Experiment 1 and result (knock-down experiments)
Knock down of CRAF, but not BRAF in the HCT116 (KRAS-MT) cells:
Reverse the phospho-MEK induction after inhibitor treatment
Dual knock down of ARAF and CRAF:
Have a synergistic effect on phospho-MEK level in HCT116 (KRAS-MT) lines.
Conclusion: This shows CRAF plays the major role in MEK signaling.
Experiment 2 and result
Through RAF inhibitors treatment, kinase activities of BRAF and CRAF increase via a dose dependent manner (in non-BRAF (V600E) lines):
Kinase activity after GDC-0879: increase depending on dosage of inhibitor
Kinase activity after PLX4720: increase following a dosage manner but more modest.
Technique used: Immunopreciptation assay
Technique used: Immunopreciptation assay
Experiment 3 and result
RAF activation is stimulated by dimerization, either through heterodimer or homodimer.
GDC-0879 induces A-B, B-C heterodimer as well as A-, B-, and CRAF kinase activity.
Experiment 4 and result
Do we need BRAF to activate CRAF in heterodimer and MEK phosphorylation?
Isogenic BRAF+/+ and BRAF-/- cell lines are utilised:
After treatment of RAF inhibitor, a similar pattern of induction of CRAF specific activity and pMEK/pERK level.
Conclusion: BRAF is not essential.
Experiment 5 and result
Further mechanism for priming of CRAF activity (conditions: 1. Other isoforms are not expressed. 2. Heterodimer cannot be formed.):
Conclusion: CRAF homodimer induction after RAF inhibitor treatment
Experiment 6 and result
To test whether inhibitor binding effect activation of CRAF and downstream signaling.
A gatekeeper mutant CRAF (T421N), which do not bind to either GDC-0879 or PLX4720 illustrates that pMEK level induction is similar to untreated control.
Conclusion: Inhibitor binding to the active site is essential to CRAF kinase activity and downstream activity.
Experiment 7 and result
To test whether CRAF kinase activity was required for pMEK induction (cell hyper-proliferation) by RAF inhibitor
Chemical used: AZ-628, a chemically unrelated RAF inhibitor can permanently bind to the kinase active site. (High potency against CRAF)
We assume that the prolonged binding will stop the induction of pMEK level if CRAF kinase activity is required.
Result: AZ-628 treated cell: no in vitro kinase activity, no pMEK induction, and no hyper-proliferation.
Conclusion: CRAF kinase activity required.
