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

TY - JOUR

T1 - Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation

AU - James, Stephen R.

AU - Downes, C. Peter

AU - Gigg, Roy

AU - Grove, Simon J. A.

AU - Holmes, Andrew B.

AU - Alessi, Dario R.

PY - 1996/5/1

Y1 - 1996/5/1

N2 - 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 , PtdIns(3,3)P and PtdIns(4,5)P 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 , PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P 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 was submicromolar and that PtdIns(3,4)P and PtdIns(4,5)P bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P 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 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.

AB - 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 , PtdIns(3,3)P and PtdIns(4,5)P 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 , PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P 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 was submicromolar and that PtdIns(3,4)P and PtdIns(4,5)P bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P 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 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.

UR - https://www.scopus.com/pages/publications/0029993517

U2 - 10.1042/bj3150709

DO - 10.1042/bj3150709

M3 - Article

C2 - 8645147

AN - SCOPUS:0029993517

SN - 0264-6021

VL - 315

SP - 709

EP - 713

JO - Biochemical Journal

JF - Biochemical Journal

IS - 3

ER -