Constitutive activation of protein kinase B and phosphorylation of p47phox by a membrane-targeted phosphoinositide 3-kinase

Curr Biol. 1996 Oct 1;6(10):1271-8. doi: 10.1016/s0960-9822(02)70713-6.

Abstract

Background: Phosphoinositide 3-kinase (PI 3-kinase) activity is required for mitogenic signaling and for secretory responses. Cell activation is presumed to cause the translocation of PI 3-kinase from the cytosol to the plasma membrane where the kinase interacts with its substrate phosphatidylinositol (4,5)-bisphosphate. Thus, a membrane-targeted and therefore constitutively active kinase could help elucidate the role of PI 3-kinase in intracellular signaling.

Results: The membrane-targeting sequence of Ha-Ras, containing the consensus sequence for palmitoylation and farnesylation, was fused to the carboxyl terminus of p110 alpha, the catalytic subunit of PI 3-kinase. The lipid anchor directed PI 3-kinase to the membrane and led to constitutively elevated phosphatidylinositol (3,4,5)-trisphosphate levels in transfected cells. Expression of membrane-targeted PI 3-kinase resulted in the continuous activation of downstream effectors, such as protein kinase B (PKB, also known as Akt/RAC), which was recently shown to regulate glycogen synthase kinase-3. The constitutive activation of PKB was abolished by the specific PI 3-kinase inhibitor wortmannin, and PKB activation was marginal in transfectants expressing non-membrane-targeted PI 3-kinase. Multiple phosphorylation of the cytosolic factor p47phox is required for the rapid assembly of the phagocyte NADPH oxidase upon stimulation with agonists of G-protein-coupled receptors. We show here that the expression of membrane-targeted PI 3-kinase in the monoblastic cell line GM-1 results in a wortmannin-sensitive continuous phosphorylation of p47phox.

Conclusions: Targeting of PI 3-kinase to the site of its preferred substrate leads to constitutive stimulus-independent enhanced catalysis and is sufficient to regulate different signal transduction pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Androstadienes / pharmacology
  • Animals
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism
  • Cell Line
  • Cell Membrane / enzymology
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Glycogen Synthase Kinases
  • Humans
  • Mice
  • Molecular Sequence Data
  • Monocytes / metabolism
  • NADPH Oxidases / biosynthesis
  • Phosphatidylinositol 3-Kinases
  • Phosphatidylinositol Phosphates / metabolism
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor) / antagonists & inhibitors
  • Phosphotransferases (Alcohol Group Acceptor) / genetics
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism*
  • Protein-Serine-Threonine Kinases*
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-akt
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Signal Transduction / physiology*
  • Transfection
  • Wortmannin

Substances

  • Androstadienes
  • Enzyme Inhibitors
  • Phosphatidylinositol Phosphates
  • Phosphoproteins
  • Proto-Oncogene Proteins
  • phosphatidylinositol 3,4,5-triphosphate
  • NADPH Oxidases
  • neutrophil cytosolic factor 1
  • Phosphatidylinositol 3-Kinases
  • Phosphotransferases (Alcohol Group Acceptor)
  • Glycogen Synthase Kinases
  • AKT1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Calcium-Calmodulin-Dependent Protein Kinases
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)
  • Wortmannin