Mirzaiebadizi, A. | bioRxiv Science

bioRxiv2025

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

The RAC1P29S 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 RAC1P29S 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, RAC1P29S 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 RAC1P29S hyperactivation by prolonging its GTP-bound form. RAC1P29S 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 RAC1P29S-driven signaling. In serum-starved cells, RAC1P29S predominantly adopts an active GTP-bound state. RAC1P29S 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/637030v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2385c2org.highwire.dtl.DTLVardef@229a31org.highwire.dtl.DTLVardef@1b93011org.highwire.dtl.DTLVardef@102b427_HPS_FORMAT_FIGEXP M_FIG C_FIG A model of RAC1P29S activation and signaling in cancer cells. RAC1P29S remains in an inactive GDP-bound state in the cytoplasm where GDI1 prevents its membrane association. Upon stimulation, GEFs, primarily DOCK2, activate RAC1P29S by promoting GDP-GTP exchange, facilitating its transition to the active GTP-bound state and initiating downstream signaling. RAC1P29S binds preferentially to IQGAP1 over PAK1, reflecting a shift in effector interactions. IQGAP1 acts as a scaffolding protein, spatially modulating RAC1P29S-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 RAC1P29S 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.

cancer biology↗

bioRxiv2023

Characterization of the small Arabidopsis thaliana GTPase and ADP-ribosylation factor-like 2 protein TITAN5

Small GTPases function by conformational switching ability between GDP- and GTP-bound states in rapid cell signaling events. The ADP-ribosylation factor (ARF) family is involved in vesicle trafficking. Though evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. Here, we characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5/HALLIMASCH/ARL2/ARLC1 (hereafter termed TTN5) from Arabidopsis thaliana. Two TTN5 variants were included in the study with point mutations at conserved residues, suspected to be functional for nucleotide exchange and GTP hydrolysis, TTN5T30N and TTN5Q70L. We found that TTN5 had a very rapid intrinsic nucleotide exchange capacity with a conserved nucleotide switching mechanism. TTN5 acted as a non-classical small GTPase with a remarkably low GTP hydrolysis activity, suggesting it is likely present in GTP-loaded active form in the cell. We analyzed signals from yellow fluorescent protein (YFP)-tagged TTN5 and from in situ immunolocalization of hemagglutine-tagged HA3-TTN5 in Arabidopsis seedlings and in a transient expression system. Together with colocalization using endomembrane markers and pharmacological treatments the microscopic analysis suggests that TTN5 can be present at the plasma membrane and dynamically associated with membranes of vesicles, Golgi stacks and multivesicular bodies. While the TTN5Q70L variant showed similar GTPase activities and localization behavior as wild-type TTN5, the TTN5T30N mutant differed in some aspects. Hence, the unusual capacity of rapid nucleotide exchange activity of TTN5 is linked with cell membrane dynamics, likely associated with vesicle transport pathways in the endomembrane system. HighlightsO_LIThe small ARF-like GTPase TTN5 has a very rapid intrinsic nucleotide exchange capacity with a conserved nucleotide switching mechanism C_LIO_LIBiochemical data classified TTN5 as a non-classical small GTPase, likely present in GTP-loaded active form in the cell C_LIO_LIYFP-TTN5 is dynamically associated with vesicle transport and different processes of the endomembrane system, requiring the active form of TTN5 C_LI

plant biology↗