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Implication of alpha4 phosphoprotein and the rapamycin-sensitive mammalian target-of-rapamycin pathway in prolactin

R T M Boudreau1, S M Sangster, L M Johnson

  • 1Department of Biochemistry and Molecular Biology, Faculty of Medicine, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.

The Journal of Endocrinology
|June 18, 2002
PubMed

Insights

Prolactin (PRL) signaling regulates rat alpha4 phosphoprotein, a mammalian target-of-rapamycin (mTOR) pathway component. PRLR signaling requires alpha4 downregulation and phosphorylation for interferon regulatory factor-1 promoter activity in Nb2 cells.

Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Rat alpha4 phosphoprotein, a homolog of yeast Tap42, is involved in the mammalian target-of-rapamycin (mTOR) signaling pathway.
  • This pathway regulates translation initiation and cell cycle progression in response to growth factors and nutrients.

Purpose of the Study:

  • To obtain the full-length rat alpha4 cDNA and characterize its expression and regulation by prolactin (PRL).
  • To investigate the role of alpha4 in PRL receptor (PRLr) signaling and cell proliferation.

Main Methods:

  • 5'-RACE for full-length cDNA isolation.
  • mRNA expression analysis via Northern blotting.
  • Western blot analysis for protein detection.
  • Enzymatic deglycosylation and phosphorylation studies.
  • Co-immunoprecipitation to identify binding partners.
  • Subcellular localization studies.
  • Functional assays involving rapamycin treatment and transient alpha4 overexpression.

Main Results:

  • Full-length rat alpha4 cDNA (1023 bp) predicted a 340 amino acid protein. Alpha4 mRNA was downregulated by PRL in PRL-dependent Nb2 cells but constitutively expressed in PRL-independent Nb2-Sp cells.
  • Western analysis detected a ~45 kDa protein, which, after deglycosylation, yielded the predicted 39 kDa protein. PRL and TPA induced alpha4 phosphorylation, enhanced by their combination.
  • Alpha4 associated with protein phosphatase 2A catalytic subunit and localized to nuclear fractions. Rapamycin inhibited Nb2 cell proliferation. Overexpression of alpha4 inhibited PRL-stimulated interferon regulatory factor-1 promoter activity.

Conclusions:

  • PRL downregulation of alpha4 expression and/or PRL-inducible phosphorylation are crucial for PRLr signaling to the interferon regulatory factor-1 promoter in Nb2 cells.
  • These findings suggest cross-talk between mTOR and PRLr signaling pathways during Nb2 cell proliferation.

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