induced a rapid, transient increase in phosphotyrosine which paralleled the binding of Stat5 to its cognate recognition sequence in the β-casein gene promoter. Lactogenic hormone treatment of the HC11 cells also led to a rapid activation of the mitogen-activated protein (MAP) kinase pathway. We examined the role of this pathway in β-casein transcription using a specific MAP kinase kinase inhibitor, PD98059. Concentrations of PD98059 which completely abrogated lactogen-induced MAP kinase activation did not affect the phosphorylation state of Stat5, its DNA binding activity, or transcriptional activation of a β-casein reporter construct. This indicates that the MAP kinase pathway does not contribute to lactogenic hormone induction of the β-casein gene.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Stat

signal transducer and activator of transcription

GAS

interferon-γ activated site

MAP kinase

mitogen-activated protein kinase

MEK

MAP kinase kinase

EGF

epidermal growth factor

PAGE

polyacrylamide gel electrophoresis

EMSA

electrophoretic mobility shift assay

ERK

extracellular signal-regulated kinase

ECM

extracellular matrix

IFN

interferon.
↵² M. Wartmann, unpublished results.
↵³ N. Cella, unpublished results.
↵⁴ N. Cella and I. Beuvink, unpublished results.
- Received July 1, 1996.
- Revision received August 20, 1996.