Identification of 14-3-3zeta as a protein kinase B/Akt substrate

J Biol Chem. 2002 Jun 14;277(24):21639-42. doi: 10.1074/jbc.M203167200. Epub 2002 Apr 15.

Abstract

Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3zeta, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3zeta and endogenous 14-3-3zeta from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3zeta in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3zeta. Based on a motif search of 14-3-3zeta, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3zeta with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3zeta dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3zeta both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.

MeSH terms

  • 14-3-3 Proteins
  • Amino Acid Motifs
  • Cell Line
  • Dimerization
  • Glutathione Transferase / metabolism
  • Humans
  • Phosphorylation
  • Protein Binding
  • Protein-Serine-Threonine Kinases*
  • Proto-Oncogene Proteins / chemistry*
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt
  • Recombinant Fusion Proteins / metabolism
  • Serine / chemistry
  • Substrate Specificity
  • Tyrosine 3-Monooxygenase / chemistry*
  • Tyrosine 3-Monooxygenase / metabolism

Substances

  • 14-3-3 Proteins
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Serine
  • Tyrosine 3-Monooxygenase
  • Glutathione Transferase
  • AKT1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt