Cell Signaling Technology

Product Pathways - Akt Signaling

Akt2 Kinase #7503

Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.

Description

Purified recombinant human Akt2 kinase (Ala107-Glu481), supplied as a GST fusion protein.

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing human Akt2 (Ala107-Glu481) (GenBank Accession No. NM_001626.2) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Gel Staining

Gel Staining

Figure 1. The purity of the GST-Akt2 fusion protein was analyzed using SDS/PAGE followed by anti-Akt2 Western blot (A) or Silver stain (B).

Kinase Assay - Radiometric

Kinase Assay - Radiometric

Figure 2. Akt2 kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20.000, Substrate: R11-GSK3 (14-27), 5 µg/50 µl and 100 ng/50 µl Recombinant Akt2.

Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure 3. Dose dependence curve of Akt2 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) (C9C3) Rabbit mAb #9570 to detect phosphorylation of substrate peptide (#1133) by Akt2 kinase. In a 50 µl reaction, increasing amounts of Akt2 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)


Quality Control

The theoretical molecular weight of the GST-Akt2 fusion protein is 73 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. Akt2 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Akt2 activity using HTScan™ Akt2 Kinase Assay Kit #7504 [Fig.3].

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 by phosphorylating and inactivating 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). Inhibition of mTOR stops the protein synthesis machinery due to inactivation of its effector, p70 S6 kinase and activation of the eukaryotic initiation factor 4E binding protein 1 (4E-BP1), an inhibitor of translation (18,19).

  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. Nave, 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.

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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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