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.
To devise the criteria for the success of the two-hybrid screen, 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 of 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
interactions at once, 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.