NRas Nanoclusters Mediate Crosstalk Between BRAF/ERK and PI3K/AKT Signaling in Melanoma Cells

Melanocyte signaling through the MAPK pathway is orchestrated by NRas and relayed downstream via multiple effectors, such as RAF, Ral, and PI3K. In spite of their significance, the molecular mechanisms of signaling relay by NRas, their dynamics, and their potential as therapeutic targets remain unclear. Using multi-color single molecule localization microscopy (PALM and dSTORM), we resolved the mutual nanoscale organization of NRas, PI3K, and BRAF at the plasma membrane of fixed and live melanoma cells. Surprisingly, NRas and its oncogenic mutation Q61R colocalized with PI3K in mutual nanoclusters, where BRAF was also frequently present. In live cells, NRas and PI3K co-clustering declined, yet persisted over minutes. Clinically relevant perturbations revealed unexpected crosstalk within these nanoclusters and consequently, between the MAPK and PI3K pathways. Specifically, overexpression of the Ras binding domain (RBD) and PI3K inhibition by wortmannin disrupted NRAS-PI3K interactions, and reduced both pAKT and pERK levels and cancer cell proliferation. MEK inhibition with trametinib resulted in similar, yet more pronounced effects. Thus, our findings provide novel insights into NRAS-mediated signaling through nanoscale clusters and underscore their potential as therapeutic targets.