Implication of α4 phosphoprotein and the rapamycin-sensitive mammalian target-of-rapamycin pathway in prolactin receptor signalling

A prolactin (PRL)-responsive 3′-end cDNA encoding rat α4 phosphoprotein was previously isolated from a rat lymphoma cDNA library. Rat α4 is a homologue of yeast Tap42 and is a component of the mammalian target-of-rapamycin (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 α4 cDNA was obtained by 5′-RACE and the 1023 bp open reading frame predicted a 340 amino acid protein of 39.1 kDa. The α4 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, PRL-independent Nb2-Sp cells showed constitutive expression of α4 that was not affected by PRL. Western analysis of Nb2 cell lysates or of V5-tagged-α4 expressed in COS-1 cells detected a single immunoreactive band of ∼45 kDa. Enzymatic deglycosylation of affinity-purified 45 kDa α4 yielded the predicted 39 kDa protein. Phosphorylation of Nb2 α4 was induced by PRL or 2-O-tetradecanoyl-phorbol-13-acetate (TPA) and further enhanced by a combination of PRL and TPA. The Nb2 α4 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 α4 in COS-1 cells inhibited PRL stimulation of the immediate-early gene interferon regulatory factor-1 promoter activity. Therefore, PRL downregulation of α4 expression and/or PRL-inducible phosphorylation of α4 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.