IkappaB kinase epsilon and TANK-binding kinase 1 activate AKT by direct phosphorylation

Abstract

AKT activation requires phosphorylation of the activation loop (T308) by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and the hydrophobic motif (S473) by the mammalian target of rapamycin complex 2 (mTORC2). We recently observed that phosphorylation of the AKT hydrophobic motif was dramatically elevated, rather than decreased, in mTOR knockout heart tissues, indicating the existence of other kinase(s) contributing to AKT phosphorylation. Here we show that the atypical IκB kinase ε and TANK-binding kinase 1 (IKKε/TBK1) phosphorylate AKT on both the hydrophobic motif and the activation loop in a manner dependent on PI3K signaling. This dual phosphorylation results in a robust AKT activation in vitro. Consistently, we found that growth factors can induce AKT (S473) phosphorylation in Rictor(-/-) cells, and this effect is insensitive to mTOR inhibitor Torin1. In IKKε/TBK1 double-knockout cells, AKT activation by growth factors is compromised. We also observed that TBK1 expression is elevated in the mTOR knockout heart tissues, and that TBK1 is required for Ras-induced mouse embryonic fibroblast transformation. Our observations suggest a physiological function of IKKε/TBK1 in AKT regulation and a possible mechanism of IKKε/TBK1 in oncogenesis by activating AKT.

Fig. 1.

Overexpression of IKKε and TBK1 activates AKT. (A) Overexpression of TBK1 activates AKT. HeLa cells stably expressing WT TBK1, KM TBK1, or vector control (V) were tested for AKT signaling. Western blots with various antibodies are indicated. pi, phospho-antibody. (B) Overexpression of IKKε activates AKT. The experiments were similar to those shown in A.

Fig. 2.

IKKε and TBK1 are important for proper AKT activation. (A) IKKε and TBK1 double-knockout MEFs are defective in AKT phosphorylation. Both heterozygous [D(^+/−)] and homozygous knockout [D(^−/−)] MEFs were serum-starved for 16 h and then stimulated with insulin (200 nM) or EGF (50 ng/mL) for the indicated times. Western blots with various antibodies are shown. (B) IKKε and TBK1 double-knockout MEFs are defective in AKT signaling. The experiments were similar to those shown in A, except that cells were treated with Wortmannin (100 nM), TNFα (50 ng/mL), EGF (50 ng/mL), PDGF (50 ng/mL), and insulin (200 nM) for 30 min. L.E., longer exposure.

Fig. 3.

IKKε and TBK1 directly phosphorylate and activate AKT. (A) IKKε and TBK1 phosphorylate AKT on both S473 and T308 in vitro. IKKε, TBK1, IKK1, and Rictor plus mTOR were transfected into HEK293 cells and immunoprecipitated with HA antibody as indicated. The immunoprecipitates were used to phosphorylate the inactive 6XHis-AKT in vitro. Phosphorylation of AKT was determined by phospho antibodies. IB, immunoblotting; IP, immunoprecipitation. (B) Phosphorylation of AKT by endogenous TBK1. Endogenous IKK1, mTOR, PDK1, and TBK1 were immunoprecipitated from HeLa cells and then used for in vitro AKT phosphorylation as in A. (C) IKKε activates AKT in vitro. Tagged IKKε (WT and KM) were immunoprecipitated with anti-HA agarose-conjugated beads from transfected HEK293 cells and then incubated with inactive 6XHis-AKT to active the AKT kinase (IKKε assay). The IKKε immunoprecipitates were then removed by centrifugation. The activated AKT in the supernatant was subsequently assayed for its ability to phosphorylate TSC2 (AKT assay). (D) Activation of AKT by IKKε. The pi-TSC2(S939) and GST-TSC2 immunoblotting signals from two independent experiments were quantified using ImageJ. AKT activity was defined as the signal ratio between pi-TSC2(S939) and GST-TSC2.

Fig. 4.

TBK1 and mTORC2 act independently in AKT activation. (A) mTORC2-independent activation of AKT in Rictor^−/− MEF. Rictor^+/+ or Rictor^−/− cells were pretreated with different doses of Torin1 for 30 min, then stimulated with or without the combination of EGF (50 ng/mL), insulin (200 nM), PDGF (50 ng/mL) (EGF + insulin + PDGF) for 20 min. Cell lysates were blotted with antibodies as indicated. (B) PDGF activates AKT in Rictor^−/− cells in an mTOR-independent manner. Rictor^+/+, Rictor^−/−, and TBK1 stably expressed Rictor^−/− cells were pretreated or not treated with mTOR inhibitor Torin1 at different concentrations for 30 min, then treated or not treated with PDGF for 20 min. Cell lysates were blotted with different antibodies as indicated. (C) TBK1 contributes to AKT (S473) phosphorylation in the Rictor^−/− MEF cells. Control (Scrab.) and two different lentiviral-based shRNAs targeting Tbk1 (Tbk1#1 and Tbk1#2) were used to knock down TBK1 in the Rictor^−/− MEF cells. To detect AKT (S473) phosphorylation, cells were treated with or without 50 ng/mL of EGF for 5 min, and AKT was immunoprecipitated from lysate of 10-cm dishes, AKT(S473) phosphorylation was detected in the AKT-concentrated immunoprecipitates, and Tbk1 levels were determined in the cell lysate. (D) TBK1 expression does not affect mTORC2 activity. TBK1, Rictor or mTOR was immunoprecipitated from TBK1-expressing WT or TBK1-expressing KM HeLa cells and assayed for AKT (S473) phosphorylation in vitro. The post-IP supernatants were also blotted with various antibodies, as indicated. (E) mTORC2 does not affect TBK1 activity. Rictor or mTOR was down-regulated by shRNA in HeLa cells. Endogenous TBK1 was immunoprecipitated, and activity was determined by in vitro AKT phosphorylation assays.

Fig. 5.

PI3K signaling is required for AKT activation by TBK1. (A) Inhibition of PI3K abolishes TBK1-dependent AKT phosphorylation. TBK1-expressing Rictor^−/− MEF cells were treated with LY429002 (25 μM) or Wortmannin (100 nM) as indicated. Phosphorylation of AKT was determined by Western blot analysis. (B) TBK1 activity is insensitive to PI3K inhibitor treatment. HeLa cells were treated with LY249002 (25 μM) or Wortmannin (100 nM) for 30 min or rapamycin (50 nM) for 16 h as indicated. TBK1 and mTOR were immunoprecipitated, and kinase activity was determined using AKT as a substrate. (Lower) Phosphorylation of AKT in cell lysate was determined as well. (C) Both IKKε and TBK1 are membrane-associated. HeLa cells (serum-starved for 16 h or not) and MDCK2 cells were fractionated into nuclear, cytosolic, and membrane fractions. The distributions of IKKε and TBK1 in different subcellular fractions were determined by Western blot analysis along with fractional markers.

Fig. 6.

TBK1 is activated in mTOR knockout heart tissue and is required for Ras-induced MEF transformation. (A) AKT phosphorylation is elevated in cardiac-specific mTOR knockout myocardium. Heart homogenate from control mice (WT-Cre) and mTOR cardiac knockout (cKO) mice at 1 mo after tamoxifen injection were prepared and probed for AKT phosphorylation and other antibodies as indicated. Two representative animals of each genotype are shown. (B) TBK1 expression and signaling are elevated in mTOR (cKO) myocardium. The same samples as in A were probed for TBK1 protein levels, Ser172 phosphorylation, and IRF3 phosphorylation. (C) TBK1 is required for K-Ras (V12)-induced focus formation in MEFs. IKKε/TBK1 double-knockout MEFs with TBK1 re-expression or vector control (the same cells as in Fig. S2B) were infected with K-Ras (V12). Cells were cultured for 4 wk, and cell foci were stained by crystal violet.