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

Growth factor induced activation of phosphoinositide 3-kinase and protein k inase B (PKB) leads to increased activity of the mammalian target of rapamy cin (mTOR). This subsequently leads to increased phosphorylation of eIF4E b inding 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 deprive d of amino acids and this is associated with the attenuation of 4EBP1 phosp horylation and p70S6K 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 fou nd that PKB directly phosphorylates mTOR and, using phosphospecific antibod ies, we have shown this phosphorylation occurs at Ser(2448). Insulin also i nduces phosphorylation on Ser(2448) and this effect is blocked by wortmanni n but not rapamycin, consistent with the effect being mediated by PKB. Amin o-acid starvation rapidly attenuated the reactivity of the Ser(2448) phosph ospecific antibody with mTOR and this could not be restored by either insul in stimulation of cells or incubation with PKB in vitro. Our findings demon strate 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 t he counteracting regulatory effects of growth factors and amino acid levels .