IκB kinase ε and TANK-binding kinase 1 activate AKT by direct phosphorylation

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.

[1]  M. Latronico,et al.  MTORC1 regulates cardiac function and myocyte survival through 4E-BP1 inhibition in mice. , 2010, The Journal of clinical investigation.

[2]  J. Testa,et al.  AKT signaling in normal and malignant cells , 2005, Oncogene.

[3]  Ben S. Wittner,et al.  Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1 , 2009, Nature.

[4]  Survival , 2004 .

[5]  P. Cohen,et al.  Role of Translocation in the Activation and Function of Protein Kinase B* , 1997, The Journal of Biological Chemistry.

[6]  Osamu Takeuchi,et al.  The Roles of Two IκB Kinase-related Kinases in Lipopolysaccharide and Double Stranded RNA Signaling and Viral Infection , 2004, The Journal of experimental medicine.

[7]  D. Guertin,et al.  Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. , 2006, Developmental cell.

[8]  P. Cohen,et al.  Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B , 1995, Nature.

[9]  E. Casanova,et al.  Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy. , 2008, Cell metabolism.

[10]  K. Inoki,et al.  TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling , 2002, Nature Cell Biology.

[11]  P. Vogt,et al.  The akt kinase: molecular determinants of oncogenicity. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  D. Guertin,et al.  Rictor, a Novel Binding Partner of mTOR, Defines a Rapamycin-Insensitive and Raptor-Independent Pathway that Regulates the Cytoskeleton , 2004, Current Biology.

[13]  B. Hemmings,et al.  PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. , 2008, Molecular cell.

[14]  G. Mills,et al.  A new mutational AKTivation in the PI3K pathway. , 2007, Cancer cell.

[15]  D. Guertin,et al.  Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex , 2005, Science.

[16]  P. Vogt,et al.  PI 3-kinase and cancer: changing accents. , 2009, Current opinion in genetics & development.

[17]  B. Hemmings,et al.  Identification of a PKB/Akt Hydrophobic Motif Ser-473 Kinase as DNA-dependent Protein Kinase*♦ , 2004, Journal of Biological Chemistry.

[18]  Eric S. Lander,et al.  Integrative Genomic Approaches Identify IKBKE as a Breast Cancer Oncogene , 2007, Cell.

[19]  L. Cantley,et al.  Targeting the PI3K-Akt pathway in human cancer: rationale and promise. , 2003, Cancer cell.

[20]  M. Greenberg,et al.  Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor , 1999, Cell.

[21]  P. Cohen,et al.  Mechanism of activation of protein kinase B by insulin and IGF‐1. , 1996, The EMBO journal.

[22]  Lili He,et al.  Deregulation of IKBKE is associated with tumor progression, poor prognosis, and cisplatin resistance in ovarian cancer. , 2009, The American journal of pathology.

[23]  Sean F. Eddy,et al.  Inducible IκB Kinase/IκB Kinase ε Expression Is Induced by CK2 and Promotes Aberrant Nuclear Factor-κB Activation in Breast Cancer Cells , 2005 .

[24]  P. Cohen,et al.  Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Bα , 1997, Current Biology.

[25]  J. Qin,et al.  SIN1/MIP1 Maintains rictor-mTOR Complex Integrity and Regulates Akt Phosphorylation and Substrate Specificity , 2006, Cell.

[26]  Lewis C Cantley,et al.  The phosphoinositide 3-kinase pathway. , 2002, Science.

[27]  Michael Karin,et al.  Regulation and Function of IKK and IKK-Related Kinases , 2006, Science's STKE.

[28]  K. Guan,et al.  Negative Regulation of the Forkhead Transcription Factor FKHR by Akt* , 1999, The Journal of Biological Chemistry.

[29]  K. Inoki,et al.  Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. , 2006, Genes & development.

[30]  D. Sabatini,et al.  Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. , 2006, Molecular cell.

[31]  M. White,et al.  RalB GTPase-Mediated Activation of the IκB Family Kinase TBK1 Couples Innate Immune Signaling to Tumor Cell Survival , 2006, Cell.

[32]  L. Schaeffer,et al.  Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy , 2009, The Journal of cell biology.

[33]  W. Schamel,et al.  Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for the Identification and Analysis of Multiprotein Complexes , 2006, Science's STKE.

[34]  J. Blenis,et al.  Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. , 2002, Molecular cell.

[35]  Mazhar Adli,et al.  IKK-i/IKKϵ Controls Constitutive, Cancer Cell-associated NF-κB Activity via Regulation of Ser-536 p65/RelA Phosphorylation* , 2006, Journal of Biological Chemistry.

[36]  D. Sabatini,et al.  An ATP-competitive Mammalian Target of Rapamycin Inhibitor Reveals Rapamycin-resistant Functions of mTORC1* , 2009, Journal of Biological Chemistry.