Phospho-Drosophila Akt (Ser505

4054:

Western Blotting

Phospho Drosophila Akt (Ser505) Antibody detects endogenous levels of Akt only when phosphorylated at serine 505. It does not recognize drosophila Akt when phosphorylated at other sites, nor does it recognize related kinases such as PKC or p70 S6 Kinase.

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Ser505 of drosophila Akt. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western blot analysis of extracts from serum-starved (6 hours) S2 cells treated with 10 ug/ml insulin with or without 1 uM wortmannin pretreatment, using Phospho-Drosophila Akt (Ser505) Antibody (top) or Akt antibody #9272 (bottom).

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 Kip (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).

Drosophila Akt (D-Akt) regulates multiple biological processes such as cell survival (20) and cell growth (21). Major sites of phosphorylation include Ser505 and Thr342. These activation sites are homologous to mammalian Ser473 and Thr308 respectively. Identified downstream targets of D-Akt include Trh (Ser665) (22) and Tsc2 (23). Like Akt, D-Akt functions in a wortmannin-sensitive pathway involving PI3 kinase (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. Staveley, B. E. et al. (1998) Curr Biol 8(10) , 599-602.
21. Scanga, S. E. et al. (2000) Oncogene 19, 3971-3977.
22. Jin, J. et al. (2001) Dev Cell 1(6) , 726-728.
23. Potter, C. J. et al. (2002) Nature Cell Biology 4, 658-665.
24. Linassier, C. et al. (1997) Biochem. J. 321, 849-856.

• Palomero, T. et al. (2007) Nat Med 13, 1203-10. Applications: IF-P (paraffin)
• Dionne, M.S. et al. (2006) Curr Biol 16, 1977-85. Applications: Western Blotting

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know !

• 4060 Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb
• 2965 Phospho-Akt (Thr308) (C31E5E) Rabbit mAb
• 4691 Akt (pan) (C67E7) Rabbit mAb
• 7071 Phototope^® -HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide

For Research Use Only. Not For Use In Diagnostic Procedures.