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Abstract
Activating mutations of codon 816 of the Kit gene have been implicated in malignant
cell growth of acute myeloid leukemia (AML), systemic mastocytosis and germ cell tumors.
Substitution of aspartic acid with valine (D816V) renders the receptor independent
of ligand for activation and signaling. Wild-type c-Kit is a tyrosine kinase receptor
that requires its ligand, stem cell factor (SCF), for activation. Several isoforms
of c-Kit exist as a result of alternative mRNA splicing, of which two are characterized
by the presence or absence of four amino acids (GNNK- and GNNK+, respectively) in
the extracellular domain. The two isoforms show differences in signal transduction
and biological activities and the shorter isoform seems to be highly expressed than
the longer isoform in human malignancies. In this study we analysed the signal transduction
downstream of the oncogenic c-Kit mutant D816V in an isoform specific context, using
the hematopoietic cell line Ba/F3 stably transfected with the different versions of
isoform and mutant receptor. Our data show that in contrast to the differences shown
in the activation of wild-type c-Kit isoforms, both isoforms of c-Kit/D816V are constitutively
phosphorylated to the same extent. By the use of Western blot analysis we investigated
the activation of different signaling proteins and found that both D816V/GNNK- and
D816V/GNNK+ constitutively phosphorylated Gab2, Shc, SHP-2 and Cbl to almost the same
extent as c-Kit/GNNK-. In addition, both isoforms of c-Kit/D816V induced SCF-independent
cell survival and proliferation equally well. This is in contrast to wild-type c-Kit,
where c-Kit/GNNK- induced better cell survival and stronger proliferation than c-Kit/GNNK+,
and both required stimulation with SCF. Taken together, these findings reveal that
the differences in downstream signal transduction and biological responses between
the two GNNK isoforms are eliminated by the D816V mutant.