Among twenty
proteins identified in the two-hybrid
screen with RAF isoforms one protein showed a significant sequence
similarity to the vast family of protein kinases and therefore was named
hA38 kinase. hA38 kinase sequence had also all necessary features
of the consensus protein kinase motif and it had the biggest similarity
to the subfamily of dual specificity protein kinases. Since it was
known from the literature that RAF protein is phosphorylated in vivo, hA38
kinase can be a putative kinase that phosphorylate RAF. The Northern
blot analysis of hA38 kinase mRNA with Clontech
cancer and normal tissue mRNA blots showed that this kinase is expressed
only in tumor cells, but not in the normal adult tissues. Since hA38
cDNA is also found in the tissue culture cells and fetal tissues it suggests
that it is a growth associated kinase. Its expression pattern is
consistent with the fact that RAF phosphorylation is increased upon stimulation
of cell growth.
To start further
characterization of hA38 kinase I have made a His-tag fusion protein and
expressed it in E. coli. I have attempted to purify hA38 in sufficient
quantities for crystallization but the protein was aggregated. Nevertheless,
purified His-hA38 kinase had enzymatic activity. As a continuation
of my attempts to determine 3D structure of the hA38 kinase I have used
the Modeler program, which is the part of MSI
Insight software package. Modeler allows 3D structure predictions
from the alignments of hA38 kinase sequence with the sequences of other
protein kinases which 3D structure have been determined.
The patent
on hA38 kinase is being filed by Novartis and its sequence can not be displayed
here.