title: Impact of feedback phosphorylation and Raf heterodimerization on normal and mutant B-Raf signaling.
abstract: The B-Raf kinase is a Ras pathway effector activated by mutation in numerous human cancers and certain developmental disorders. Here we report that normal and oncogenic B-Raf proteins are subject to a regulatory cycle of extracellular signal-regulated kinase (ERK)-dependent feedback phosphorylation, followed by PP2A- and Pin1-dependent dephosphorylation/recycling. We identify four S/TP sites of B-Raf phosphorylated by activated ERK and find that feedback phosphorylation of B-Raf inhibits binding to activated Ras and disrupts heterodimerization with C-Raf, which is dependent on the B-Raf pS729/14-3-3 binding site. Moreover, we find that events influencing Raf heterodimerization can alter the transforming potential of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity but have no significant effect on proteins with high kinase activity, such as V600E B-Raf. Mutation of the feedback sites or overexpression of the Pin1 prolyl-isomerase, which facilitates B-Raf dephosphorylation/recycling, resulted in increased transformation, whereas mutation of the S729/14-3-3 binding site or expression of dominant negative Pin1 reduced transformation. Mutation of each feedback site caused increased transformation and correlated with enhanced heterodimerization and activation of C-Raf. Finally, we find that B-Raf and C-Raf proteins containing mutations identified in certain developmental disorders constitutively heterodimerize and that their signaling activity can also be modulated by feedback phosphorylation.
authors.affiliation: Laboratory of Cell and Developmental Signaling, NCI-Frederick, Building 560, Frederick, MD 21702, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Ritt
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Monson
authors.forename: Suzanne I
authors.initials: SI
authors.lastname: Specht
authors.forename: Deborah K
authors.initials: DK
authors.lastname: Morrison
mesh_terms: D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D025461:Feedback, Physiological; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D009570:Nitriles / Q000494:pharmacology; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D055503:Protein Multimerization; D054648:Protein Phosphatase 2 / Q000378:metabolism; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D002070:Butadienes; D004791:Enzyme Inhibitors; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D009570:Nitriles; D010982:Platelet-Derived Growth Factor; C113580:U 0126; C482120:Braf protein, mouse; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; D054648:Protein Phosphatase 2; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase; C000606850:Pin1 protein, mouse