Protein Phosphatases 1 and 2A Promote Raf-1 Activation by Regulating 14-3-3 Interactions

Oncogene. 2001 Jul 5;20(30):3949-58. doi: 10.1038/sj.onc.1204526.

Abstract

Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, protein-protein and lipid-protein interactions. We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation. General serine-threonine phosphatase inhibitors such sodium fluoride, or ss-glycerophosphate and sodium pyrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I(1) or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribution of 14-3-3 and Raf-1 across different plasma membrane microdomains. These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins
  • Amino Acid Substitution
  • Animals
  • Binding Sites
  • COS Cells
  • Carrier Proteins*
  • Cell Membrane / metabolism
  • Chlorocebus aethiops
  • Diphosphates / pharmacology
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Genes, ras
  • Glycerophosphates / pharmacology
  • Intracellular Signaling Peptides and Proteins*
  • Isoenzymes / metabolism
  • Macromolecular Substances
  • Microcystins
  • Models, Biological
  • Mutation, Missense
  • Peptides, Cyclic / pharmacology
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphoprotein Phosphatases / physiology*
  • Phosphorylation
  • Protein Phosphatase 2
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Proteins / pharmacology
  • Proto-Oncogene Proteins c-raf / chemistry
  • Proto-Oncogene Proteins c-raf / genetics
  • Proto-Oncogene Proteins c-raf / metabolism*
  • Proto-Oncogene Proteins p21(ras) / metabolism
  • RNA-Binding Proteins / pharmacology
  • Sodium Fluoride / pharmacology
  • Transfection
  • Tyrosine 3-Monooxygenase / physiology*

Substances

  • 14-3-3 Proteins
  • Carrier Proteins
  • Diphosphates
  • Enzyme Inhibitors
  • Glycerophosphates
  • Intracellular Signaling Peptides and Proteins
  • Isoenzymes
  • Macromolecular Substances
  • Microcystins
  • Peptides, Cyclic
  • Proteins
  • RNA-Binding Proteins
  • protein phosphatase inhibitor-1
  • protein phosphatase inhibitor-2
  • Sodium Fluoride
  • Tyrosine 3-Monooxygenase
  • Proto-Oncogene Proteins c-raf
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2
  • Proto-Oncogene Proteins p21(ras)
  • cyanoginosin LR
  • sodium pyrophosphate
  • beta-glycerophosphoric acid