Cell Signaling Technology

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HTScan® Akt2 Kinase Assay Kit #7504

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.

Kit Includes Quantity
Phospho-eNOS (Ser1177) Antibody # 9571 30 microliters
Kinase Buffer (10X) # 9802 15 milliliters
ATP (10 mM) # 9804 1 milliliters
eNOS (Ser1177) Biotinylated Peptide # 1133 1.25 milliliters
Akt2 Kinase # 7503 5 micrograms

Description

The kit provides a means of performing kinase activity assays with recombinant human Akt2 kinase. It includes active Akt2 kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine antibody for detection of the phosphorylated form of the substrate peptide.

Molecular Weights

Peptide substrate, Biotin-peptide: 2,431 Daltons. GST-Akt2 Kinase domain: 73 kDa.

Peptide Core Sequence

TQS*FS

Kinase Assay - Radiometric

Kinase Assay - Radiometric

Figure 1. 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, recombinant Akt2: 100 ng/50 µl.

Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure 3. Dose dependence curve of Akt2 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) Antibody #9571 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.)

Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure 5. Staurosporine inhibition of Akt2 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) Antibody #9571 to detect phosphorylation of Akt2 substrate peptide (#1133) by Akt2 kinase. In a 50 µl reaction, 50 ng Akt2, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)


Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure 4. Peptide concentration dependence of Akt2 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) Antibody #9571 to detect phosphorylation of substrate peptide (#1133) by Akt2 kinase. In a 50 µl reaction, 50 ng of Akt2 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure 2. Time course of Akt2 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) Antibody #9571 to detect phosphorylation of Akt2 substrate peptide (#1133) by Akt2 kinase. In a 50 µl reaction, 50 ng Akt2 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing a fragment of 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.

Quality Control

The substrate peptide was selected using our Serine/Threonine Kinase Substrate Screening Kit #7400. Phospho-eNOS (Ser1177) Antibody #9571 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified Akt2 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the Akt2 kinase was determined using a radiometric assay [Fig.1]. Time course [Fig.2], kinase dose dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify Akt2 activity using the Akt2 substrate peptide provided in this kit. Akt2 sensitivity to the inhibitor staurosporine was measured using the Akt2 substrate peptide provided in this kit [Fig.5].

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-EP1), an inhibitor of translation (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. 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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