RASAL3 (RAS protein activator like-3) is a hematopoietic-specific GTPase-activating protein (GAP) that negatively regulates Ras family small GTPases by accelerating GTP hydrolysis, converting active RAS-GTP to inactive RAS-GDP. The protein contains a canonical GAP1 family architecture with an N-terminal PH domain, a C2 domain, and a central RasGAP catalytic domain. RASAL3 is predominantly expressed in cells of hematopoietic lineages including T cells, B cells, NKT cells, neutrophils, and dendritic cells. Functional studies demonstrate that RASAL3 regulates NKT cell number and function by negatively controlling Ras-ERK signaling - loss of RASAL3 causes decreased NKT cell numbers in liver and reduced cytokine production upon alpha-GalCer stimulation, accompanied by enhanced ERK phosphorylation indicative of dysregulated Ras signaling. RASAL3 also supports naive T cell survival and restrains neutrophil hyperactivation during inflammatory responses. In dendritic cells, RASAL3 participates in a complex with CCDC88B and ARHGEF2 that modulates RHOA activity and controls cell migration. In vitro biochemical assays indicate RASAL3 displays GAP activity toward Ras in T cells (but not Rap1GAP activity) and also preferentially stimulates GTP hydrolysis on Rac2, suggesting broader substrate specificity within hematopoietic signaling contexts.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005096 GTPase activator activity | IBA GO_REF:0000033 | ACCEPT | Summary: GTPase activator activity is the core molecular function of RASAL3, consistent with its RasGAP domain architecture and experimental validation of GAP activity toward Ras and Rac2 substrates. Reason: This annotation accurately captures the primary enzymatic function of RASAL3. The protein contains a RasGAP catalytic domain (residues 474-682) and has been demonstrated to accelerate GTP hydrolysis on Ras in T cell assays [PMID:25652366] and on Rac2 in vitro [Shin 2018, Biomedical Reports]. The IBA evidence is well-supported by phylogenetic conservation across the RasGAP family. Supporting Evidence: PMID:25652366 Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. file:human/RASAL3/RASAL3-deep-research-falcon.md In T cells, Rasal3 acts as a RasGAP to dampen Ras-ERK signaling following TCR stimulation; it does not exhibit Rap1GAP activity in those assays (Muro 2015). In vitro GAP assays using human RASAL3 GAP domain found preferential stimulation of GTP hydrolysis on Rac2. |
| GO:1902531 regulation of intracellular signal transduction | IBA GO_REF:0000033 | ACCEPT | Summary: RASAL3 regulates intracellular signal transduction by modulating Ras-ERK signaling downstream of antigen receptors in immune cells. Reason: This is an appropriate parent term that captures RASAL3's role in regulating Ras-MAPK signaling cascades in hematopoietic cells. More specific child terms like GO:0046580 (negative regulation of Ras protein signal transduction) are also annotated and provide greater specificity. Supporting Evidence: PMID:25652366 RASAL3-deficient NKT cells treated with alpha-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. file:human/RASAL3/RASAL3-deep-research-falcon.md RASAL3 dampens TCR-induced Ras to Raf to MEK to ERK signaling, supporting naive T cell survival. |
| GO:0005096 GTPase activator activity | IEA GO_REF:0000043 | ACCEPT | Summary: Computational annotation of GTPase activator activity based on UniProtKB keyword mapping. Reason: Consistent with IBA annotation and experimental evidence. The UniProt keyword-based inference is well-supported by the domain architecture (RasGAP domain) and experimental validation. Supporting Evidence: file:human/RASAL3/RASAL3-uniprot.txt Contains Ras-GAP domain (residues 474-682) with conserved arginine finger at position 500, crucial for GTP hydrolysis by stabilizing the transition state. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Cytoplasmic localization inferred from UniProtKB subcellular location vocabulary. Reason: Consistent with IDA annotation from PMID:25652366 demonstrating cytoplasmic localization. RASAL3 is a cytoplasmic protein that functions in cytosolic signaling and can also localize to membrane-proximal regions. Supporting Evidence: file:human/RASAL3/RASAL3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:25652366}. |
| GO:0005938 cell cortex | IEA GO_REF:0000044 | ACCEPT | Summary: Cell cortex localization inferred from UniProtKB subcellular location vocabulary. Reason: UniProt annotation indicates cytoplasm and cell cortex localization based on experimental data from PMID:25652366. The cell cortex localization is consistent with the protein's PH and C2 domains which mediate membrane-proximal signaling. Supporting Evidence: file:human/RASAL3/RASAL3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm, cell cortex {ECO:0000269|PubMed:25652366}. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | KEEP AS NON CORE | Summary: Protein binding annotation from high-throughput binary protein interactome mapping. Reason: This term is too vague to be informative about RASAL3's specific molecular function. The annotation derives from a high-throughput protein interactome study (HuRI) that identified multiple interaction partners. While the interactions are experimentally supported, the generic protein binding term does not distinguish between functional interactions (e.g., substrate binding) and incidental or non-physiological interactions. More specific terms would be preferable if the functional significance of particular interactions were characterized. Supporting Evidence: file:human/RASAL3/RASAL3-uniprot.txt Multiple interaction partners listed in UniProt INTERACTION section including AMOTL2, DEF6, HOMER1, PICK1, RASD1, and others, with IntAct evidence. PMID:32296183 Apr 8. A reference map of the human binary protein interactome. |
| GO:0042802 identical protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | ACCEPT | Summary: Identical protein binding indicates RASAL3 homodimerization or self-association. Reason: UniProt interaction data shows RASAL3 self-interaction (Q86YV0 with Q86YV0), suggesting homodimerization. Self-association is common among RasGAP family proteins and may be relevant to regulation or localization. Supporting Evidence: file:human/RASAL3/RASAL3-uniprot.txt Q86YV0; Q86YV0 RASAL3; NbExp=3; IntAct=EBI-3437896, EBI-3437896 PMID:32296183 Apr 8. A reference map of the human binary protein interactome. |
| GO:0005886 plasma membrane | IDA GO_REF:0000052 | ACCEPT | Summary: Plasma membrane localization based on immunofluorescence data curation from Human Protein Atlas. Reason: Plasma membrane localization is consistent with RASAL3's role in membrane-proximal Ras signaling. The PH domain (residues 197-293) mediates phospholipid binding and membrane recruitment. The annotation is supported by HPA immunofluorescence data showing membrane association. Supporting Evidence: file:human/RASAL3/RASAL3-deep-research-falcon.md Consistent with PH/C2 domains, RASAL3 is suited for membrane-proximal signaling; in DCs it colocalizes within complexes with CCDC88B/ARHGEF2 that regulate migration, implying function at or near signaling membranes. |
| GO:0098562 cytoplasmic side of membrane | IDA PMID:25652366 RASAL3, a novel hematopoietic RasGAP protein, regulates the ... | ACCEPT | Summary: Cytoplasmic side of membrane localization demonstrated experimentally in NKT cell studies. Reason: This specific localization is appropriate for a RasGAP that must access membrane-bound Ras substrates. RASAL3's PH and C2 domains facilitate membrane recruitment to regulate Ras at the cytoplasmic face of membranes. Supporting Evidence: PMID:25652366 RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling file:human/RASAL3/RASAL3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm, cell cortex |
| GO:0005737 cytoplasm | IDA PMID:25652366 RASAL3, a novel hematopoietic RasGAP protein, regulates the ... | ACCEPT | Summary: Cytoplasmic localization directly demonstrated in the NKT cell functional study. Reason: Direct experimental evidence from the primary RASAL3 characterization paper confirms cytoplasmic localization, consistent with its role as a cytosolic signaling regulator. Supporting Evidence: PMID:25652366 In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. file:human/RASAL3/RASAL3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:25652366}. |
| GO:0046580 negative regulation of Ras protein signal transduction | IMP PMID:25652366 RASAL3, a novel hematopoietic RasGAP protein, regulates the ... | ACCEPT | Summary: RASAL3 negatively regulates Ras signaling as demonstrated by enhanced ERK phosphorylation in RASAL3-deficient cells. Reason: This is the core biological process annotation for RASAL3. The IMP evidence is strong - RASAL3 knockout mice show enhanced ERK phosphorylation upon alpha-GalCer stimulation, directly demonstrating that RASAL3 normally suppresses Ras-ERK signaling. Supporting Evidence: PMID:25652366 RASAL3-deficient NKT cells treated with alpha-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. PMID:25652366 Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases. |
| GO:0051142 positive regulation of NK T cell proliferation | IMP PMID:25652366 RASAL3, a novel hematopoietic RasGAP protein, regulates the ... | MODIFY | Summary: The annotation indicates RASAL3 positively regulates NKT cell proliferation, but this appears to be inverted - RASAL3 knockout DECREASES NKT cell numbers. Reason: The annotation may represent a curator error or misinterpretation. The PMID:25652366 study shows that RASAL3-deficient mice have DECREASED NKT cells (severe decrease in NKT cells in liver). Therefore RASAL3 is required for normal NKT cell maintenance, meaning loss of RASAL3 reduces NKT cell numbers. The correct interpretation is that RASAL3 positively regulates NKT cell homeostasis or maintenance, but the mechanism is through restraining Ras-ERK signaling to appropriate levels. An alternative term might be 'regulation of NK T cell homeostasis' if available, or this annotation should be reconsidered. However, if the curator intended that RASAL3 acts as a positive regulator by keeping Ras signaling in check (preventing exhaustion or death), then the annotation could stand but needs clarification. Proposed replacements: positive regulation of natural killer cell activation negative regulation of activated T cell proliferation Supporting Evidence: PMID:25652366 We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. PMID:25652366 The treatment of RASAL3-deficient mice with alpha-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-gamma from NKT cells. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-5658231 | ACCEPT | Summary: Cytosolic localization from Reactome pathway annotation for RAS GAPs stimulating RAS GTPase activity. Reason: Consistent with experimental evidence showing cytoplasmic localization. The Reactome pathway places RASAL3 in the cytosol where it encounters and inactivates membrane-associated Ras. Supporting Evidence: file:human/RASAL3/RASAL3-uniprot.txt Reactome pathway R-HSA-5658442 Regulation of RAS by GAPs includes RASAL3. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-5658435 | ACCEPT | Summary: Cytosolic localization from Reactome pathway annotation for RAS GAPs binding RAS:GTP. Reason: Duplicate of above cytosol annotation from different Reactome reaction. Both are valid TAS evidence supporting cytosolic localization where RASAL3 binds to and inactivates RAS-GTP. Supporting Evidence: file:human/RASAL3/RASAL3-uniprot.txt Reactome pathway annotation for RAS GAP function. |
| GO:0016020 membrane | HDA PMID:19946888 Defining the membrane proteome of NK cells. | ACCEPT | Summary: Membrane localization from high-throughput proteomics study of NK cell membrane proteome. Reason: The membrane annotation from HDA evidence is consistent with RASAL3's function at or near membranes where it accesses Ras substrates. The parent term 'membrane' is appropriately general for high-throughput data. Supporting Evidence: file:human/RASAL3/RASAL3-deep-research-falcon.md RASAL3 is suited for membrane-proximal signaling consistent with PH/C2 domains. PMID:19946888 Defining the membrane proteome of NK cells. |
| GO:0070062 extracellular exosome | HDA PMID:20458337 MHC class II-associated proteins in B-cell exosomes and pote... | REMOVE | Summary: Extracellular exosome localization from high-throughput proteomics of B-cell exosomes. Reason: This annotation likely represents non-specific detection in high-throughput exosome proteomics rather than a physiologically relevant localization. Cytoplasmic proteins frequently appear as contaminants in exosome preparations. There is no evidence that exosomal localization is functionally relevant for RASAL3, which functions intracellularly to regulate Ras signaling. The core function of RASAL3 is clearly cytoplasmic/membrane-proximal regulation of Ras-MAPK signaling in immune cells. Supporting Evidence: file:human/RASAL3/RASAL3-deep-research-falcon.md RASAL3 functions intracellularly in T cells, NKT cells, neutrophils, and dendritic cells to regulate Ras-ERK signaling. No evidence for functional exosomal localization. PMID:20458337 2010 May 11. MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis. |
Loading supporting content…
Download this section (compressed HTML)Q: What is the mechanistic basis for RASAL3 substrate specificity toward Ras versus Rac2?
Q: How does the CCDC88B-RASAL3-ARHGEF2 complex coordinate Ras and Rho GTPase signaling in dendritic cells?
Q: Does RASAL3 phosphorylation at serine residues in the disordered N-terminus regulate its GAP activity or localization?
Experiment: In vitro GAP assays with purified RASAL3 against a panel of Ras and Rho family GTPases to define complete substrate specificity.
Hypothesis: RASAL3 may have broader substrate specificity beyond Ras and Rac2 within the small GTPase superfamily.
Experiment: Structure determination of RASAL3 RasGAP domain in complex with Ras substrate to understand catalytic mechanism.
Hypothesis: The arginine finger at R500 is critical for transition state stabilization during GTP hydrolysis.
Experiment: Phosphomimetic and phosphodeficient mutant analysis to determine role of TCR-induced phosphorylation in RASAL3 regulation.
Hypothesis: Phosphorylation at N-terminal serine residues may regulate RASAL3 membrane recruitment or GAP activity in response to antigen receptor signaling.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)