BIOLUMINESCENCE RESONANCE ENERGY TRANSFER (BRET) ASSAY OF THE RAS/RAF INTERACTION

Abstract

The ERK/MAPK cascade plays a central role in the regulation of cellular growth, proliferation, and differentiation, and is dependent upon the binding of the Raf kinases to activated, GTP-bound Ras. The three Ras family members, HRas, KRas, and NRas, share 85% sequence identity, and yet show unique distribution within different cancer subtypes—suggesting potentially unique roles for the funily members. Furthermore, the dominant oncogenic codon substitution varies amongst the Ras proteins, and within the cancer subtypes. To better elucidate the differences amongst the Ras family members and mutational variants, oncogenic HRas, KRas4B and NRas were investigated for their interaction with the effectors A-Raf, B-Raf and C-Raf, using the live-cell BRET system. Here we show that the oncogenic Ras family members differentially interact with the downstream Raf effectors, and that the Ras hypervariable region (HVR) governs some of these preferences.