We show here that BRaf inhibitors paradoxically activate CRaf and MAPK signaling in Ras transformed cells by profoundly dysregulating Ras nanocluster dynamics. Specifically, BRaf inhibitors selectively enhance the plasma membrane nanoclustering of oncogenic K-Ras and N-Ras but have no effect on H-Ras nanoclustering. Raf inhibitors are known to drive the formation of stable BRaf-CRaf and CRaf-CRaf dimers. Our results demonstrate that the presence of two Ras-binding domains in a single Raf dimer is sufficient and required to increase Ras nanoclustering, indicating that Raf dimers promote K- and N-Ras nanocluster formation by crosslinking constituent Ras proteins. Ras crosslinking increases the fraction of K-Ras and N-Ras in their cognate nanoclusters, leading to an increase in MAPK output from the plasma membrane. Intriguingly, increased MAPK signaling?in BRaf inhibited cells is accompanied by significantly decreased Akt activation. We show that this signal pathway crosstalk results from a novel mechanism of competition between stabilized Raf dimers and p110¦Á for recruitment to Ras nanoclusters.
Our findings reveal that BRaf inhibitors disrupt Ras nanocluster dynamics with significant, yet divergent, consequences for MAPK and PI3K signaling.