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
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Research Support, Non-U.S. Gov't
MeSH terms
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14-3-3 Proteins
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Amino Acid Substitution
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Animals
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Binding Sites
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COS Cells
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Carrier Proteins*
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Cell Membrane / metabolism
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Chlorocebus aethiops
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Diphosphates / pharmacology
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Enzyme Activation
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Enzyme Inhibitors / pharmacology
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Genes, ras
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Glycerophosphates / pharmacology
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Intracellular Signaling Peptides and Proteins*
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Isoenzymes / metabolism
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Macromolecular Substances
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Marine Toxins
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Microcystins
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Models, Biological
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Mutation, Missense
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Peptides, Cyclic / pharmacology
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Phosphoprotein Phosphatases / antagonists & inhibitors
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Phosphoprotein Phosphatases / physiology*
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Phosphorylation
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Protein Phosphatase 2
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Protein Processing, Post-Translational
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Protein Structure, Tertiary
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Proteins / pharmacology
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Proto-Oncogene Proteins c-raf / chemistry
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Proto-Oncogene Proteins c-raf / genetics
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Proto-Oncogene Proteins c-raf / metabolism*
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Proto-Oncogene Proteins p21(ras) / metabolism
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RNA-Binding Proteins / pharmacology
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Sodium Fluoride / pharmacology
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Transfection
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Tyrosine 3-Monooxygenase / physiology*
Substances
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14-3-3 Proteins
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Carrier Proteins
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Diphosphates
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Enzyme Inhibitors
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Glycerophosphates
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Intracellular Signaling Peptides and Proteins
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Isoenzymes
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Macromolecular Substances
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Marine Toxins
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Microcystins
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Peptides, Cyclic
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Proteins
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RNA-Binding Proteins
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protein phosphatase inhibitor-1
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protein phosphatase inhibitor-2
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Sodium Fluoride
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Tyrosine 3-Monooxygenase
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Proto-Oncogene Proteins c-raf
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Phosphoprotein Phosphatases
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Protein Phosphatase 2
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Proto-Oncogene Proteins p21(ras)
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cyanoginosin LR
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sodium pyrophosphate
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beta-glycerophosphoric acid