Cell Signaling Technology

Product Pathways - PI3K / Akt Signaling

Phospho-Akt2 (Ser474) (D3H2) Rabbit mAb (Akt2 Specific) #8599

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP H M R Endogenous 60 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation
Reactivity Key:  H=Human  M=Mouse  R=Rat
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Phospho-Akt2 (Ser474) (D3H2) Rabbit mAb (Akt2 Specific) recognizes endogenous levels of Akt2 protein only when phosphorylated at Ser474. This antibody does not cross-react with Akt1 protein when phosphorylated at Ser473 or with Akt3 protein when phosphorylated at Ser472.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser474 of human Akt2 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from Akt1 (-/-) mouse embryonic fibroblasts (MEF) or Akt2 (-/-) MEF, untreated (-) or treated with Human Platelet-Derived Growth Factor AA (hPDGF-AA) #8913 (100 ng/ml, 15 min; +), using Phospho-Akt2 (Ser474) (D3H2) Rabbit mAb (upper), Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (middle), or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from LNCaP cells, transfected with a construct expressing Akt1 shRNA or Akt2 shRNA, uninduced (-) or doxycycline-induced for the indicated times, using Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) #9018 (top), Phospho-Akt2 (Ser474) (D3H2) Rabbit mAb (Akt2 Specific) (2nd from top), Akt1 (C73H10) Rabbit mAb #2938 (middle), Akt2 (D6G4) Rabbit mAb #3063 (2nd from bottom), or PI3 Kinase p85 (19H8) Rabbit mAb #4257 (bottom). (Data kindly provided by Drs. Rebecca Chin and Alex Toker, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA).

Background

Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).

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  2. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
  3. Franke, T.F. et al. (1995) Cell 81, 727-36.
  4. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
  5. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
  6. Jacinto, E. et al. (2006) Cell 127, 125-37.
  7. Cardone, M.H. et al. (1998) Science 282, 1318-21.
  8. Brunet, A. et al. (1999) Cell 96, 857-68.
  9. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
  10. Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
  11. Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
  12. Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.
  13. Cross, D.A. et al. (1995) Nature 378, 785-9.
  14. Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.
  15. Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.
  16. Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.
  17. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
  18. Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.
  19. Manning, B.D. et al. (2002) Mol Cell 10, 151-62.

Application References

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