Phosphorylation of Mammalian Target of Rapamycin (mTOR) at Ser-2448 IsMediated by p70S6 Kinase

Abstract

The mammalian target of rapamycin (mTOR) coordinates cell growth with the growth factor and nutrient/ energy status of the cell. The phosphatidylinositol 3-kinase-AKT pathway is centrally involved in the transmission of mitogenic signals to mTOR. Previous studies have shown that mTOR is a direct substrate for the AKT kinase and identified Ser-2448 as the AKT target site in mTOR. In this study, we demonstrate that rapamycin, a specific inhibitor of mTOR function, blocks serum-stimulated Ser-2448 phosphorylation and that this drug effect is not explained by the inhibition of AKT. Furthermore, the phosphorylation of Ser-2448 was dependent on mTOR kinase activity, suggesting that mTOR itself or a protein kinase downstream from mTOR was responsible for the modification of Ser-2448. Here we show that p70S6 kinase phosphorylates mTOR at Ser-2448 in vitro and that ectopic expression of rapamycin-resistant p70S6 kinase restores Ser-2448 phosphorylation in rapamycin-treated cells. In addition, we show that cellular amino acid status, which modulates p70S6 kinase (S6K1) activity via the TSC/Rheb pathway, regulates Ser-2448 phosphorylation. Finally, small interfering RNA-mediated depletion of p70S6 kinase reduces Ser-2448 phosphorylation in cells. Taken together, these results suggest that p70S6 kinase is a major effector of mTOR phosphorylation at Ser-2448 in response to both mitogen-and nutrient-derived stimuli.The mammalian target of rapamycin (mTOR) 1 is a member of the phosphatidylinositol 3 (PI-3)-kinase-related kinase family (PIKK), which includes ATM, ATR, hSMG-1, and DNA-PK (1-3). These large Ser/Thr protein kinases play essential roles in cellular responses to growth factors and stress. In particular, mTOR plays a critical role in coordinating cell growth with growth factor inputs as well as cellular nutrient and energy status.The bacterially derived macrolide ester, rapamycin, is clinically approved as an immunosuppressant and shows promising anti-tumor activity. Rapamycin, when complexed with FKBP-12 (FK506-binding protein, 12 kDa), binds specifically to mTOR at a conserved stretch of ϳ100 amino acids termed the FKBP-12⅐ra-pamycin binding domain (4). Mutation of a critical serine residue (Ser-2035) in the FKBP-12⅐rapamycin binding domain to a more bulky amino acid, such as isoleucine, abrogates FKBP-12⅐rapa-mycin binding to mTOR, and generates a rapamycin-resistant form of mTOR (4, 5). The mechanism by which rapamycin inhibits mTOR function remains poorly understood.Recent studies have provided significant insights into the growth factor and nutrient signaling pathway(s) upstream of mTOR. Stimulation of many growth factor receptors leads to activation of the PI-3 kinase-AKT pathway, and it appears that this pathway is centrally involved in the coupling of mitogenic stimuli to mTOR. Moreover, loss of the tumor suppressor PTEN provides a powerful signal for tumor progression and simultaneously confers increased sensitivity to the anti-proliferative effect of rapamycin (6, 7). Nutrient/energy status also regulat...

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“…The level of p-p70S6K on Thr421/Ser424, showed a pattern similar to p-p70S6K on Thr389. We also found that the phosphorylation of mTOR at Ser-2448, which was reported to be the target of p70S6 kinase, 18 was reduced as well. Propionate also inhibited mTOR pathway in SW480 cells (Supplementary Figure S3).…”

EGFR-dependent and independent activation of Akt/mTOR cascade in bone and soft tissue tumors

“…This lack of clear correlation in activation among mTOR cassette proteins presumably arises from the convergence of multiple signaling pathways involving mTOR. 9 For example, mTOR can be phosphorylated by other protein kinases, in addition to Akt, 12,34 and the mTOR substrates, S6K and 4E-BP1, can be phosphorylated by Erk1/2. 35 Moreover, signaling pathways are often not unidirectional and may be subject to feedback signaling.…”

“…35 Moreover, signaling pathways are often not unidirectional and may be subject to feedback signaling. Examples include mTOR activation by p-S6K, 34 and Akt inhibition by mTOR. 10 It is also important to note that activation of these signaling molecules may be transient and simultaneous activation of linked kinases may be difficult to detect in many tissue specimens.…”

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“…One downstream molecule of mammalian target of rapamycin (mTOR) is ribosomal S6 kinase (p70S6K). This kinase regulates the efficiency of translation of certain mRNAs and also functions in a negative feedback loop to control Akt activity (Chiang and Abraham, 2005;Inoki et al, 2005;Martelli et al, 2007). Akt, mTOR and p70S6K activation have been associated with a more severe prognosis in breast and other cancers (Bose et al, 2002;Clark et al, 2002;DeGraffenried et al, 2004;Nagata et al, 2004;Lin et al, 2005;Tsutsui et al, 2005a, b;Klos et al, 2006;Tokunaga et al, 2006;Martelli et al, 2007).…”