Prolactin Activates Tyrosyl Phosphorylation of Insulin Receptor Substrate 1 and Phosphatidylinositol-3-OH Kinase

doi:10.1074/jbc.272.4.2050

Prolactin Activates Tyrosyl Phosphorylation of Insulin Receptor Substrate 1 and Phosphatidylinositol-3-OH Kinase*

From the ^‡ Departamento de Biologia Molecular, Universidad Autonoma de Madrid, Facultad de Ciencias, 28049 Madrid, Spain, the
^§ Dipartimento di Farmacologia Sperimentale, Università degli Studi di Napoli “Federico II,” 80131 Napoli, Italy, and
INSERM U.344, Endocrinologie Moléculaire, Faculté de Médecine Necker, 75730 Paris Cedex 15, France

^¶ To whom correspondence should be addressed:
INSERM U.344, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
. Tel.: 33-1-40-61-53-10; Fax: 33-1-43-06-04-43.

Abstract

Prolactin (PRL) has been demonstrated to induce tyrosine phosphorylation and activation of the cytoplasmic tyrosine kinase JAK2. The present study represents an initial effort to identify the phosphorylation repertoire of the PRL receptor (PRLR). For this purpose we have modified the rat PRLR cDNA to encode an additional N-terminal epitope specifically designed to allow the rapid purification of the PRLR and associated proteins from transfected cells. The Flag-tagged PRLR was stably expressed in the human 293 cell line. PRL induced tyrosine phosphorylation of proteins of 85, 95, and 185 kDa from 10 to 30 min after PRL stimulation. Immunoblot analysis of immunoprecipitation indicates that p85 corresponds to the 85-kDa regulatory subunit of phosphatidylinositol (PI)-3′ kinase, p95 to PRLR, and p185 to insulin receptor substrate 1 (IRS-1). Both PI-3′ kinase and IRS-1 appear to associate with PRLR in a PRL-dependent manner. These results thus indicate that kinases other than JAK2, namely PI-3′ kinase, are activated by PRL.

Footnotes

↵* This work was supported by INSERM and the Association pour la Recherche sur le Cancer and the Ministerio de Educacion y Ciencia and Universidad Autonoma de Madrid (to J. José Berlanga). 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:

PRL

prolactin

PRLR

prolactin receptor

FPRLR

Flag prolactin receptor

oPRL

ovine prolactin

PI

phosphatidylinositol

IRS-1

insulin receptor substrate-1

GH

growth hormone

IL

interleukin

DMEM

Dulbecco's modified Eagle's medium.
- Received August 9, 1996.
- Revision received November 20, 1996.