Abstract
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 we show that Akt can be phosphorylated at the hydrophobic motif site (Ser-473) in the absence of mTORC2. First, increasing the levels of PIP(3) in Sin1(-/-) MEFs by (i) expression of a constitutively active PI3K or (ii) relief of a negative feedback loop on PI3K by prolonged inhibition of mTORC1 or S6K is sufficient to rescue hydrophobic motif phosphorylation of Akt. The resulting accumulation of PIP(3) at the plasma membrane results in Ser-473 phosphorylation. Second, constructs of Akt in which the PH domain is constitutively disengaged from the kinase domain are phosphorylated at the hydrophobic motif site in Sin1(-/-) MEFs; both myristoylated-Akt and Akt lacking the PH domain are phosphorylated at Ser-473. Thus, disruption of the interface between the PH and kinase domains of Akt bypasses the requirement for mTORC2. In summary, these data support a model in which Akt can be phosphorylated at Ser-473 and activated in the absence of mTORC2 by mechanisms that depend on removal of the PH domain from the kinase domain.
Highlights
The Akt/protein kinase B Ser/Thr protein kinases play a central role in signaling downstream of phosphatidylinositol 3-kinase (PI3K)2 [1]
Akt Can Be Phosphorylated at Ser-473 in the Absence of mammalian target of rapamycin (mTOR) Kinase Activity—To determine whether phosphorylation of Akt on Ser-473 absolutely depends on mTOR kinase activity, we asked whether phosphorylation at this site was abolished by Torin1, a selective ATP-competitive inhibitor of mTOR that blocks the activity of both mTOR complexes [25]. 293T cells were treated with 50 nM Torin1 and the relative phosphorylation of Akt at each of its three regulatory sites (Thr-450, Thr-308, Ser-473) was monitored using phosphospecific antibodies (Fig. 1)
Torin1 treatment caused the dephosphorylation of the hydrophobic motif (Ser-473) and activation loop (Thr-308) with kinetics that mirrored the inactivation of mTOR complex 1 (mTORC1) (Fig. 1, graph)
Summary
The Akt/protein kinase B Ser/Thr protein kinases play a central role in signaling downstream of phosphatidylinositol 3-kinase (PI3K)2 [1]. Increasing the levels of PIP3 in Sin1؊/؊ MEFs by (i) expression of a constitutively active PI3K or (ii) relief of a negative feedback loop on PI3K by prolonged inhibition of mTORC1 or S6K is sufficient to rescue hydrophobic motif phosphorylation of Akt. The resulting accumulation of PIP3 at the plasma membrane results in Ser-473 phosphorylation.
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