Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation.
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.
James
S R
SR
Department of Biochemistry, University of Dundee, Scotland.
Downes
C P
CP
Gigg
R
R
Grove
S J
SJ
Holmes
A B
AB
Alessi
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008081:Liposomes; D008954:Models, Biological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats
D008081:Liposomes; D010446:Peptide Fragments; D018129:Phosphatidylinositol Phosphates; D011518:Proto-Oncogene Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt