New insights into the classification of the RAC1 P29S hotspot mutation in melanoma as an oncogene
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The RAC1 P29S hotspot mutation, prevalent in melanoma, drives tumorigenesis by enhancing molecular interactions and hyperactivating key signaling pathways, making it a compelling target for cancer therapy. This study provides a comprehensive biochemical characterization of RAC1 P29S compared to wild-type RAC1 and mutations T17N and F28L. The P29S mutation significantly impairs nucleotide binding to guanosine triphosphate (GTP) and guanosine diphosphate, accelerating intrinsic nucleotide exchange. While minimally affecting regulation by guanosine dissociation inhibitor 1, RAC1 P29S exhibits reduced activation via diffuse B-cell lymphoma family guanine nucleotide exchange factors but retains effective activation by dedicator of cytokinesis 2. Critically, the P29S mutation severely impairs GTPase-activating protein-stimulated GTP hydrolysis, most likely contributing to RAC1 P29S hyperactivation by prolonging its GTP-bound form. RAC1 P29S displays a stronger binding affinity for IQ motif-containing GTPase-activating protein 1 than for p21-activated kinase 1, highlighting the role of the former in scaffolding RAC1 P29S -driven signaling. In serum-starved cells, RAC1 P29S predominantly adopts an active GTP-bound state. RAC1 P29S overexpression activates key cancer-associated pathways, including extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, reinforcing its role as an oncogenic driver in melanoma. These insights suggest potential therapeutic targets for melanoma treatment, including RAC1 regulators and modulators.
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.
