Prolactin activates mammalian target-of-rapamycin through phosphatidylinositol 3-kinase and stimulates phosphorylation of p70S6K and 4E-binding protein-1 in lymphoma cells.
Mitogens activate the mammalian target-of-rapamycin (mTOR) pathway through phosphatidylinositol 3-kinase (PI3K). The activated mTOR kinase phosphorylates/ activates ribosomal protein S6 kinase (p70S6K) and phosphorylates/inactivates eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), resulting in the initiation of translation and cell-cycle progression. The prolactin receptor signaling cascade has been implicated in crosstalk with the mTOR pathway, but whether prolactin (PRL) directly activates mTOR is not known. This study showed that PRL stimulated the phosphorylation of mTOR, p70S6K, Akt, and Jak2 kinases in a dose- and time-dependent manner in PRL-dependent rat Nb2 lymphoma cells. PRL-stimulated phosphorylation of mTOR was detected as early as 10 min, closely following the phosphorylation of Akt (upstream of mTOR), but preceding that of the downstream p70S6K. PRL activation of mTOR was inhibited by rapamycin (mTOR inhibitor), LY249002, and wortmannin (P13K inhibitors), but not by AG490 (Jak2 inhibitor), indicating that it was mediated by the P13K/Akt, but not Jak2, pathway. PRL also stimulated phosphorylation of 4E-BP1 in Nb2 cells. PRL-induced phosphorylation of p70S6K and 4E-BP1 was inhibited by rapamycin, but not by okadaic acid (inhibitor of protein phosphatase, PP2A). PRL induced a transient interaction between p70S6K and the catalytic subunit of PP2A (PP2Ac) in 1 and 2 h, whereas a PP2Ac-4E-BP1 complex was constitutively present in quiescent and PRL-treated Nb2 cells. These results suggested that p70S6K and 4E-BP1 were substrates of PP2A and the inhibition of mTOR promoted their dephosphorylation by PP2A. In summary, PRL-stimulated phosphorylation of mTOR is mediated by PI3K. PRL-activated mTOR may phosphorylate p70S6K and 4E-BP1 by restraining PP2A.
Bishop
Jessica D
JD
Department of Biochemistry and Molecular Biology, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, B3H 1X5 Canada.
Nien
Wei Lun
WL
Dauphinee
Shauna M
SM
Too
Catherine K L
CK
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D008223:Lymphoma / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism*; D011388:Prolactin / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D013268:Stimulation, Chemical; D058570:TOR Serine-Threonine Kinases; D000077191:Wortmannin
D000730:Androstadienes; D002352:Carrier Proteins; D002867:Chromones; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D019319:Okadaic Acid; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D011388:Prolactin; D011494:Protein Kinases; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D000077191:Wortmannin; C499526:Eif4ebp1 protein, rat; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C546845:mTOR protein, rat