Implication of phosphatidylinositol 3-kinase membrane recruitment in hydrogen peroxide-induced activation of PI3K and Akt

Biochemistry. 2003 Mar 18;42(10):2995-3003. doi: 10.1021/bi0205911.

Abstract

The effect of tyrosine phosphorylation of PI3K on its enzymatic activity is quite controversial, and the molecular mechanism by which ROS trigger PI3K membrane relocation is unclear. Therefore, we investigated the regulatory mechanism of hydrogen peroxide-induced PI3K activation in DT40 cells, utilizing genetic and pharmacological approaches. Our results revealed that hydrogen peroxide induced tyrosine phosphorylation of the p110 but not the p85 subunit of PI3K in DT40 cells. This phosphorylation was intact in Btk- and Cbl-deficient DT40 cells, but was drastically suppressed in Lyn, Syk, or BCAP-deficient DT40 cells. Tyrosine phosphorylation of p110 did not alter its catalytic activity, and hydrogen peroxide stimulation did not cause an increase in the intrinsic PI3K activity; however, hydrogen peroxide stimulation did induce PI(3,4,5)P3 accumulation and activate Akt. The activation of Akt, as monitored by its ability to phosphorylate GSK-3alpha/beta and by its S473 phosphorylation, was strictly dependent on PI3K activity. Under our conditions, hydrogen peroxide-induced PI3K and Akt activation was independent of Lyn, Syk, Cbl, BCAP, or Ras when each was eliminated individually either by mutation or by a specific inhibitor. In comparison, Akt activation by B cell receptor cross-linking was dependent on BCAP. In addition, hydrogen peroxide treatment caused an increase in the amount of p85 PI3K associated with the particulate fraction. Together, these results indicate that the hydrogen peroxide-induced PI3K and Akt activation in DT40 cells was achieved through PI3K membrane recruitment to its substrate site, thereby enabling PI3K to maximize its catalytic efficiency.

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Agammaglobulinaemia Tyrosine Kinase
  • Animals
  • Carrier Proteins / physiology
  • Cell Line
  • Chickens
  • Enzyme Activation
  • Enzyme Precursors / metabolism
  • Enzyme Precursors / physiology
  • Hydrogen Peroxide / antagonists & inhibitors
  • Hydrogen Peroxide / pharmacology*
  • Intracellular Signaling Peptides and Proteins
  • Oncogene Protein v-cbl
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Phosphatidylinositol Phosphates / metabolism
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphorylation
  • Protein Subunits / antagonists & inhibitors
  • Protein Subunits / metabolism
  • Protein Transport
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases / metabolism
  • Protein-Tyrosine Kinases / physiology
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-akt
  • Receptors, Antigen, B-Cell / physiology
  • Retroviridae Proteins, Oncogenic / antagonists & inhibitors
  • Retroviridae Proteins, Oncogenic / metabolism
  • Retroviridae Proteins, Oncogenic / physiology
  • Syk Kinase
  • Tyrosine / metabolism
  • ras Proteins / physiology
  • src-Family Kinases / deficiency
  • src-Family Kinases / metabolism
  • src-Family Kinases / physiology

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Enzyme Precursors
  • Intracellular Signaling Peptides and Proteins
  • Oncogene Protein v-cbl
  • PIK3AP1 protein, human
  • Phosphatidylinositol Phosphates
  • Phosphoinositide-3 Kinase Inhibitors
  • Protein Subunits
  • Proto-Oncogene Proteins
  • Receptors, Antigen, B-Cell
  • Retroviridae Proteins, Oncogenic
  • phosphatidylinositol 3,4,5-triphosphate
  • Tyrosine
  • Hydrogen Peroxide
  • Protein-Tyrosine Kinases
  • Agammaglobulinaemia Tyrosine Kinase
  • BTK protein, human
  • SYK protein, human
  • Syk Kinase
  • lyn protein-tyrosine kinase
  • src-Family Kinases
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • ras Proteins