Splice Form Specific Signaling of the Hematopoietic Growth Factor Receptor c-Kit.

Abstract

c-Kit is a member of the receptor tyrosine kinase family class III. Upon stimulation with its ligand, stem cell factor, c-Kit becomes activated and phosphorylated on tyrosine residues. The receptor activation leads to cellular responses such as proliferation, differentiation, survival and migration. c-Kit is known to be involved in the pathogenesis of several forms of cancer, e.g. gastrointestinal stroma tumors, leukemias and small-cell lung cancer. c-Kit exists in different isoforms due to alternative mRNA splicing. We are studying one pair of isoforms which varies by the presence or absence of four amino acids GNNK, in the juxtamembrane region just outside the plasma membrane. They are denoted GNNK− and GNNK+, respectively, and have previously been shown by our group to display functional differences in the activation of signaling pathways and cellular responses in the fibroblast cell line NIH3T3. The GNNK− splice form binds and activates Src family kinases more efficiently than the GNNK+ splice form. This leads to stronger phosphorylation of ShcA, extracellular signal regulated kinase 1/2 (Erk) and Cbl compared to the GNNK+ form. In order to study the two GNNK spliceforms in a more physiological context, we have retrovirally transduced two hematopoietic cell lines, 32D and BaF/3, which we now use as our model system. We could demonstrate that SCF stimulation induced a kinetically faster and stronger tyrosine phosphorylation pattern in the GNNK− splice form than the c-Kit GNNK+ splice form. Moreover, the phosphorylation of Erk and Shc is stronger in the c-Kit GNNK− splice form. These results are in line with the results described for NIH3T3 fibroblasts and previously published by our group. We are investigating the SCF-induced proliferative response in the hematopoietic cell lines 32D and BaF/3. Having established these model cell lines, we now aim to use Affymetrix microarray technology to detect differences in gene expression between the two splice forms upon ligand stimulation for different periods of time. As these different splice forms show remarkable differences in their signaling capacity we expect to find significant differences, but also similarities, in gene induction/repression mediated by the c-Kit splice forms. The idea is to link these findings to the biological responses induced by the two splice forms. We will more thoroughly investigate these findings using different c-Kit mutants and a battery of inhibitors of signaling proteins of interest available in the lab. An increased knowledge of c-Kit signaling in general and of the two different c-Kit splice forms in particular will help to find rational treatment for the unfavorable effect of c-Kit in cancer.