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

Leukemia. 2000 Apr;14(4):642-56. doi: 10.1038/sj.leu.2401720.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Apoptosis / physiology*
  • Autocrine Communication
  • Cell Division / drug effects
  • Cell Line
  • Cell Line, Transformed
  • Cell Transformation, Neoplastic / genetics
  • Dimethyl Sulfoxide / pharmacology
  • Enzyme Activation / drug effects
  • Estradiol / pharmacology
  • Granulocyte-Macrophage Colony-Stimulating Factor / biosynthesis
  • Granulocyte-Macrophage Colony-Stimulating Factor / genetics
  • Granulocyte-Macrophage Colony-Stimulating Factor / pharmacology*
  • Hematopoietic Stem Cells / cytology*
  • Interleukin-3 / pharmacology*
  • MAP Kinase Kinase 1
  • MAP Kinase Signaling System / physiology*
  • Mice
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinase Kinases / physiology*
  • Mitogen-Activated Protein Kinases / metabolism
  • Multigene Family*
  • Phosphorylation
  • Protein Isoforms / genetics
  • Protein Isoforms / physiology*
  • Protein Processing, Post-Translational
  • Protein-Serine-Threonine Kinases / physiology*
  • Proto-Oncogene Proteins c-raf / genetics
  • Proto-Oncogene Proteins c-raf / physiology*
  • Recombinant Fusion Proteins / physiology
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transfection

Substances

  • Interleukin-3
  • Protein Isoforms
  • Recombinant Fusion Proteins
  • Estradiol
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases
  • MAP Kinase Kinase 1
  • Map2k1 protein, mouse
  • Mitogen-Activated Protein Kinase Kinases
  • Tetradecanoylphorbol Acetate
  • Dimethyl Sulfoxide