The pro-survival kinase Akt requires phosphorylation at two conserved residues, the activation loop site (Thr-308) and the hydrophobic motif site (Ser-473), for maximal activation. Previous reports indicate that mTORC2 is necessary for phosphorylation of the hydrophobic motif and that this site is not phosphorylated in cells lacking components of the mTORC2 complex, such as Sin1. Here The Akt/protein kinase B Ser/Thr protein kinases play a central role in signaling downstream of phosphatidylinositol 3-kinase (PI3K) 2 (1). The three isoforms (Akt1, Akt2, and Akt3) share a similar domain structure: an N-terminal pleckstrin homology (PH) domain, followed by an ␣-helical linker, and a C-terminal kinase domain that is controlled by phosphorylation (2). Once activated by phosphorylation, Akt phosphorylates defined substrates throughout the cell, ultimately inducing pro-proliferation and anti-apoptotic signaling pathways (1). The activation state of Akt is tightly controlled, and its dysregulation is implicated in the development of a variety of diseases, most notably cancer (3).One key regulator of Akt phosphorylation is the mammalian target of rapamycin (mTOR), an evolutionarily conserved Ser/ Thr protein kinase that forms two distinct protein complexes in cells. These complexes are differentially regulated and perform distinct cellular functions (4). The mTOR complex 1 (mTORC1) is composed of mTOR, Raptor, and mLST8 and is sensitive to inhibition by rapamycin. mTORC1 is a crucial regulator of cell growth in response to nutrients, stress, and growth factors (5, 6). A second complex, mTOR complex 2 (mTORC2), consists of mTOR, Rictor, mLST8, and Sin1 and is generally considered to be rapamycin-insensitive (7,8). It is this complex, mTORC2, that regulates Akt and, additionally, protein kinase C (9, 10).Upon biosynthesis, the nascent Akt polypeptide is phosphorylated at the ribosome by mTORC2 (10 -12) at a conserved C-terminal site originally identified in protein kinase C and named the turn motif site (13). Phosphorylation of this residue (Thr-450 in Akt1) (14) is important for the stability of AGC kinases (15,16). Thus, Akt is heavily ubiquitinated and degraded in cells lacking mTORC2 because the turn motif site is not phosphorylated (10, 11). This processing phosphorylation of Akt is constitutive and its dephosphorylation in cells has not been reported.Once processed by phosphorylation at the turn motif site, Akt localizes to the cytosol, where it is maintained in an inactive conformation through the interaction between its PH and kinase domains (17). In the presence of proliferative signals, PI3K is activated and generates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). Akt is subsequently recruited from the cytosol to the plasma membrane through binding of its PH domain to PIP 3 , resulting in a conformational change that separates the PH and kinase domains and unmasks two key regulatory residues whose phosphorylations are required for maximal activation of the kinase. The first site,...