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

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.