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Chapter 9,443

Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation

James, S.R.; Downes, C.P.; Gigg, R.; Grove, S.J.; Holmes, A.B.; Alessi, D.R.

Biochemical Journal 315: 709-713

1996

ISSN/ISBN: 0264-6021
PMID: 8645147
DOI: 10.1042/bj3150709
Accession: 009442016
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)P-3, PtdIns(3,4)P-2 and PtdIns(4,5)P-2 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)P-2, PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P-3 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)P-3 was submicromolar and that PtdIns(3,4)P-3 and PtdIns(4,5)P-2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P-3 did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which PtdIns(3,4,5)P-3 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.

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Related References

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