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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.

journal_name

Leukemia

journal_title

Leukemia

authors

Hoyle PE,Moye PW,Steelman LS,Blalock WL,Franklin RA,Pearce M,Cherwinski H,Bosch E,McMahon M,McCubrey JA

doi

10.1038/sj.leu.2401720

subject

Has Abstract

pub_date

2000-04-01 00:00:00

pages

642-56

issue

eissn

0887-6924

issn

1476-5551

journal_volume

pub_type

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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.