To test: RAF activation sequence in response to inhibitor priming
Membrane targeting is a crucial step, which induce dimerization, RAF activation, and downstream signaling.
Membrane targeting is a crucial step, which induce dimerization, RAF activation, and downstream signaling.
Role of RAS-GTP—membrane targeting
Experiment 1 and result
1. Fractionation experiments show that membrane targeting of BRAF and CRAF have a dose dependent manner of RAF inhibitor treatment (except PLX4720):
- In non-BRAF (V600E) cell lines, CRAF phosphorylation increases.
- Membrane and cytosolic pMEK level increases when CRAF translocation.
Experiment 2 and result
Dominant-negative KRAS (S17N) shows retention of CRAF after treating inhibitor.
KRAS-WT lines show membrane localization of CRAF.
Conclusion: Depend on RAS-GTP, CRAF get activated through membrane translocation, Ser 338 phosphorylation and dimerization. Due to the activation of CRAF, pMEK level in membrane and cytosol increases after binding of RAF inhibitor.
Another factor affect membrane targeting: Conformation of the ATP-binding pocket
Experiment 3 and result
Gatekeeper threonine mutant CRAF (T421N)(not bind to GDC-0879 and weakly to AZ-628) remains in the cytoplasm
Conclusion: inhibitor occupancy of the ATP binding pocket induces membrane targeting of CRAF.
Experiment 4 and result
Conclusion: inhibitor occupancy of the ATP binding pocket induces membrane targeting of CRAF.
Experiment 4 and result
CRAF (D486A), a mutant devoid of kinase activity can translocate to membrane after inhibitor binding.
Conclusion: No kinase activity needed for membrane targeting.
Conclusion: No kinase activity needed for membrane targeting.
