Dr. Wennogle
was interested in the function of RAF protein, because RAF was a drug target
at Novartis corporation. To elucidate further the mechanism of RAF
regulation I have decided to use the two-hybrid screen method. However,
I have faced the same problem as during the two-hybrid
screen with the CTD: how to sort out functionally important interactions.
To solve this problem I took advantage of the fact that RAF protein has
three very homologous isoforms in mammalian genome. Three RAF isoforms
are conserved in evolution from mouse to human and therefore must have
isoform-specific functions. I have reasoned that the isoform specific
functions must be achieved through isoform-specific protein-protein interactions
and therefore started looking for the proteins that will specifically bind
to only one RAF isoform.
I have fused
A-RAF,
B-RAF
and C-RAF
N-terminal domains to GAL4-DNA binding domain and performed saturated two-hybrid
screen with A-RAF and C-RAF. B-RAF fusion was self-active and two-hybrid
screen with it was impossible. In total, I found 20 different RAF
interacting proteins. Some of them were A-RAF or C-RAF specific,
and other interacted with both isoforms. Since it was impossible
to validate all found interaction I decided to focus only on the most interesting
of them: A-RAF specific interaction with putative
mammalian mitochondria import receptors and novel
protein kinase.
The sequences
for all twenty RAF interacting proteins are being patented by Novartis
and can not be displayed here.