Recent evidence has suggested that activation of phosphoinositide 3-kinase (PI 3-kinase) is required for the activation of Akt-1 by growth factors and insulin. Here we demonstrate by two independent methods that Akt-1 from L6 myotubes binds to PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns(4,5)P2 when presented against a background of phosphatidylserine (PtdSer) or a 1:1 mixture of PtdSer and phosphatidylcholine (PtdCho). No binding was observed with the lipids PtdIns(3,5)P2, PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P3 in a similar manner as inactive Akt-1. Quantitative analysis using surface plasmon resonance showed that the equilibrium association constant for the binding of Akt-1 to PtdIns(3,4,5)P3 was submicromolar and that PtdIns(3,4)P2 and PtdIns(4,5)P2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P3 did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which PtdIns(3,4,5)P3 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.
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Research Article|
May 01 1996
Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation
Stephen R. JAMES;
Stephen R. JAMES
∥
*Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee, Scotland DD1 4HN, U.K.
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C. Peter DOWNES;
C. Peter DOWNES
*Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee, Scotland DD1 4HN, U.K.
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Roy GIGG;
Roy GIGG
†National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K.
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Simon J. A. GROVE;
Simon J. A. GROVE
‡Cambridge Centre for Molecular Recognition, Department of Chemistry, Lensfield Road, Cambridge CB2 1EW, U.K.
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Andrew B. HOLMES;
Andrew B. HOLMES
‡Cambridge Centre for Molecular Recognition, Department of Chemistry, Lensfield Road, Cambridge CB2 1EW, U.K.
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Dario R. ALESSI
§MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee DD1 4HN, U.K.
¶To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
January 22 1996
Revision Received:
February 26 1996
Accepted:
March 04 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 315 (3): 709–713.
Article history
Received:
January 22 1996
Revision Received:
February 26 1996
Accepted:
March 04 1996
Citation
Stephen R. JAMES, C. Peter DOWNES, Roy GIGG, Simon J. A. GROVE, Andrew B. HOLMES, Dario R. ALESSI; Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation. Biochem J 1 May 1996; 315 (3): 709–713. doi: https://doi.org/10.1042/bj3150709
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