Kinetic signatures of RAS-BRAF engagement reveal isoform selectivity, oncogenic amplification, and mutation-dependent inhibitor resistance.

Abstract

The RAS-RAF-MEK-ERK (MAPK) signaling cascade is a central regulator of cellular proliferation and differentiation, and its dysregulation is a frequent driver of oncogenesis. RAF activation requires recruitment by GTP-bound RAS at the plasma membrane; however, the precise molecular determinants governing RAS-RAF engagement remain incompletely understood. Here, we show that the BRAF N-terminal region forms a cooperative BSR-CRD autoinhibitory gate that restricts RAS engagement and encodes isoform-specific kinetic behavior. Using Open Surface Plasmon Resonance (OpenSPR) and Bio-Layer Interferometry (BLI), together with NanoBiT cellular assays, we reveal a kinetic encoding mechanism in which KRAS and NRAS define distinct regimes of BRAF engagement: KRAS exhibits stability-driven, long-lived complex formation, whereas NRAS displays frequency-driven, transient interactions. Oncogenic mutations reshape these regimes by selectively stabilizing RAS-BRAF association without uniformly increasing affinity, amplifying isoform-specific kinetic signatures. Pharmacological profiling further reveals isoform-dependent sensitivity of RAS-RAF disruption governed by nucleotide state and compatibility with the BSR-CRD gate. Together, these findings establish that KRAS and NRAS operate through distinct kinetic regimes of BRAF engagement governed by a structurally gated N-terminal regulatory architecture encoding temporal and pharmacological specificity in MAPK signaling.