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

P. E. Hoyle, P. W. Moye, L. S. Steelman, W. L. Blalock, R. A. Franklin, M. Pearce, H. Cherwinski, E. Bosch, M. McMahon, J. A. McCubrey

Research output: Contribution to journalArticlepeer-review

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 (Δ) of all three Raf kinases were fused to the hormone-binding domain of the estrogen receptor (ER), rendering their activities conditionally dependent upon exogenous β-estradiol. The vast majority of ΔRaf:ER-infected FDC-P1 cells remained cytokine-dependent; however, cells were obtained at low frequency in which expression of ΔRaf:ER abrogated cytokine dependency. Isoform specific differences between the Raf kinases were observed as cytokine-independent cells were obtained more frequently from ΔA-Raf:ER than either ΔRaf-1:ER or ΔB-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 ΔB-Raf:ER and ΔRaf-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 ΔRaf:ER-transformed cells, and their presence was dependent upon a functional MEK1 protein. The cytokine-independent phenotype required the continued activity of the ΔRaf:ER proteins as removal of β-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.

Original languageEnglish
Pages (from-to)642-656
Number of pages15
JournalLeukemia
Volume14
Issue number4
Publication statusPublished - 2000

Keywords

  • Cytokines
  • Hematopoietic cells
  • Oncogenes
  • Raf
  • Signal transduction

ASJC Scopus subject areas

  • Hematology
  • Cancer Research

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