Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells

Raf is a key serine-threonine protein kinase which participates in the tran smission of growth, anti-apoptotic and differentiation messages. These sign als can be initiated after receptor ligation and are transmitted to members of the MAP kinase cascade that subsequently activate transcription factors controlling gene expression. Raf is a member of a multigene family which i ncludes: Raf-1, A-Raf and B-Raf. The roles that individual Raf kinases play in the regulation of normal and malignant hematopoietic cell growth are no t clear. The following studies show that all three Raf kinases are function ally present in certain human hematopoietic cells, and their aberrant expre ssion can result in abrogation of cytokine dependency. Cytokine-dependent T F-1 cells were infected with retroviruses encoding aminoterminal deleted (D elta) A-Raf, B-Raf and Raf-l proteins. These Raf proteins were conditionall y inducible as they were fused to the hormone-binding domain of the estroge n receptor (ER). A hierarchy in the abilities of Raf containing retroviruse s to abrogate cytokine dependency was observed as Delta A-Raf:ER was 20- to 200-fold more efficient than either Delta Raf-1:ER or Delta B-Raf:ER, resp ectively. This result was unexpected as A-Raf is an intrinsically weaker ki nase than either Raf-l or B-Raf. The activated Raf proteins induced downstr eam MEK and MAP (ERK1 and ERK2) kinase activities in the cells which prolif erated in response to Raf activation. Furthermore, a functional MEK signali ng pathway was necessary as treatment of the cells with a MEK1-inhibitor su ppressed Raf-mediated proliferation. To determine whether the regulatory ph osphorylation residues contained in the modified Raf oncoproteins were nece ssary for transformation, they were altered by site-directed mutagenesis. S ubstitution of the regulatory phosphorylation tyrosine residues with phenyl alanine in either A-Raf or Raf-l reduced the capacity of these oncoproteins to abrogate cytokine dependency. In contrast, changing the critical aspart ic acid residues of B-Raf to either tyrosine or phenylalanine increased the frequency of estradiol-responsive cells. Thus, the amino acids present in the regulatory residues modulated the capability of Raf proteins to abrogat e the cytokine dependency of TF-1 cells. Differences in the levels of Raf a nd downstream kinase activities were observed between cytokine-dependent an d estradiol-responsive Delta Raf:ER-infected cells as estradiol-responsive cells usually expressed more Raf and MEK activity than GM-CSF-dependent, De lta Raf:ER-infected cells. Abrogation of cytokine dependency by the activat ed Delta Raf:ER proteins was associated with autocrine growth factor synthe sis which was sufficient to promote the growth of uninfected TF-1 cells. In summary, these observations indicate that the aberrant expression of certa in activated Delta Raf:ER oncoproteins can alter the cytokine dependency of human hematopoietic TF-I cells. These cells will be useful in evaluating t he roles of the individual Raf oncoproteins in signal transduction, cell cy cle progression, autocrine transformation, regulation of apoptosis and diff erentiation. Moreover, these Raf-infected cells may be important in evaluat ing the efficacy of novel anticancer drugs designed to inhibit Raf and down stream signal transduction molecules.