Mechanism of activation of protein kinase B by insulin and IGF-1

EMBO J. 1996 Dec 2;15(23):6541-51.

Abstract

Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Enzyme Activation
  • Humans
  • Insulin / pharmacology*
  • Insulin-Like Growth Factor I / pharmacology*
  • Kinetics
  • L Cells
  • Mice
  • Molecular Sequence Data
  • Phosphorylation
  • Protein-Serine-Threonine Kinases*
  • Proto-Oncogene Proteins / drug effects
  • Proto-Oncogene Proteins / isolation & purification
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-akt
  • Recombinant Proteins / drug effects
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Serine
  • Threonine
  • Transfection

Substances

  • Insulin
  • Proto-Oncogene Proteins
  • Recombinant Proteins
  • Threonine
  • Serine
  • Insulin-Like Growth Factor I
  • AKT1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt