New insights into the classification of the RAC1 P29S hotspot mutation in melanoma as an oncogene.

The RAC1^P29S hotspot mutation, which is prevalent in melanoma, drives tumorigenesis by promoting the persistent activation of RAC1. This mutation enhances molecular interactions, and hyperactivates key signaling pathways, making RAC1^P29S a promising target for cancer therapy. This study provides a comprehensive biochemical and cell-based characterization of RAC1^P29S, as well as comparisons with wild-type RAC1 and the T17N and F28L mutants. The P29S substitution significantly impairs nucleotide binding while accelerating intrinsic nucleotide exchange. While it minimally affects regulation by guanosine dissociation inhibitor 1 (GDI1), RAC1^P29S exhibits reduced activation via DBL family guanine nucleotide exchange factors (GEFs) but retains effective activation by dedicator of cytokinesis 2 (DOCK2). Importantly, the P29S mutation severely impairs GTPase-activating protein-stimulated GTP hydrolysis, which most likely contributes to RAC1^P29S hyperactivation by prolonging its GTP-bound active form. This mutation displays a stronger binding affinity for the IQ motif-containing GTPase-activating protein 1 (IQGAP1) than for the p21-activated kinase 1 (PAK1), indicating altered effector interactions that modulate downstream signaling spatially. These biochemical findings are consistent with the fact that RAC1^P29S predominantly adopts an active GTP-bound state under serum-starved conditions. IGR1 human melanoma cells harboring endogenous RAC1^P29S exhibit persistent RAC1^P29S•GTP accumulation, even without upstream GEF activation. Furthermore, the pharmacological inhibition of DOCK2 with CPYPP significantly reduces RAC1^P29S activation in these cells, which confirms the pivotal role of DOCK2 in sustaining RAC1^P29S-driven signaling. Overexpression of RAC1^P29S activates key oncogenic pathways, including ERK1/2 and p38 MAPK, highlighting its role as a constitutively active driver mutation. Together, these results imply that targeting upstream regulators such as DOCK2 and downstream effectors, such as IQGAP1, could be effective therapeutic strategies for counteracting RAC1^P29S-mediated melanoma progression and resistance to targeted therapies. A model of RAC1^P29S activation and signaling in cancer cells. RAC1^P29S remains in an inactive GDP-bound state in the cytoplasm where GDI1 prevents its membrane association. Upon stimulation, GEFs, primarily DOCK2, activate RAC1^P29S by promoting GDP-GTP exchange, facilitating its transition to the active GTP-bound state and initiating downstream signaling. RAC1^P29S binds preferentially to IQGAP1 over PAK1, reflecting a shift in effector interactions. IQGAP1 acts as a scaffolding protein, spatially modulating RAC1^P29S-driven signaling and amplifying its effects. Under normal conditions, GAPs such as p50GAP regulate RAC1 by accelerating GTP hydrolysis, thereby maintaining its dynamic activation cycle. However, the P29S mutation severely impairs p50GAP-mediated hydrolysis, leading to accumulation of RAC1^P29S in its GTP-bound state and loss of temporal regulation. This persistent activation hyperactivates downstream effectors and promotes cancer-associated pathways, including ERK and p38 MAPK, which drive cell growth, survival, invasion and metastasis.