Determination of Akt/PKB Signaling

Roumen Pankov1

1 National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.6
DOI:  10.1002/0471143030.cb1406s22
Online Posting Date:  May, 2004
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Abstract

This unit provides a basic protocol for assaying the level of activity, as well as the activation capacity and dynamics of inhibition of Akt/PKB in cultured cells. All these data can be obtained in a single nonradioactive experiment by standard techniques including immunoblotting and immunodetection with phosphospecific antibodies.

This unit also provides a support protocol for assaying the membrane translocation of Akt.

Keywords: Akt; protein kinase B (PKB); okadaic acid; phosphospecific antibodies

     
 
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Table of Contents

  • Basic Protocol 1: Determination of Akt/PKB Signaling in Cultured Cells
  • Support Protocol 1: Determination of Akt/PKB Translocation to the Plasma Membrane
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determination of Akt/PKB Signaling in Cultured Cells

  Materials
  • GD25 cells obtained from R. Fassler, Max Planck Institute of Biochemistry, Martinsried, Germany, and expressing wild‐type β1 integrin (β1 cells) or integrin mutant W/A (mutant cells)
  • Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (DMEM/10% FBS; appendix 2A)
  • Starvation medium: DMEM containing 1% bovine serum albumin (DMEM/1% BSA)
  • 0.5 µM okadaic acid (OA; 1 mM stock solution in DMSO, appendix 1B) in DMEM/1% BSA
  • Platelet‐derived growth factor‐BB (PDGF‐BB), freshly prepared at 10 µg/ml in 10 mM acetic acid
  • AG 1433 (e.g., Calbiochem) PDGF kinase inhibitor, 10 mM stock solution in dimethyl sulfoxide (DMSO)
  • PBS ( appendix 2A), ice cold
  • Modified radioimmunoprecipitation assay (mRIPA) buffer (see recipe), ice cold
  • 2× SDS sample buffer ( appendix 2A)
  • 10% separating gels with 4% stacking gels (unit 6.1)
  • Pre‐stained protein standards (e.g., Novex)
  • Transfer buffer (unit 6.2)
  • Ponceau S solution (unit 6.2)
  • Tris‐buffered saline with 0.1% Tween 20 (TTBS, appendix 2A)
  • Blocking solution: TTBS containing 5% dry nonfat milk (TTBS/milk)
  • Primary antibodies: polyclonal anti‐phospho Akt (Ser 473) (e.g., Cell Signaling, Biosource), monoclonal anti‐actin (e.g., Sigma)
  • Dry nonfat milk
  • Secondary horseradish peroxidase (HRP)–conjugated anti‐rabbit or anti‐mouse antibodies (e.g., Amersham Biosciences)
  • Enhanced chemiluminescence (ECL) detection reagent (unit 14.2)
  • 35‐mm tissue culture dishes
  • Plastic cell scraper (rubber policeman)
  • 1.5‐ml microcentrifuge tubes, prechilled
  • Sonicator/ultrasonic processor
  • Boiling water bath
  • Two nitrocellulose membranes cut to gel size
  • Four Whatman 3MM filter papers cut to gel size
  • SDS‐PAGE/transfer apparatus (e.g., Bio‐Rad, Novex)
  • Constant‐voltage/current power supply (e.g., Bio‐Rad)
  • Flat containers for washing gels and membranes
  • Shaker
  • Heat‐sealable plastic bags
  • Heat sealer
  • X‐ray film (e.g., Hyperfilm; Amersham Biosciences)
  • Film cassette for X‐ray film
  • X‐ray film developer
  • Additional reagents and equipment for tissue culture (unit 1.1), SDS‐PAGE (unit 6.1), and immunoblotting (unit 6.2)
NOTE: All tissue culture incubations should be performed in a humidified 37°C, 10% CO 2 incubator. Use pre‐warmed cell culture medium for all treatments.

Support Protocol 1: Determination of Akt/PKB Translocation to the Plasma Membrane

  • Cytosolic buffer (see recipe)
  • Membrane buffer (see recipe)
  • Dry ice
  • 60‐mm dishes
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Figures

Videos

Literature Cited

Literature Cited
   Andjelkovic, M., Alessi, D.R., Meier, R., Fernandez, A., Lamb, N.J.C., Frech, M., Cron, P., Cohen, P., Lucoc, J.M., and Hemmings, B.A. 1997. Role of translocation in the activation and function of protein kinase B. J. Biol. Chem. 272:31515‐31524.
   Brazil, D.P. and Hemmings, B.A. 2001. Ten years of protein kinase B signalling: A hard Akt to follow. Trends Biochem. Sci. 26:657‐664.
   Chan, T.O., Rittenhouse, S.E., and Tsichlis, P.N. 1999. AKT/PKB and other D3 phosphoinositide‐regulated kinases: Kinase activation by phosphoinositide‐dependent phosphorylation. Annu. Rev. Biochem. 68:965‐1014.
   Datta, S.R., Brunet, A., and Greenberg, M.E. 1999. Cellular survival: A play in three Akts. Genes Dev. 13:2905‐2927.
   Fassler, R., Pfaff, M., Murphy, J., Noegel, A., Johansson, S., Timpl, R., and Albrecht, R. 1995. Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts. J. Cell Biol. 128:979‐988.
   Franke, T.F., Kaplan, D.R., and Cantley, L.C. 1997. PI3K: Downstream AKT ion blocks apoptosis. Cell 88:435‐437.
   Hemmings, B.A. 1997. Akt signaling: Linking membrane events to life and death decisions. Science 275:628‐603.
   Hill, M.M. and Hemmings, B.A. 2002. Inhibition of protein kinase B/Akt. Implications for cancer therapy. Pharmacol. Ther. 93:243‐251.
   Kobayashi, S., Shirai, T., Kiyokawa, E., Mochizuki, N., Matsuda, M., and Fukui, Y. 2001. Membrane recruitment of DOCK180 by binding to PtdIns(3,4,5)P3. Biochem. J. 354:73‐78.
   Maira, S.M., Galetic, I., Brazil, D.P., Kaech, S., Ingley, E., Thelen, M., and Hemmings, B.A. 2001. Carboxyl‐terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v‐Akt at the plasma membrane. Science 294:374‐380.
   Marte, B.M. and Downward, J. 1997. PKB/Akt: Connecting phosphoinositide 3‐kinase to cell survival and beyond. Trends Biochem. Sci. 22:355‐358
   Meier, R. and Hemmings, B.A. 1999. Regulation of protein kinase B. J. Recept. Signal Transduct. Res. 19:121‐128.
   Millward, T.A., Zolnierowicz, S., and Hemmings, B.A. 1999. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem. Sci. 24:186‐191.
   Pankov, R., Cukierman, E., Clark, K., Matsumoto, K., Hahn, C., Poulin, B., and Yamada, K.M. 2003. Specific beta 1 integrin site selectively regulates Akt/protein kinase B signaling via local activation of protein phosphatase 2A. J. Biol. Chem. 278:18671‐18681.
   Persad, S. and Dedhar, S. 2003. The role of integrin‐linked kinase (ILK) in cancer progression. Cancer Metastasis Rev. 22:375‐384.
   Scheid, M.P. and Woodgett, J.R. 2003. Unravelling the activation mechanisms of protein kinase B/Akt. FEBS Lett. 546:108‐112.
   Testa, J.R. and Bellacosa, A. 1997. Membrane translocation and activation of the Akt kinase in growth factor‐stimulated hematopoietic cells. Leuk. Res. 21:1027‐1031.
   Vanhaesebroeck, B. and Alessi, D.R. 2000. The PI3K‐PDK1 connection: More than just a road to PKB. Biochem. J. 346:561‐576.
   Yamada, K.M. and Araki, M. 2001. Tumor suppressor PTEN: Modulator of cell signaling, growth, migration and apoptosis. J. Cell Sci. 114:2375‐2382.
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