GNG2-MRAS interaction regulates SHOC2-dependent RAF activation and MAPK signaling

Dysregulation of the mitogen-activated protein kinase (MAPK) signaling pathway is a hallmark of many human cancers and RASopathies. The SHOC2-MRAS-PP1Cα; (SMP) holophosphatase complex is a crucial regulatory node of this pathway. It activates the MAPK pathway by dephosphorylating the inhibitory phosphoserine on RAF kinases and thereby relieving RAF autoinhibition. Although GNG2, the γ-subunit of heterotrimeric G proteins, has recently emerged as a negative modulator of MAPK signaling, its molecular mechanism of action remains incompletely understood. Here, we identify GNG2 and its truncated variants as inhibitors of SMP complex assembly by directly targeting MRAS. Using NanoBiT live-cell bioluminescence, co-immunoprecipitation (Co-IP), biolayer interferometry (BLI), and molecular dynamics simulation studies, we demonstrate that full-length GNG2 (residues 1-71aa) and two helical fragments (Δ1, residues 1-29aa and Δ2, residues 30-71aa) bind MRAS, disrupt SMP complex formation, and therefore prevent RAF dephosphorylation. Expression of the truncated GNG2 variants significantly reduced phosphorylation of ERK in cells, consistent with inhibition of pathway activation. Furthermore, a short peptide synthesized from the GNG2 Δ2 sequence effectively disrupted SMP complex formation in both purified protein systems and live cells and potently attenuated MAPK signaling. Molecular dynamics simulation studies revealed that GNG2 variants engage with the Switch I/II regions of MRAS, sterically interfering with SHOC2-PP1Cα association. Together, these findings establish GNG2 as a direct regulator of SMP complex assembly and demonstrate that GNG2-derived peptides represent a promising strategy for disrupting SHOC2-MRAS interactions and suppressing MAPK signaling in RAS/RAF-driven pathologies.