title: A Raf-1 mutant that dissociates MEK/extracellular signal-regulated kinase activation from malignant transformation and differentiation but not proliferation.
abstract: It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.
authors.affiliation: The Beatson Institute for Cancer Research, CR-UK Beatson Laboratories, Garscube Estate, Bearsden, Glasgow G61 1BD, Scotland, UK. A.Dhillon@beatson.gla.ac.uk
authors.forename: Amardeep S
authors.initials: AS
authors.lastname: Dhillon
authors.forename: Sharon
authors.initials: S
authors.lastname: Meikle
authors.forename: Carole
authors.initials: C
authors.lastname: Peyssonnaux
authors.forename: Joan
authors.initials: J
authors.lastname: Grindlay
authors.forename: Christian
authors.initials: C
authors.lastname: Kaiser
authors.forename: Helge
authors.initials: H
authors.lastname: Steen
authors.forename: Peter E
authors.initials: PE
authors.lastname: Shaw
authors.forename: Harald
authors.initials: H
authors.lastname: Mischak
authors.forename: Alain
authors.initials: A
authors.lastname: Eychène
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
mesh_terms: D048948:14-3-3 Proteins; D016475:3T3 Cells / Q000378:metabolism / Q000637:transplantation / Q000648:ultrastructure; D021581:Active Transport, Cell Nucleus; D000483:Alleles; D000818:Animals; D001665:Binding Sites; D019556:COS Cells / Q000378:metabolism / Q000648:ultrastructure; D002454:Cell Differentiation / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002522:Chlorocebus aethiops; D003260:Contact Inhibition; D004789:Enzyme Activation; D025461:Feedback, Physiological; D017930:Genes, Reporter; D048728:MAP Kinase Kinase Kinase 1; D020935:MAP Kinase Signaling System; D051379:Mice; D016513:Mice, SCID; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells / Q000166:cytology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics* / Q000502:physiology; D051381:Rats; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D014446:Tyrosine 3-Monooxygenase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482578:Map3k1 protein, mouse