title: Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism.
abstract: In this study, the abilities of constitutive and conditional forms of the three Raf kinases to abrogate the cytokine dependency of FDC-P1 cells were examined. The constitutively active forms (delta) of all three Raf kinases were fused to the hormone-binding domain of the estrogen receptor (ER), rendering their activities conditionally dependent upon exogenous beta-estradiol. The vast majority of deltaRaf:ER-infected FDC-P1 cells remained cytokine-dependent; however, cells were obtained at low frequency in which expression of deltaRaf:ER abrogated cytokine dependency. Isoform specific differences between the Raf kinases were observed as cytokine-independent cells were obtained more frequently from deltaA-Raf:ER than either deltaRaf-1:ER or deltaB-Raf:ER infected cells. To determine whether the regulatory phosphorylation sites in the Raf proteins were necessary for abrogation of cytokine dependency, they were changed by site-directed mutagenesis. Substitution with phenylalanine eliminated the transforming ability of the deltaB-Raf:ER and deltaRaf-1:ER kinases. However, a similar substitution in A-Raf did not extinguish its transforming activity. The activated Raf proteins induced essential downstream MEK1 activity as treatment with the MEK1 inhibitor, PD98059, suppressed Raf-mediated growth. Activated MAP kinases (ERK1 and ERK2) were detected in deltaRaf:ER-transformed cells, and their presence was dependent upon a functional MEK1 protein. The cytokine-independent phenotype required the continued activity of the deltaRaf:ER proteins as removal of beta-estradiol caused the cells to stop growing and undergo apoptosis. The Raf-responsive cells were found to express autocrine growth factors, which promoted their growth. Constitutive activation of the Raf-1 oncogene resulted in malignant transformation as cytokine-independent FDC-P1 cells infected with a retrovirus encoding an activated Raf-1 protein formed tumors upon injection of immunocompromised mice. In summary, Raf kinases can abrogate cytokine dependency, prevent apoptosis and induce the tumorigenicity of a certain subpopulation of FDC-P1 cells by a MEK1-dependent mechanism.
authors.affiliation: Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27858, USA.
authors.forename: P E
authors.initials: PE
authors.lastname: Hoyle
authors.forename: P W
authors.initials: PW
authors.lastname: Moye
authors.forename: L S
authors.initials: LS
authors.lastname: Steelman
authors.forename: W L
authors.initials: WL
authors.lastname: Blalock
authors.forename: R A
authors.initials: RA
authors.lastname: Franklin
authors.forename: M
authors.initials: M
authors.lastname: Pearce
authors.forename: H
authors.initials: H
authors.lastname: Cherwinski
authors.forename: E
authors.initials: E
authors.lastname: Bosch
authors.forename: M
authors.initials: M
authors.lastname: McMahon
authors.forename: J A
authors.initials: JA
authors.lastname: McCubrey
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019898:Autocrine Communication; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004958:Estradiol / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D007377:Interleukin-3 / Q000494:pharmacology*; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D005810:Multigene Family; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
chemical_list: D007377:Interleukin-3; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D004958:Estradiol; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; 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; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide