Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation

Abstract

Growth factor induced activation of phosphoinositide 3-kinase and protein kinase B (PKB) leads to increased activity of the mammalian target of rapamycin (mTOR). This subsequently leads to increased phosphorylation of eIF4E binding protein-1 (4EBP1) and activation of p70 ribosomal S6 protein kinase (p70(S6K)), both of which are important steps in the stimulation of protein translation. The stimulation of translation is attenuated in cells deprived of amino acids and this is associated with the attenuation of 4EBP1 phosphorylation and p70(S6K) activation. It has been suggested that PKB regulates mTOR function by phosphorylation although direct phosphorylation of mTOR by PKB has not been demonstrated previously. In the present work, we have found that PKB directly phosphorylates mTOR and, using phosphospecific antibodies, we have shown this phosphorylation occurs at Ser(2448). Insulin also induces phosphorylation on Ser(2448) and this effect is blocked by wortmannin but not rapamycin, consistent with the effect being mediated by PKB. Amino-acid starvation rapidly attenuated the reactivity of the Ser(2448) phosphospecific antibody with mTOR and this could not be restored by either insulin stimulation of cells or incubation with PKB in vitro. Our findings demonstrate that mTOR is a direct target for PKB and support the conclusion that regulation of phosphorylation of Ser(2448) is a point of convergence for the counteracting regulatory effects of growth factors and amino acid levels.

The effects of graded levels of calorie restriction: IX. Global metabolomic screen reveals modulation of carnitines, sphingolipids and bile acids in the liver of C57BL/6 mice

“…The regulation of the IGF‐1/PI3K/Akt pathways is pertinent to nutritional lifespan studies as direct and indirect targets of Akt include mTOR and FOXO. Inhibition of mTOR increases lifespan in several species and can be reduced through CR (Navé et al ., 1999; Johnson et al ., 2013). …”

The Molecular Target of Rapamycin (mTOR) as a Therapeutic Target Against Cancer

“…2). 47,48 PI3K is a heterodimeric lipid enzyme that play a central role in cellular proliferation, motility, neovascularization, viability, and senescence, and has been demonstrated to be upregulated in many types of malignant cells. Its principal physiological function is to phosphorylate the D3 portion of membrane phosphoinositols.…”

Essential Amino Acids Regulate Both Initiation and Elongation of mRNA Translation Independent of Insulin in MAC-T Cells and Bovine Mammary Tissue Slices

“…mTOR is a protein kinase regulating cell growth and PS when it resides in the rapamycin-sensitive mTOR complex 1 (12,13). Activated mTOR signaling is generally characterized by phosphorylation of mTOR at Ser2448, ribosomal protein S6 kinase 1 (S6K1, Thr389), and eIF4 binding protein 1 (4EBP1) at Thr37/46 (14)(15)(16). Although eEF2 is not considered a direct substrate of mTOR, an inverse relationship between phosphorylation of mTOR and eEF2 has been reported (17,18).…”