cyanoginosin-lr and sodium-pyrophosphate

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.

Topics: 14-3-3 Proteins; Amino Acid Substitution; Animals; Binding Sites; Carrier Proteins; Cell Membrane; Chlorocebus aethiops; COS Cells; Diphosphates; Enzyme Activation; Enzyme Inhibitors; Genes, ras; Glycerophosphates; Intracellular Signaling Peptides and Proteins; Isoenzymes; Macromolecular Substances; Marine Toxins; Microcystins; Models, Biological; Mutation, Missense; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Structure, Tertiary; Proteins; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras); RNA-Binding Proteins; Sodium Fluoride; Transfection; Tyrosine 3-Monooxygenase