Cell Signaling Technology

Product Pathways - PI3K / Akt Signaling

Akt (E17K Mutant Specific) (D1T7P) Rabbit mAb #14702

No. Size Price
14702S 100 µl ( 10 western blots ) ¥3,100.00 现货查询 购买询价
14702 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W 1:1000 Human, Transfected Only 87 (Akt-GFP) Rabbit IgG
IP 1:50
IF-IC 1:800

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, IP=Immunoprecipitation, IF-IC=Immunofluorescence (Immunocytochemistry),

Homology

Species predicted to react based on 100% sequence homology: Mouse, Rat,

Specificity / Sensitivity

Akt (E17K Mutant Specific) (D1T7P) Rabbit mAb recognizes levels of transfected E17K mutant Akt1 and Akt2 protein. This antibody weakly recognizes transfected E17K mutant Akt3.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to E17K mutant sequence of human Akt1 protein.

IF-IC

IF-IC

Confocal immunofluorescent analysis of 293T cells transiently transfected with GFP-tagged wild-type Akt1 (upper left), GFP-tagged mutant E17K Akt1 (upper right), GFP-tagged wild-type Akt2 (lower left), or GFP-tagged mutant E17K Akt2 (lower right) using Akt (E17K Mutant Specific) (D1T7P) Rabbit mAb (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Note: Green - GFP-tagged wild-type Akt and E17K mutant; Red - Akt (E17K mutant); Yellow - overlay of GFP and Akt (E17K mutant).

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, untransfected (lane 1) or transfected with GFP tagged wild-type Akt1 (lane 2), E17K mutant Akt1 (lane 3), wild-type Akt2 (lane 4), or mutant E17K Akt2 (lane 5), using Akt (E17K Mutant Specific) (D1T7P) Rabbit mAb (upper) and GFP (D5.1) XP® Rabbit mAb #2956 (lower).

IP

IP

Immunoprecipitation of Akt (E17K) from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Akt (E17K Mutant Specific) (D1T7P) Rabbit mAb. Western blot analysis was performed using Akt (E17K Mutant Specific) (D1T7P) Rabbit mAb.

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).

Mutation of the glutamic acid at residue 17 to lysine (E17K) of Akt was initially identified in human breast, colorectal, and ovarian cancers (20). This conserved glutamic acid residue is located at the lipid-binding pocket of the Akt1 plextrin homology domain. The E17K mutation increases the affinity between Akt1 and phospholipids at the plasma membrane, leading to increased Akt1 recruitment, super-activation of the Akt pathway, cellular transformation, and tumor formation (20,21). Additional studies detect the presence of the Akt1 (E17K) mutation in multiple cancers, including lung cancer, prostate cancer, and endometrial carcinoma (22,23). The presence of mutant Akt3 (E17K) protein has also been seen in cases of melanoma (24).

  1. Franke, T.F. et al. (1997) Cell 88, 435-7.
  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.
  20. Carpten, J.D. et al. (2007) Nature 448, 439-44.
  21. Landgraf, K.E. et al. (2008) Biochemistry 47, 12260-9.
  22. Malanga, D. et al. (2008) Cell Cycle 7, 665-9.
  23. Cohen, Y. et al. (2010) Gynecol Oncol 116, 88-91.
  24. Davies, M.A. et al. (2008) Br J Cancer 99, 1265-8.

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