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

Abstract

A prolactin (PRL)-responsive 3 -end cDNA encoding rat 4 phosphoprotein was previously isolated from a rat lymphoma cDNA library. Rat 4 is a homologue of yeast Tap42 and is a component of the mammalian target-ofrapamycin (mTOR) signalling pathway that stimulates translation initiation and G1 progression in response to nutrients and growth factors. In the present study, the full-length rat 4 cDNA was obtained by 5 -RACE and the 1023 bp open reading frame predicted a 340 amino acid protein of 39·1 kDa. The 4 mRNA was expressed in quiescent PRL-dependent Nb2 lymphoma cells deprived of PRL for up to 72 h but expression was downregulated within 4 h of PRL treatment. In contrast, PRLindependent Nb2-Sp cells showed constitutive expression of 4 that was not affected by PRL. Western analysis of Nb2 cell lysates or of V5-tagged-4 expressed in COS-1 cells detected a single immunoreactive band of 45 kDa. Enzymatic deglycosylation of affinity-purified 45 kDa 4 yielded the predicted 39 kDa protein. Phosphorylation of Nb2 4 was induced by PRL or 2-O-tetradecanoylphorbol-13-acetate (TPA) and further enhanced by a combination of PRL and TPA. The Nb2 4 associated with the catalytic subunit of protein phosphatase 2A and localized predominantly in Nb2 nuclear fractions with trace amounts in the cytosol. The immunosuppressant drug rapamycin inhibited proliferation of Nb2 cells in response to PRL or interleukin-2, but had no effect on Nb2-Sp cells. Furthermore, transient overexpression of 4 in COS-1 cells inhibited PRL stimulation of the immediate-early gene interferon regulatory factor-1 promoter activity. Therefore, PRL downregulation of 4 expression and/or PRL-inducible phosphorylation of 4 may be necessary for PRL receptor (PRLr) signalling to the interferon regulatory factor-1 promoter in the Nb2 cells and, furthermore, implicates cross-talk between the mTOR and PRLr signalling cascades during Nb2 cell mitogenesis.

Prolactin activates mammalian target-of-rapamycin through phosphatidylinositol 3-kinase and stimulates phosphorylation of p70S6K and 4E-binding protein-1 in lymphoma cells

“…by rapamycin pointed to a possible interaction between the PRLR and mTOR pathways. We have shown that rapamycin partially inhibited Nb2 cell proliferation, but crosstalk between the PRL and mTOR pathways was further supported by our observation that overexpressed a4 phosphoprotein, a component in the mTOR pathway, inhibited PRL-stimulated IRF-1 promoter activity in transfected COS-1 cells (Boudreau et al 2002). In the murine L2 clonal T-cell line, which requires both interleukin 2 (IL2) and PRL for proliferation, rapamycin, and PI3K inhibitors (wortmannin and LY294002), markedly inhibited IL2-induced cell proliferation and nuclear translocation of PRL (Belkowski et al 1999).…”

“…We have observed that rapamycin alone did not completely abolish, but only partially inhibited the PRL stimulation of Nb2 cell proliferation by 50% (Boudreau et al 2002). PRL has also been reported to stimulate the activity of MAPK before that of S6K (i.e.…”

“…Rapamycin inhibited PRLstimulated Nb2 cells proliferation and growth inhibition was maximal at 50%. We also showed that transient overexpression of a4 in COS-1 cells inhibited PRL stimulation of interferon regulatory factor-1 (IRF-1) promoter activity (Boudreau et al 2002). IRF-1 is an immediate-early PRL-responsive gene in lymphoid cells (Yu-Lee et al 1990).…”

“…We showed that a component of the mTOR pathway, the a4 phosphoprotein, was downregulated by prolactin (PRL) in the PRL-dependent rat Nb2 lymphoma cells (Boudreau et al 2002). Rapamycin inhibited PRLstimulated Nb2 cells proliferation and growth inhibition was maximal at 50%.…”

mTOR Signaling in Lymphangioleiomyomatosis

“…[136][137][138][139] Separate studies suggested that prolactin stimulates mTOR, and modifies STAT1 activity in mTOR-dependent fashion. 140,141 Further studies are required to better understand how mTORCs interact with androgen or estrogen signaling pathways.…”