Background: The Raf-l kinase, a 72-kDa cytoplasmic serine-threonine ki
nase, plays a central role as a second messenger in signal transductio
n. After ligand binding to a variety of transmembrane tyrosine kinase
growth factor receptors including epidermal growth factor (EGF) recept
or, the 72-kDa kinase is activated through phosphorylation to a 74-kDa
phosphoprotein. The Raf-l kinase is constitutively activated in many
transformed cells either directly, by mutations within its amino-termi
nus regulatory region, or indirectly, due to overstimulation by autocr
ine growth factors or activated proximal oncogenes. The role of Raf-l
kinase in breast cancer has not been studied. Methods: To investigate
the role of Raf-l kinase expression and its activation in breast cance
r, we studied three human breast cancer cell lines expressing varying
amounts of EGF receptor to determine the level of Raf-l protein and th
e proportion expressed in the higher molecular weight form. Effects of
serum starvation and stimulation with EGF on the Raf-l protein were s
tudied in T47D, BT474, and MDA-MB231 cells by precipitation of cell ly
sates with an anti-Raf-1 antibody followed by immunoblotting. [H-3]Thy
midine incorporation by these cells after EGF stimulation was also det
ermined as a measure of DNA synthesis. Results: In ah three breast can
cer cell lines studied, the Raf-l protein was identified in a 70- and
a 74-kDa form. The level of Raf-1 was similar in all three cell lines
and appeared unrelated to EGF receptor expression on the cell surface.
The majority of the protein was found in the 74-kDa form even after s
erum starvation. A minor shift from the lower to higher molecular weig
ht form of Raf-1 was apparent in cells treated with EGF, and increased
[H-3]thymidine incorporation could be demonstrated in two of the cell
lines after EGF stimulation. Conclusion: Baseline expression of the 7
4-kDa or activated form of the Raf-l kinase appeared to be elevated in
the breast cancer cells studied, indicating constitutive activation.
Further investigation into the role of Raf-l protein in the pathogenes
is of breast cancer is indicated.