Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions.
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.
Jaumot
M
M
Laboratory of Experimental Oncology, Department of Pathology, University of Queensland Medical School, Herston Road, Queensland 4006, Australia.
Hancock
J F
JF
D048948:14-3-3 Proteins; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D011756:Diphosphates / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011905:Genes, ras; D005994:Glycerophosphates / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D046911:Macromolecular Substances; D008387:Marine Toxins; D052998:Microcystins; D008954:Models, Biological; D020125:Mutation, Missense; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000502:physiology*; D000097847:Protein Phosphatase Inhibitory Proteins
D048948:14-3-3 Proteins; D002352:Carrier Proteins; D011756:Diphosphates; D004791:Enzyme Inhibitors; D005994:Glycerophosphates; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D046911:Macromolecular Substances; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D011506:Proteins; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras); D016601:RNA-Binding Proteins; D012969:Sodium Fluoride; D014446:Tyrosine 3-Monooxygenase; C021042:protein phosphatase inhibitor-1; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins; C057862:cyanoginosin LR; C003319:sodium pyrophosphate; C031463:beta-glycerophosphoric acid