{"gene":"RALB","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2006,"finding":"RalB directly binds its effector Sec5 (a component of the exocyst complex), and the RalB/Sec5 complex recruits and activates the atypical IκB kinase TBK1, suppressing apoptosis in cancer cells and mediating innate immune signaling upon viral infection.","method":"Co-immunoprecipitation, protein complex analysis, kinase assays, loss-of-function (siRNA), cell transformation assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, kinase activity assays, loss-of-function with defined phenotypic readouts, replicated in multiple cell contexts","pmids":["17018283"],"is_preprint":false},{"year":2011,"finding":"RalB localizes to nascent autophagosomes and is activated upon nutrient deprivation. Through direct binding to its effector Exo84, RalB induces assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, driving autophagosome formation and isolation membrane maturation.","method":"Subcellular fractionation/live imaging, co-immunoprecipitation, loss-of-function (siRNA/shRNA), epistasis (constitutively active RalB rescue), in vitro complex reconstitution","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including localization, complex reconstitution, loss-of-function with defined phenotype, and constitutively active rescue","pmids":["21241894"],"is_preprint":false},{"year":2006,"finding":"RalB (but not RalA) is required for cell migration; RalB promotes exocyst (Sec6/8 complex) assembly and its localization to the leading edge of migrating cells, with loss of RalB impairing vectorial cell motility.","method":"siRNA loss-of-function, immunofluorescence localization, exocyst assembly assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with defined migration phenotype, localization experiment, exocyst assembly assay, two orthogonal methods in one study","pmids":["16382162"],"is_preprint":false},{"year":2013,"finding":"Ubiquitylation of RalB at Lys47 switches its effector preference: ubiquitylation sterically inhibits RalB binding to Exo84 (blocking autophagy) while facilitating its interaction with Sec5 (promoting TBK1-innate immunity signaling). The deubiquitylase USP33 removes this ubiquitin mark upon nutrient starvation, relocalizing to RalB-positive vesicles and enabling RalB-Exo84-Beclin1 complex formation for autophagy.","method":"Ubiquitylation mapping, mutagenesis (Lys47 mutants), co-immunoprecipitation, USP33 knockdown/overexpression, localization studies","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — site-specific mutagenesis, reciprocal Co-IP, identification of writer/eraser, multiple orthogonal methods in one study","pmids":["24056301"],"is_preprint":false},{"year":2008,"finding":"RalB (but not RalA) is required for abscission and completion of cytokinesis through recruitment of the exocyst to the midbody; distinct RalGEF proteins provide upstream input to RalB specifically at this step, whereas RalA acts earlier at the cytokinetic furrow.","method":"siRNA knockdown, live-cell imaging, subcellular localization, epistasis with RalGEF mutants","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA with defined cytokinesis phenotype, localization experiments, epistasis with RalGEF, two orthogonal methods","pmids":["18756269"],"is_preprint":false},{"year":2006,"finding":"RalB is required for invasion and metastasis of pancreatic cancer cells, while RalA (but not RalB) is required for anchorage-independent growth and tumor initiation; both are more commonly activated in pancreatic tumors than other Ras effector pathways.","method":"siRNA knockdown, in vitro invasion assays, tail-vein metastasis assays in mice, panel of 10 cell lines","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function validated across 9–10 cell lines and in vivo metastasis model, well-controlled with isoform-specific siRNAs","pmids":["17174914"],"is_preprint":false},{"year":2014,"finding":"In the unliganded state, integrin αvβ3 recruits both KRAS and RalB to the tumor cell plasma membrane, leading to activation of TBK1 and NF-κB; this αvβ3-KRAS-RalB-NF-κB pathway is necessary and sufficient for tumor initiation, anchorage independence, self-renewal, and erlotinib resistance.","method":"Co-immunoprecipitation, plasma membrane fractionation, loss-of-function (siRNA/shRNA), gain-of-function, patient-derived xenografts, pharmacological inhibition","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vivo PDX models, multiple cell lines, gain- and loss-of-function experiments","pmids":["24747441"],"is_preprint":false},{"year":2010,"finding":"Protein kinase C (PKC) phosphorylates RalB at Ser198 in its C-terminal membrane-targeting sequence, causing RalB translocation from plasma membrane to perinuclear regions; this phosphorylation is necessary for actin cytoskeletal organization, anchorage-independent growth, cell migration, and experimental lung metastasis.","method":"In vitro kinase assay, mass spectrometry phospho-site mapping, site-directed mutagenesis, phosphosite-specific antibodies, phorbol ester stimulation, siRNA rescue experiments, subcellular fractionation","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, MS-based site mapping, mutagenesis with functional rescue, in vivo metastasis assay, multiple orthogonal methods in one study","pmids":["20940393"],"is_preprint":false},{"year":2012,"finding":"PKCα phosphorylation of RalB at Ser198 results in enhanced RalB endomembrane accumulation, decreased association with exocyst component Sec5, regulation of v- and t-SNARE interactions, control of vesicular trafficking of α5-integrin to the cell surface, and modulation of cell attachment to fibronectin.","method":"Mutagenesis, co-immunoprecipitation, subcellular fractionation, vesicle trafficking assays, cell adhesion assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis, co-IP for effector interaction, vesicle trafficking assay, cell biological readouts, multiple orthogonal methods","pmids":["22393054"],"is_preprint":false},{"year":2012,"finding":"RalB mediates invadopodium formation in KRAS mutant pancreatic cancer cells through RalBP1/RLIP76 (but not Sec5 or Exo84), and this function requires the ATPase activity of RalBP1 rather than its canonical GAP activity toward Rho GTPases.","method":"siRNA knockdown, dominant-negative/constitutively active mutants, ATPase-deficient RLIP76 mutants, invadopodium formation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — isoform-specific knockdown, effector-selective mutants, ATPase-domain mutagenesis, defined cellular phenotype readout","pmids":["22331470"],"is_preprint":false},{"year":2015,"finding":"RalB (but not RalA) is required for TGFβ-induced EMT-driven cell dissemination by acting via the RhoGEF GEF-H1, which associates with the exocyst complex. Uncoupling of exocyst subunit Sec5 from GEF-H1 impairs RhoA activation and traction force generation.","method":"siRNA knockdown, co-immunoprecipitation (Sec5-GEF-H1 interaction), traction force microscopy, 3D invasion assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with defined invasion/force phenotype and Co-IP of GEF-H1 with exocyst, but single lab","pmids":["26152517"],"is_preprint":false},{"year":2018,"finding":"Active RalB at the plasma membrane promotes recruitment of the Wave Regulatory Complex (WRC) via the exocyst, inducing membrane protrusions and invasion; Ras signals to RalB through RalGEFs RGL1 and RGL2 to drive invasiveness.","method":"Optogenetic light-controlled RalB activation, co-immunoprecipitation, siRNA knockdown, invasion assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — novel optogenetic activation combined with Co-IP and loss-of-function, multiple orthogonal methods in one study","pmids":["30320548"],"is_preprint":false},{"year":2019,"finding":"SIRT2 acts as a deacylase for RalB: RalB undergoes lysine fatty acylation predominantly at Lys200, which enhances plasma membrane localization and recruitment of its effectors Sec5 and Exo84 to the plasma membrane; SIRT2 removes this acylation and affects cell migration.","method":"Biochemical acylation assays, mutagenesis (Lys200), co-immunoprecipitation, subcellular fractionation, trans-well migration assays, SIRT2 knockdown","journal":"ACS chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical acylation characterization, site-specific mutagenesis, localization assays, Co-IP for effectors; single lab","pmids":["31433161"],"is_preprint":false},{"year":2009,"finding":"NMR solution structure of RalB bound to GTP analogue GMPPNP revealed that the switch regions predominantly adopt state 1 (non-effector-binding competent) in the unbound form; 31P NMR of RalB.GTP shows both states 1 and 2 are sampled, and addition of an effector only partially stabilizes state 2, revealing dynamic properties of the effector-binding switches.","method":"NMR spectroscopy (solution structure determination), 31P NMR, backbone dynamics measurements","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with functional dynamics validation (31P NMR + effector binding), rigorous structural method","pmids":["19166349"],"is_preprint":false},{"year":2010,"finding":"The crystal structure of RalB in complex with the Ral-binding domain of RLIP76 reveals a coiled-coil binding motif that contacts both nucleotide-sensitive switch regions of RalB; this mode of binding is distinct from the Sec5 and Exo84 exocyst interactions, and Sec5, Exo84, and RLIP76 bind Ral proteins competitively with similar affinities in vitro.","method":"X-ray crystallography (structure of RalB-RLIP76 complex), affinity measurements with RalB mutants, competitive binding assays","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with mutagenesis-based interface validation and quantitative binding competition assays","pmids":["20696399"],"is_preprint":false},{"year":2007,"finding":"RalB (and RalA) are exclusively geranylgeranylated; inhibition of geranylgeranylation by GGTIs mediates the proapoptotic and anti-anchorage-dependent growth effects specifically through RalB (whereas inhibition of anchorage-independent growth goes through RalA). GGTI treatment of RalB suppresses survivin and induces p27Kip1.","method":"Farnesylated GGTI-resistant RalB mutants, radiolabeled prenylation assays, colony formation assays, Western blot for survivin and p27","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — rescue experiments with GGTI-resistant mutants, biochemical prenylation assays, two orthogonal readouts; single lab","pmids":["17875936"],"is_preprint":false},{"year":2007,"finding":"RalB (and RalA) localize to dense core vesicles in neuroendocrine PC12 cells and function specifically as GTP sensors required for GTP-dependent exocytosis of dense core vesicles, but are dispensable for Ca2+-dependent exocytosis or vesicle docking.","method":"Stable shRNA knockdown of RalA and RalB, GTP-dependent exocytosis assays, Ca2+-dependent exocytosis assays, electron microscopy (docking), immunolocalization on vesicles, rescue transfection","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Moderate — double knockdown with rescue, functional exocytosis assays, EM for docking, localization; multiple orthogonal methods in one study","pmids":["17202486"],"is_preprint":false},{"year":2011,"finding":"RalA and RalB have opposing roles in tight junction development: RalA knockdown increases paracellular permeability and reduces TJ component incorporation, while RalB knockdown decreases paracellular permeability and increases TJ component incorporation; both activities are mediated through the exocyst complex.","method":"siRNA knockdown, paracellular permeability assays, immunofluorescence of TJ components, exocyst loss-of-function epistasis","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with defined TJ phenotypes, epistasis with exocyst knockdown; single lab","pmids":["22013078"],"is_preprint":false},{"year":2019,"finding":"RalB and its activator RGL2 co-localize at early and recycling endosomes (and to lesser extent at autophagosomes); RalB signaling is active at these endomembrane compartments basally, and RalB activity increases at autophagosomes upon nutrient starvation. RGL2 is required for both invasion and autophagy.","method":"Quantitative automated image analysis (Endomapper), FRET-based RalB biosensor, siRNA knockdown, subcellular fractionation","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET biosensor for activity, quantitative localization analysis; single lab, no full mechanistic reconstitution","pmids":["31222145"],"is_preprint":false},{"year":2019,"finding":"RalA and RalB both relocalize to depolarized mitochondria in a clathrin-mediated endocytosis-dependent manner; genetic and pharmacologic inhibition of RalA and RalB increases TBK1 activity basally and in response to mitochondrial depolarization, suggesting a model where Ral proteins at depolarized mitochondria facilitate TBK1 activation by releasing inhibition.","method":"Live-cell imaging (relocalization upon depolarization), clathrin inhibition, siRNA/genetic knockdown, TBK1 kinase activity assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — localization experiments with functional consequence (TBK1 activity), multiple perturbations; single lab","pmids":["30995277"],"is_preprint":false},{"year":2016,"finding":"RALB signaling is required for AML cell survival downstream of RAS; knockdown of RALB leads to decreased phosphorylation of TBK1 and reduced BCL2 expression, inducing apoptosis and phenocopying suppression of oncogenic RAS.","method":"Genetic knockdown (shRNA), NRAS-inducible mouse AML model, phospho-TBK1 Western blot, BCL2 Western blot, apoptosis assays, patient-derived AML samples","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — shRNA knockdown with defined molecular (TBK1, BCL2) and cellular (apoptosis) readouts, patient samples; single lab","pmids":["27556501"],"is_preprint":false},{"year":2016,"finding":"Ras-oncogene-independent activation of RALB (via CDK5-mediated activation) drives AML relapse; pharmacological inhibition of CDK5 with dinaciclib suppresses RALB activity and RALB-dependent TBK1 phosphorylation, inducing anti-leukemic effects.","method":"Mouse NRAS(V12)-inducible AML relapse model, RALB expression/activity assays, CDK5 inhibitor (dinaciclib) treatment, patient-derived xenografts, TBK1 phosphorylation assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo relapse model plus PDX model, pharmacologic and genetic perturbation of CDK5-RALB axis; single lab","pmids":["27991934"],"is_preprint":false},{"year":2025,"finding":"RalB (but not RalA) is required for regulated exocytosis of Weibel-Palade bodies (WPBs) in endothelial cells; unlike typical GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting cells. Upon stimulation, exocyst is uncoupled from RalB-GTP, enabling WPB tethering and exocytosis. PKC-dependent phosphorylation of RalB C-terminal HVR promotes exocyst binding, and dephosphorylation (or nonphosphorylatable mutant expression) disengages exocyst and augments WPB exocytosis.","method":"siRNA knockdown, constitutively active/dominant-negative RalB mutants, phosphorylation-site mutagenesis (nonphosphorylatable mutant), exocyst binding assays in GDP vs GTP states, PKC inhibition, live-cell exocytosis assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis of phosphorylation and nucleotide state, biochemical binding assays, loss-of-function with defined exocytosis phenotype, multiple orthogonal methods","pmids":["40172988"],"is_preprint":false},{"year":2004,"finding":"In Xenopus early development, RalB signals to the actin cytoskeleton via RLIP (RalBP1); membrane targeting of RLIP recapitulates activated RalB phenotype (cortical actin disruption), and overexpression of the RLIP Ral-binding domain competitively blocks RalB-induced actin effects.","method":"Xenopus microinjection, dominant-negative competition (RalBD overexpression), constitutively active RalB (G23V), cortical actin/phenotype analysis","journal":"Mechanisms of development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — competition/rescue approach in Xenopus with defined actin phenotype; ortholog study (Xenopus), single lab","pmids":["15511640"],"is_preprint":false},{"year":2020,"finding":"RALB depletion in KRAS mutant colorectal cancer cells induces Caspase-8-dependent cell death through upregulation of the death receptor DR5 (TRAIL-R2) by preventing its lysosomal degradation; TRAIL treatment causes association of RALB with the death-inducing signaling complex (DISC).","method":"siRNA/shRNA knockdown, proteomic analysis, DR5 knockout/knockdown epistasis, lysosomal degradation assays, Co-IP (RALB-DISC interaction), apoptosis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic epistasis (DR5 KO rescue), Co-IP of RALB with DISC, proteomics, multiple methods; single lab","pmids":["33122623"],"is_preprint":false},{"year":2015,"finding":"Thermodynamic mapping of RalB-effector interfaces using panels of RalB and RLIP76 mutants revealed distinct energetic landscapes for RalB-RLIP76 versus RalB-Sec5 complexes, providing a physical basis for effector-selective mutations; despite identical contact residues, RalA and RalB show different energetic profiles in RLIP76 binding.","method":"Affinity measurements (ITC/SPR), site-directed mutagenesis panels of RalB and RLIP76, structure-guided interface analysis","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — quantitative thermodynamic measurements with systematic mutagenesis; single lab, no cellular functional validation","pmids":["25621740"],"is_preprint":false},{"year":1996,"finding":"RalA and RalB are geranylgeranylated (not farnesylated) in vitro, and both proteins distribute to the particulate fraction of human platelets, with RalB also detectable in the platelet cytosol, indicating differential subcellular distribution between the isoforms.","method":"[3H]-mevalonolactone prenylation assay with geranylgeranyl pyrophosphate inhibition, subcellular fractionation, Western blot with isoform-specific antibodies","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — biochemical prenylation assay and fractionation; single lab, limited mechanistic follow-up","pmids":["8972729"],"is_preprint":false},{"year":2025,"finding":"Active Merlin (NF2 tumor suppressor isoforms 1 and 2) directly binds RalA and RalB in a PIP2-dependent manner at the plasma membrane, co-localizing with RalA/B. Merlin loss results in aberrant activation of RalA and RalB. Merlin competitively inhibits RalB binding to its exocyst effectors Sec5 and Exo84, and regulates the kinetics of exocytosis in a RalB-dependent manner.","method":"Proximity biotinylation, direct binding assays, co-localization, competitive binding assays (Merlin vs Sec5/Exo84 for RalB), exocytosis kinetics assays, loss-of-function (Merlin KO)","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct binding assays and competitive binding established, proximity biotinylation; preprint, single lab, not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"RALB is a RAS-superfamily small GTPase that cycles between GDP- and GTP-bound states and functions as a molecular switch downstream of RAS; in its active (GTP-bound) form it engages two mutually exclusive exocyst effectors—Sec5 (promoting TBK1 activation and innate immune/anti-apoptotic signaling) and Exo84 (driving autophagosome assembly via ULK1 and Beclin1-VPS34 complexes)—with the choice between effectors controlled by ubiquitylation at Lys47 (added by an uncharacterized E3, removed by USP33), while PKCα-mediated phosphorylation at Ser198 in the C-terminal hypervariable region regulates plasma membrane vs. endomembrane localization, Sec5 interaction, vesicle trafficking and exocytosis; additionally, RALB–exocyst promotes cell migration via leading-edge exocyst targeting, invasion via RalBP1/RLIP76 ATPase-dependent invadopodium formation and WRC recruitment, cytokinesis abscission at the midbody, and GTP-dependent dense-core vesicle exocytosis, with lysine fatty acylation (reversed by SIRT2) providing an additional layer of membrane targeting and effector recruitment control."},"narrative":{"mechanistic_narrative":"RALB is a RAS-superfamily small GTPase that operates as a nucleotide-dependent molecular switch downstream of RAS, coordinating membrane trafficking, innate immune/anti-apoptotic signaling, autophagy, and tumor cell motility through the exocyst complex [PMID:17018283, PMID:21241894, PMID:17174914]. In its active state RALB engages two mutually exclusive exocyst-associated effectors: binding the exocyst subunit Sec5 recruits and activates the kinase TBK1 to suppress apoptosis and mediate innate immune signaling [PMID:17018283], while binding Exo84 nucleates catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst to drive autophagosome formation upon nutrient deprivation [PMID:21241894]. The choice between these effector outputs is set by ubiquitylation at Lys47, which sterically blocks Exo84 while favoring Sec5; the deubiquitylase USP33 removes this mark during starvation to permit RALB-Exo84-Beclin1 assembly and autophagy [PMID:24056301]. RALB membrane targeting and effector engagement are further tuned by post-translational modification of its C-terminal hypervariable region: PKCα phosphorylation at Ser198 shifts RALB from the plasma membrane to endomembranes, decreasing Sec5 association and controlling SNARE interactions, integrin trafficking, and adhesion [PMID:20940393, PMID:22393054], and lysine fatty acylation at Lys200 (reversed by SIRT2) enhances plasma-membrane localization and Sec5/Exo84 recruitment [PMID:31433161]. Distinct from RALA, RALB drives vectorial cell migration via leading-edge exocyst targeting [PMID:16382162], cytokinetic abscission at the midbody [PMID:18756269], and invasion/metastasis through both the RalBP1/RLIP76 ATPase-dependent invadopodium pathway [PMID:22331470] and exocyst-mediated recruitment of the WAVE Regulatory Complex [PMID:30320548]. In RAS-mutant cancers, an integrin αvβ3-KRAS-RALB axis activates TBK1 and NF-κB to support tumor initiation and drug resistance [PMID:24747441], and RALB-TBK1-BCL2 signaling sustains AML survival, including a RAS-independent CDK5-driven mode at relapse [PMID:27556501, PMID:27991934]. Structural and thermodynamic analyses establish that RALB switch regions sample distinct conformational states and that Sec5, Exo84, and RLIP76 bind competitively through structurally distinct interfaces [PMID:19166349, PMID:20696399, PMID:25621740].","teleology":[{"year":2006,"claim":"Established that RALB acts through the exocyst subunit Sec5 to activate TBK1, defining a direct effector route by which RALB suppresses apoptosis and mediates innate immunity, and separately that RALB uniquely supports directed cell migration via exocyst targeting to the leading edge.","evidence":"Reciprocal Co-IP, TBK1 kinase assays, and isoform-specific siRNA with migration/exocyst-assembly readouts in cancer and migrating cells","pmids":["17018283","16382162"],"confidence":"High","gaps":["Did not define how RALB-Sec5 relieves TBK1 autoinhibition","Upstream activator selecting the Sec5 versus Exo84 output not identified"]},{"year":2006,"claim":"Resolved a division of labor between Ral isoforms in pancreatic cancer, showing RALB is required for invasion and metastasis whereas RALA drives anchorage-independent growth and tumor initiation, linking RALB specifically to the invasive program downstream of RAS.","evidence":"Isoform-specific siRNA across a 10-cell-line panel, in vitro invasion assays, and tail-vein metastasis assays in mice","pmids":["17174914"],"confidence":"High","gaps":["Effector mediating the invasion phenotype not pinpointed in this study","Mechanism of isoform-selective function unexplained"]},{"year":2007,"claim":"Showed RALB is geranylgeranylated and that this prenylation underlies its proapoptotic and growth-suppressive responses to GGTIs, and identified dense-core vesicles as a site where RALB acts as a GTP sensor for regulated exocytosis.","evidence":"GGTI-resistant prenylation-mutant rescue with survivin/p27 readouts; shRNA knockdown with GTP- versus Ca2+-dependent exocytosis assays and EM in PC12 cells","pmids":["17875936","17202486","8972729"],"confidence":"Medium","gaps":["Direct membrane-anchoring contribution of geranylgeranylation versus HVR modifications not deconvolved","Effectors at dense-core vesicles not identified"]},{"year":2008,"claim":"Defined a RALB-specific role in the final step of cytokinesis, recruiting the exocyst to the midbody for abscission while RALA acts earlier at the furrow, with distinct RalGEFs providing the temporal input.","evidence":"Isoform-specific siRNA, live-cell imaging, midbody localization, and epistasis with RalGEF mutants","pmids":["18756269"],"confidence":"High","gaps":["Identity of the RalGEF acting at the midbody not fully resolved","Membrane cargo delivered for abscission not defined"]},{"year":2009,"claim":"Provided the structural and dynamic basis of RALB effector binding, revealing that even GTP-loaded RALB samples a non-effector-competent state-1 conformation, so effector engagement is conformationally gated rather than a simple GTP-on switch.","evidence":"NMR solution structure, 31P NMR conformational analysis, and backbone dynamics of RALB-GMPPNP with effector titration","pmids":["19166349"],"confidence":"High","gaps":["How cellular modifications bias the state-1/state-2 equilibrium unknown","Conformational selection by individual effectors not quantified in cells"]},{"year":2010,"claim":"Mapped the competitive architecture of RALB effector interfaces, showing RLIP76 binds the switch regions via a coiled-coil mode distinct from Sec5/Exo84 and that the three effectors compete for the active GTPase with similar affinities.","evidence":"X-ray crystallography of the RalB-RLIP76 complex, interface mutagenesis, and competitive binding assays","pmids":["20696399"],"confidence":"High","gaps":["How effector choice is biased in vivo beyond affinity competition not addressed","Did not resolve regulatory inputs that partition effectors spatially"]},{"year":2010,"claim":"Identified PKC phosphorylation of RALB Ser198 in the membrane-targeting HVR as a switch that drives plasma-membrane-to-perinuclear relocalization and is required for actin organization, anchorage-independent growth, migration, and lung metastasis.","evidence":"In vitro kinase assay, MS phospho-site mapping, phosphosite mutagenesis with siRNA rescue, and in vivo metastasis assay","pmids":["20940393"],"confidence":"High","gaps":["Counteracting phosphatase not identified here","Link between relocalization and metastatic output mechanistically incomplete"]},{"year":2012,"claim":"Resolved the downstream consequences of Ser198 phosphorylation and a RLIP76-dependent invasion route, showing PKCα phosphorylation reduces Sec5 binding and reroutes integrin trafficking, while invadopodium formation requires RLIP76 ATPase activity rather than its RhoGAP function.","evidence":"Phospho-site mutagenesis, Co-IP, vesicle trafficking and adhesion assays; effector-selective and ATPase-deficient RLIP76 mutants in invadopodium assays","pmids":["22393054","22331470"],"confidence":"High","gaps":["Substrate of RLIP76 ATPase activity in invadopodia unknown","How phosphorylation-driven trafficking integrates with effector competition unclear"]},{"year":2013,"claim":"Established ubiquitylation at Lys47 as the molecular switch governing effector choice, sterically blocking Exo84 (autophagy) while favoring Sec5 (immunity), with USP33 acting as the starvation-induced eraser that licenses autophagy.","evidence":"Ubiquitylation site mapping, Lys47 mutagenesis, reciprocal Co-IP, and USP33 perturbation with localization studies","pmids":["24056301"],"confidence":"High","gaps":["E3 ligase that writes the Lys47 ubiquitin mark not identified","Signals controlling USP33 relocalization to RalB vesicles incomplete"]},{"year":2014,"claim":"Placed RALB within an integrin αvβ3-KRAS module at the membrane that activates TBK1-NF-κB to confer tumor initiation, self-renewal, and erlotinib resistance, linking RALB effector signaling to drug-tolerant cancer states.","evidence":"Reciprocal Co-IP, plasma-membrane fractionation, gain/loss-of-function, and patient-derived xenografts with pharmacologic inhibition","pmids":["24747441"],"confidence":"High","gaps":["How unliganded integrin recruits the KRAS-RALB complex structurally unresolved","Relative contribution of Sec5 versus other effectors not dissected"]},{"year":2015,"claim":"Extended the invasion and effector-interface picture, showing RALB drives TGFβ-induced EMT dissemination via a GEF-H1-exocyst-RhoA traction axis, and that RALB-RLIP76 versus RALB-Sec5 interfaces have distinct energetic landscapes enabling effector-selective mutations.","evidence":"siRNA with traction-force microscopy and 3D invasion assays plus Sec5-GEF-H1 Co-IP; ITC/SPR thermodynamic mutagenesis panels","pmids":["26152517","25621740"],"confidence":"Medium","gaps":["GEF-H1-exocyst pathway tested in a single lab","Thermodynamic mapping lacked cellular functional validation"]},{"year":2016,"claim":"Demonstrated that RALB-TBK1-BCL2 signaling is a survival dependency in AML downstream of RAS, and that RAS-independent CDK5-mediated RALB activation drives relapse, nominating the CDK5-RALB axis as a therapeutic target.","evidence":"shRNA knockdown with phospho-TBK1/BCL2 and apoptosis readouts in NRAS-inducible AML and PDX models; CDK5 inhibition with dinaciclib","pmids":["27556501","27991934"],"confidence":"Medium","gaps":["Mechanism by which CDK5 activates RALB not biochemically defined","Single-lab in vivo models"]},{"year":2018,"claim":"Directly linked plasma-membrane RALB activity to the actin nucleation machinery, showing optogenetically activated RALB recruits the WAVE Regulatory Complex via the exocyst to induce protrusions and invasion, with RGL1/RGL2 relaying RAS input.","evidence":"Optogenetic RalB activation, Co-IP, siRNA knockdown, and invasion assays","pmids":["30320548"],"confidence":"High","gaps":["Direct WRC-exocyst contact interface not mapped","How WRC recruitment is coordinated with other effector outputs unclear"]},{"year":2019,"claim":"Refined the localization and modification logic of RALB, mapping active RALB and RGL2 to early/recycling endosomes and autophagosomes, identifying SIRT2-reversible Lys200 fatty acylation as a plasma-membrane targeting determinant, and implicating Ral proteins in TBK1 control at depolarized mitochondria.","evidence":"FRET biosensor and quantitative imaging with RGL2 knockdown; biochemical acylation assays and Lys200 mutagenesis with SIRT2 knockdown; live-cell relocalization and TBK1 activity assays under mitochondrial depolarization","pmids":["31222145","31433161","30995277"],"confidence":"Medium","gaps":["Acyltransferase adding the Lys200 modification not identified","Mitochondrial TBK1 model rests on single-lab localization/activity correlation"]},{"year":2020,"claim":"Uncovered a pro-survival function in which RALB restrains apoptosis by preventing lysosomal degradation of the death receptor DR5, so RALB loss triggers Caspase-8-dependent death and RALB associates with the TRAIL DISC.","evidence":"siRNA/shRNA knockdown, proteomics, DR5 KO epistasis, lysosomal degradation assays, and RALB-DISC Co-IP in KRAS-mutant colorectal cells","pmids":["33122623"],"confidence":"Medium","gaps":["Mechanism by which RALB routes DR5 to lysosomes undefined","Effector mediating DR5 trafficking not identified"]},{"year":2025,"claim":"Revealed a non-canonical exocyst engagement in endothelial Weibel-Palade body exocytosis, where RALB binds exocyst in the GDP state at rest and uncouples upon activation, with PKC phosphorylation of the HVR promoting exocyst binding and dephosphorylation augmenting exocytosis.","evidence":"siRNA knockdown, nucleotide-state and phospho-site mutants, GDP/GTP-state exocyst binding assays, PKC inhibition, and live-cell WPB exocytosis assays","pmids":["40172988"],"confidence":"High","gaps":["Reconciliation with canonical GTP-dependent effector engagement incomplete","Phosphatase reversing HVR phosphorylation not identified"]},{"year":null,"claim":"The full regulatory circuit that integrates ubiquitylation, phosphorylation, and acylation of the RALB HVR with conformational state-switching to select among competing exocyst effectors in a given compartment remains to be unified, and the writer enzymes (Lys47 E3 ligase, Lys200 acyltransferase) and HVR phosphatase are unidentified.","evidence":"","pmids":[],"confidence":"Low","gaps":["No E3 ligase identified for Lys47 ubiquitylation","Acyltransferase for Lys200 acylation unknown","Quantitative model coupling PTMs to conformational state and effector partitioning absent"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[13,14,16,22]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,6,1]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[16]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,6,7,8,12]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[16,18,3]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[18]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2,23,7]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[1,3,18]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,6,19]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,6,11]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[16,8,22]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[4]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5,6,20,21]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,24,20]}],"complexes":["exocyst"],"partners":["EXOC2/SEC5","EXOC8/EXO84","RALBP1/RLIP76","TBK1","USP33","SIRT2","ARHGEF2/GEF-H1","NF2/MERLIN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P11234","full_name":"Ras-related protein Ral-B","aliases":[],"length_aa":206,"mass_kda":23.4,"function":"Multifunctional GTPase involved in a variety of cellular processes including gene expression, cell migration, cell proliferation, oncogenic transformation and membrane trafficking (PubMed:10393179, PubMed:17875936, PubMed:18756269). Accomplishes its multiple functions by interacting with distinct downstream effectors. Acts as a GTP sensor for GTP-dependent exocytosis of dense core vesicles (By similarity). Required both to stabilize the assembly of the exocyst complex and to localize functional exocyst complexes to the leading edge of migrating cells (By similarity). Required for suppression of apoptosis (PubMed:17875936). In late stages of cytokinesis, upon completion of the bridge formation between dividing cells, mediates exocyst recruitment to the midbody to drive abscission (PubMed:18756269). 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assignments","url":"https://pubmed.ncbi.nlm.nih.gov/19636902","citation_count":1,"is_preprint":false},{"pmid":"39345530","id":"PMC_39345530","title":"RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39345530","citation_count":0,"is_preprint":false},{"pmid":"41239602","id":"PMC_41239602","title":"Cross-tissue transcriptome-wide association and Mendelian randomization identify RALB as a susceptibility gene for breast hypertrophy.","date":"2025","source":"Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41239602","citation_count":0,"is_preprint":false},{"pmid":"41833635","id":"PMC_41833635","title":"Chromatin-binding protein HMGN1 promotes HCC tumorigenesis via histone methylation-induced RALB transcriptional suppression.","date":"2026","source":"Journal of genetics and genomics = Yi chuan xue bao","url":"https://pubmed.ncbi.nlm.nih.gov/41833635","citation_count":0,"is_preprint":false},{"pmid":"32810308","id":"PMC_32810308","title":"Retracted: microRNA-495 alleviates endothelial cell inflammatory injury in coronary heart disease via blockade of the NF-κB pathway by targeting RALB.","date":"2020","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/32810308","citation_count":0,"is_preprint":false},{"pmid":"18798517","id":"PMC_18798517","title":"[Preparation and characterization of monoclonal antibodies against RalB].","date":"2008","source":"Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition","url":"https://pubmed.ncbi.nlm.nih.gov/18798517","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.13.659557","title":"Active Merlin Binds RalB to Regulate Exocytosis","date":"2025-06-19","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.13.659557","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":28887,"output_tokens":7099,"usd":0.096573,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16040,"output_tokens":6169,"usd":0.117212,"stage2_stop_reason":"end_turn"},"total_usd":0.213785,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"RalB directly binds its effector Sec5 (a component of the exocyst complex), and the RalB/Sec5 complex recruits and activates the atypical IκB kinase TBK1, suppressing apoptosis in cancer cells and mediating innate immune signaling upon viral infection.\",\n      \"method\": \"Co-immunoprecipitation, protein complex analysis, kinase assays, loss-of-function (siRNA), cell transformation assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, kinase activity assays, loss-of-function with defined phenotypic readouts, replicated in multiple cell contexts\",\n      \"pmids\": [\"17018283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RalB localizes to nascent autophagosomes and is activated upon nutrient deprivation. Through direct binding to its effector Exo84, RalB induces assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, driving autophagosome formation and isolation membrane maturation.\",\n      \"method\": \"Subcellular fractionation/live imaging, co-immunoprecipitation, loss-of-function (siRNA/shRNA), epistasis (constitutively active RalB rescue), in vitro complex reconstitution\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including localization, complex reconstitution, loss-of-function with defined phenotype, and constitutively active rescue\",\n      \"pmids\": [\"21241894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RalB (but not RalA) is required for cell migration; RalB promotes exocyst (Sec6/8 complex) assembly and its localization to the leading edge of migrating cells, with loss of RalB impairing vectorial cell motility.\",\n      \"method\": \"siRNA loss-of-function, immunofluorescence localization, exocyst assembly assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with defined migration phenotype, localization experiment, exocyst assembly assay, two orthogonal methods in one study\",\n      \"pmids\": [\"16382162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Ubiquitylation of RalB at Lys47 switches its effector preference: ubiquitylation sterically inhibits RalB binding to Exo84 (blocking autophagy) while facilitating its interaction with Sec5 (promoting TBK1-innate immunity signaling). The deubiquitylase USP33 removes this ubiquitin mark upon nutrient starvation, relocalizing to RalB-positive vesicles and enabling RalB-Exo84-Beclin1 complex formation for autophagy.\",\n      \"method\": \"Ubiquitylation mapping, mutagenesis (Lys47 mutants), co-immunoprecipitation, USP33 knockdown/overexpression, localization studies\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — site-specific mutagenesis, reciprocal Co-IP, identification of writer/eraser, multiple orthogonal methods in one study\",\n      \"pmids\": [\"24056301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RalB (but not RalA) is required for abscission and completion of cytokinesis through recruitment of the exocyst to the midbody; distinct RalGEF proteins provide upstream input to RalB specifically at this step, whereas RalA acts earlier at the cytokinetic furrow.\",\n      \"method\": \"siRNA knockdown, live-cell imaging, subcellular localization, epistasis with RalGEF mutants\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with defined cytokinesis phenotype, localization experiments, epistasis with RalGEF, two orthogonal methods\",\n      \"pmids\": [\"18756269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RalB is required for invasion and metastasis of pancreatic cancer cells, while RalA (but not RalB) is required for anchorage-independent growth and tumor initiation; both are more commonly activated in pancreatic tumors than other Ras effector pathways.\",\n      \"method\": \"siRNA knockdown, in vitro invasion assays, tail-vein metastasis assays in mice, panel of 10 cell lines\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function validated across 9–10 cell lines and in vivo metastasis model, well-controlled with isoform-specific siRNAs\",\n      \"pmids\": [\"17174914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In the unliganded state, integrin αvβ3 recruits both KRAS and RalB to the tumor cell plasma membrane, leading to activation of TBK1 and NF-κB; this αvβ3-KRAS-RalB-NF-κB pathway is necessary and sufficient for tumor initiation, anchorage independence, self-renewal, and erlotinib resistance.\",\n      \"method\": \"Co-immunoprecipitation, plasma membrane fractionation, loss-of-function (siRNA/shRNA), gain-of-function, patient-derived xenografts, pharmacological inhibition\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vivo PDX models, multiple cell lines, gain- and loss-of-function experiments\",\n      \"pmids\": [\"24747441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Protein kinase C (PKC) phosphorylates RalB at Ser198 in its C-terminal membrane-targeting sequence, causing RalB translocation from plasma membrane to perinuclear regions; this phosphorylation is necessary for actin cytoskeletal organization, anchorage-independent growth, cell migration, and experimental lung metastasis.\",\n      \"method\": \"In vitro kinase assay, mass spectrometry phospho-site mapping, site-directed mutagenesis, phosphosite-specific antibodies, phorbol ester stimulation, siRNA rescue experiments, subcellular fractionation\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, MS-based site mapping, mutagenesis with functional rescue, in vivo metastasis assay, multiple orthogonal methods in one study\",\n      \"pmids\": [\"20940393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PKCα phosphorylation of RalB at Ser198 results in enhanced RalB endomembrane accumulation, decreased association with exocyst component Sec5, regulation of v- and t-SNARE interactions, control of vesicular trafficking of α5-integrin to the cell surface, and modulation of cell attachment to fibronectin.\",\n      \"method\": \"Mutagenesis, co-immunoprecipitation, subcellular fractionation, vesicle trafficking assays, cell adhesion assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis, co-IP for effector interaction, vesicle trafficking assay, cell biological readouts, multiple orthogonal methods\",\n      \"pmids\": [\"22393054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RalB mediates invadopodium formation in KRAS mutant pancreatic cancer cells through RalBP1/RLIP76 (but not Sec5 or Exo84), and this function requires the ATPase activity of RalBP1 rather than its canonical GAP activity toward Rho GTPases.\",\n      \"method\": \"siRNA knockdown, dominant-negative/constitutively active mutants, ATPase-deficient RLIP76 mutants, invadopodium formation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isoform-specific knockdown, effector-selective mutants, ATPase-domain mutagenesis, defined cellular phenotype readout\",\n      \"pmids\": [\"22331470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RalB (but not RalA) is required for TGFβ-induced EMT-driven cell dissemination by acting via the RhoGEF GEF-H1, which associates with the exocyst complex. Uncoupling of exocyst subunit Sec5 from GEF-H1 impairs RhoA activation and traction force generation.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation (Sec5-GEF-H1 interaction), traction force microscopy, 3D invasion assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with defined invasion/force phenotype and Co-IP of GEF-H1 with exocyst, but single lab\",\n      \"pmids\": [\"26152517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Active RalB at the plasma membrane promotes recruitment of the Wave Regulatory Complex (WRC) via the exocyst, inducing membrane protrusions and invasion; Ras signals to RalB through RalGEFs RGL1 and RGL2 to drive invasiveness.\",\n      \"method\": \"Optogenetic light-controlled RalB activation, co-immunoprecipitation, siRNA knockdown, invasion assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — novel optogenetic activation combined with Co-IP and loss-of-function, multiple orthogonal methods in one study\",\n      \"pmids\": [\"30320548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SIRT2 acts as a deacylase for RalB: RalB undergoes lysine fatty acylation predominantly at Lys200, which enhances plasma membrane localization and recruitment of its effectors Sec5 and Exo84 to the plasma membrane; SIRT2 removes this acylation and affects cell migration.\",\n      \"method\": \"Biochemical acylation assays, mutagenesis (Lys200), co-immunoprecipitation, subcellular fractionation, trans-well migration assays, SIRT2 knockdown\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical acylation characterization, site-specific mutagenesis, localization assays, Co-IP for effectors; single lab\",\n      \"pmids\": [\"31433161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NMR solution structure of RalB bound to GTP analogue GMPPNP revealed that the switch regions predominantly adopt state 1 (non-effector-binding competent) in the unbound form; 31P NMR of RalB.GTP shows both states 1 and 2 are sampled, and addition of an effector only partially stabilizes state 2, revealing dynamic properties of the effector-binding switches.\",\n      \"method\": \"NMR spectroscopy (solution structure determination), 31P NMR, backbone dynamics measurements\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with functional dynamics validation (31P NMR + effector binding), rigorous structural method\",\n      \"pmids\": [\"19166349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The crystal structure of RalB in complex with the Ral-binding domain of RLIP76 reveals a coiled-coil binding motif that contacts both nucleotide-sensitive switch regions of RalB; this mode of binding is distinct from the Sec5 and Exo84 exocyst interactions, and Sec5, Exo84, and RLIP76 bind Ral proteins competitively with similar affinities in vitro.\",\n      \"method\": \"X-ray crystallography (structure of RalB-RLIP76 complex), affinity measurements with RalB mutants, competitive binding assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with mutagenesis-based interface validation and quantitative binding competition assays\",\n      \"pmids\": [\"20696399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"RalB (and RalA) are exclusively geranylgeranylated; inhibition of geranylgeranylation by GGTIs mediates the proapoptotic and anti-anchorage-dependent growth effects specifically through RalB (whereas inhibition of anchorage-independent growth goes through RalA). GGTI treatment of RalB suppresses survivin and induces p27Kip1.\",\n      \"method\": \"Farnesylated GGTI-resistant RalB mutants, radiolabeled prenylation assays, colony formation assays, Western blot for survivin and p27\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — rescue experiments with GGTI-resistant mutants, biochemical prenylation assays, two orthogonal readouts; single lab\",\n      \"pmids\": [\"17875936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"RalB (and RalA) localize to dense core vesicles in neuroendocrine PC12 cells and function specifically as GTP sensors required for GTP-dependent exocytosis of dense core vesicles, but are dispensable for Ca2+-dependent exocytosis or vesicle docking.\",\n      \"method\": \"Stable shRNA knockdown of RalA and RalB, GTP-dependent exocytosis assays, Ca2+-dependent exocytosis assays, electron microscopy (docking), immunolocalization on vesicles, rescue transfection\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double knockdown with rescue, functional exocytosis assays, EM for docking, localization; multiple orthogonal methods in one study\",\n      \"pmids\": [\"17202486\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RalA and RalB have opposing roles in tight junction development: RalA knockdown increases paracellular permeability and reduces TJ component incorporation, while RalB knockdown decreases paracellular permeability and increases TJ component incorporation; both activities are mediated through the exocyst complex.\",\n      \"method\": \"siRNA knockdown, paracellular permeability assays, immunofluorescence of TJ components, exocyst loss-of-function epistasis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with defined TJ phenotypes, epistasis with exocyst knockdown; single lab\",\n      \"pmids\": [\"22013078\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RalB and its activator RGL2 co-localize at early and recycling endosomes (and to lesser extent at autophagosomes); RalB signaling is active at these endomembrane compartments basally, and RalB activity increases at autophagosomes upon nutrient starvation. RGL2 is required for both invasion and autophagy.\",\n      \"method\": \"Quantitative automated image analysis (Endomapper), FRET-based RalB biosensor, siRNA knockdown, subcellular fractionation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET biosensor for activity, quantitative localization analysis; single lab, no full mechanistic reconstitution\",\n      \"pmids\": [\"31222145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RalA and RalB both relocalize to depolarized mitochondria in a clathrin-mediated endocytosis-dependent manner; genetic and pharmacologic inhibition of RalA and RalB increases TBK1 activity basally and in response to mitochondrial depolarization, suggesting a model where Ral proteins at depolarized mitochondria facilitate TBK1 activation by releasing inhibition.\",\n      \"method\": \"Live-cell imaging (relocalization upon depolarization), clathrin inhibition, siRNA/genetic knockdown, TBK1 kinase activity assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — localization experiments with functional consequence (TBK1 activity), multiple perturbations; single lab\",\n      \"pmids\": [\"30995277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RALB signaling is required for AML cell survival downstream of RAS; knockdown of RALB leads to decreased phosphorylation of TBK1 and reduced BCL2 expression, inducing apoptosis and phenocopying suppression of oncogenic RAS.\",\n      \"method\": \"Genetic knockdown (shRNA), NRAS-inducible mouse AML model, phospho-TBK1 Western blot, BCL2 Western blot, apoptosis assays, patient-derived AML samples\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — shRNA knockdown with defined molecular (TBK1, BCL2) and cellular (apoptosis) readouts, patient samples; single lab\",\n      \"pmids\": [\"27556501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Ras-oncogene-independent activation of RALB (via CDK5-mediated activation) drives AML relapse; pharmacological inhibition of CDK5 with dinaciclib suppresses RALB activity and RALB-dependent TBK1 phosphorylation, inducing anti-leukemic effects.\",\n      \"method\": \"Mouse NRAS(V12)-inducible AML relapse model, RALB expression/activity assays, CDK5 inhibitor (dinaciclib) treatment, patient-derived xenografts, TBK1 phosphorylation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo relapse model plus PDX model, pharmacologic and genetic perturbation of CDK5-RALB axis; single lab\",\n      \"pmids\": [\"27991934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RalB (but not RalA) is required for regulated exocytosis of Weibel-Palade bodies (WPBs) in endothelial cells; unlike typical GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting cells. Upon stimulation, exocyst is uncoupled from RalB-GTP, enabling WPB tethering and exocytosis. PKC-dependent phosphorylation of RalB C-terminal HVR promotes exocyst binding, and dephosphorylation (or nonphosphorylatable mutant expression) disengages exocyst and augments WPB exocytosis.\",\n      \"method\": \"siRNA knockdown, constitutively active/dominant-negative RalB mutants, phosphorylation-site mutagenesis (nonphosphorylatable mutant), exocyst binding assays in GDP vs GTP states, PKC inhibition, live-cell exocytosis assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis of phosphorylation and nucleotide state, biochemical binding assays, loss-of-function with defined exocytosis phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"40172988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In Xenopus early development, RalB signals to the actin cytoskeleton via RLIP (RalBP1); membrane targeting of RLIP recapitulates activated RalB phenotype (cortical actin disruption), and overexpression of the RLIP Ral-binding domain competitively blocks RalB-induced actin effects.\",\n      \"method\": \"Xenopus microinjection, dominant-negative competition (RalBD overexpression), constitutively active RalB (G23V), cortical actin/phenotype analysis\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — competition/rescue approach in Xenopus with defined actin phenotype; ortholog study (Xenopus), single lab\",\n      \"pmids\": [\"15511640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RALB depletion in KRAS mutant colorectal cancer cells induces Caspase-8-dependent cell death through upregulation of the death receptor DR5 (TRAIL-R2) by preventing its lysosomal degradation; TRAIL treatment causes association of RALB with the death-inducing signaling complex (DISC).\",\n      \"method\": \"siRNA/shRNA knockdown, proteomic analysis, DR5 knockout/knockdown epistasis, lysosomal degradation assays, Co-IP (RALB-DISC interaction), apoptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic epistasis (DR5 KO rescue), Co-IP of RALB with DISC, proteomics, multiple methods; single lab\",\n      \"pmids\": [\"33122623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Thermodynamic mapping of RalB-effector interfaces using panels of RalB and RLIP76 mutants revealed distinct energetic landscapes for RalB-RLIP76 versus RalB-Sec5 complexes, providing a physical basis for effector-selective mutations; despite identical contact residues, RalA and RalB show different energetic profiles in RLIP76 binding.\",\n      \"method\": \"Affinity measurements (ITC/SPR), site-directed mutagenesis panels of RalB and RLIP76, structure-guided interface analysis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative thermodynamic measurements with systematic mutagenesis; single lab, no cellular functional validation\",\n      \"pmids\": [\"25621740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"RalA and RalB are geranylgeranylated (not farnesylated) in vitro, and both proteins distribute to the particulate fraction of human platelets, with RalB also detectable in the platelet cytosol, indicating differential subcellular distribution between the isoforms.\",\n      \"method\": \"[3H]-mevalonolactone prenylation assay with geranylgeranyl pyrophosphate inhibition, subcellular fractionation, Western blot with isoform-specific antibodies\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — biochemical prenylation assay and fractionation; single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"8972729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Active Merlin (NF2 tumor suppressor isoforms 1 and 2) directly binds RalA and RalB in a PIP2-dependent manner at the plasma membrane, co-localizing with RalA/B. Merlin loss results in aberrant activation of RalA and RalB. Merlin competitively inhibits RalB binding to its exocyst effectors Sec5 and Exo84, and regulates the kinetics of exocytosis in a RalB-dependent manner.\",\n      \"method\": \"Proximity biotinylation, direct binding assays, co-localization, competitive binding assays (Merlin vs Sec5/Exo84 for RalB), exocytosis kinetics assays, loss-of-function (Merlin KO)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct binding assays and competitive binding established, proximity biotinylation; preprint, single lab, not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RALB is a RAS-superfamily small GTPase that cycles between GDP- and GTP-bound states and functions as a molecular switch downstream of RAS; in its active (GTP-bound) form it engages two mutually exclusive exocyst effectors—Sec5 (promoting TBK1 activation and innate immune/anti-apoptotic signaling) and Exo84 (driving autophagosome assembly via ULK1 and Beclin1-VPS34 complexes)—with the choice between effectors controlled by ubiquitylation at Lys47 (added by an uncharacterized E3, removed by USP33), while PKCα-mediated phosphorylation at Ser198 in the C-terminal hypervariable region regulates plasma membrane vs. endomembrane localization, Sec5 interaction, vesicle trafficking and exocytosis; additionally, RALB–exocyst promotes cell migration via leading-edge exocyst targeting, invasion via RalBP1/RLIP76 ATPase-dependent invadopodium formation and WRC recruitment, cytokinesis abscission at the midbody, and GTP-dependent dense-core vesicle exocytosis, with lysine fatty acylation (reversed by SIRT2) providing an additional layer of membrane targeting and effector recruitment control.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RALB is a RAS-superfamily small GTPase that operates as a nucleotide-dependent molecular switch downstream of RAS, coordinating membrane trafficking, innate immune/anti-apoptotic signaling, autophagy, and tumor cell motility through the exocyst complex [#0, #1, #5]. In its active state RALB engages two mutually exclusive exocyst-associated effectors: binding the exocyst subunit Sec5 recruits and activates the kinase TBK1 to suppress apoptosis and mediate innate immune signaling [#0], while binding Exo84 nucleates catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst to drive autophagosome formation upon nutrient deprivation [#1]. The choice between these effector outputs is set by ubiquitylation at Lys47, which sterically blocks Exo84 while favoring Sec5; the deubiquitylase USP33 removes this mark during starvation to permit RALB-Exo84-Beclin1 assembly and autophagy [#3]. RALB membrane targeting and effector engagement are further tuned by post-translational modification of its C-terminal hypervariable region: PKCα phosphorylation at Ser198 shifts RALB from the plasma membrane to endomembranes, decreasing Sec5 association and controlling SNARE interactions, integrin trafficking, and adhesion [#7, #8], and lysine fatty acylation at Lys200 (reversed by SIRT2) enhances plasma-membrane localization and Sec5/Exo84 recruitment [#12]. Distinct from RALA, RALB drives vectorial cell migration via leading-edge exocyst targeting [#2], cytokinetic abscission at the midbody [#4], and invasion/metastasis through both the RalBP1/RLIP76 ATPase-dependent invadopodium pathway [#9] and exocyst-mediated recruitment of the WAVE Regulatory Complex [#11]. In RAS-mutant cancers, an integrin αvβ3-KRAS-RALB axis activates TBK1 and NF-κB to support tumor initiation and drug resistance [#6], and RALB-TBK1-BCL2 signaling sustains AML survival, including a RAS-independent CDK5-driven mode at relapse [#20, #21]. Structural and thermodynamic analyses establish that RALB switch regions sample distinct conformational states and that Sec5, Exo84, and RLIP76 bind competitively through structurally distinct interfaces [#13, #14, #25].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established that RALB acts through the exocyst subunit Sec5 to activate TBK1, defining a direct effector route by which RALB suppresses apoptosis and mediates innate immunity, and separately that RALB uniquely supports directed cell migration via exocyst targeting to the leading edge.\",\n      \"evidence\": \"Reciprocal Co-IP, TBK1 kinase assays, and isoform-specific siRNA with migration/exocyst-assembly readouts in cancer and migrating cells\",\n      \"pmids\": [\"17018283\", \"16382162\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how RALB-Sec5 relieves TBK1 autoinhibition\", \"Upstream activator selecting the Sec5 versus Exo84 output not identified\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved a division of labor between Ral isoforms in pancreatic cancer, showing RALB is required for invasion and metastasis whereas RALA drives anchorage-independent growth and tumor initiation, linking RALB specifically to the invasive program downstream of RAS.\",\n      \"evidence\": \"Isoform-specific siRNA across a 10-cell-line panel, in vitro invasion assays, and tail-vein metastasis assays in mice\",\n      \"pmids\": [\"17174914\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effector mediating the invasion phenotype not pinpointed in this study\", \"Mechanism of isoform-selective function unexplained\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed RALB is geranylgeranylated and that this prenylation underlies its proapoptotic and growth-suppressive responses to GGTIs, and identified dense-core vesicles as a site where RALB acts as a GTP sensor for regulated exocytosis.\",\n      \"evidence\": \"GGTI-resistant prenylation-mutant rescue with survivin/p27 readouts; shRNA knockdown with GTP- versus Ca2+-dependent exocytosis assays and EM in PC12 cells\",\n      \"pmids\": [\"17875936\", \"17202486\", \"8972729\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct membrane-anchoring contribution of geranylgeranylation versus HVR modifications not deconvolved\", \"Effectors at dense-core vesicles not identified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined a RALB-specific role in the final step of cytokinesis, recruiting the exocyst to the midbody for abscission while RALA acts earlier at the furrow, with distinct RalGEFs providing the temporal input.\",\n      \"evidence\": \"Isoform-specific siRNA, live-cell imaging, midbody localization, and epistasis with RalGEF mutants\",\n      \"pmids\": [\"18756269\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the RalGEF acting at the midbody not fully resolved\", \"Membrane cargo delivered for abscission not defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Provided the structural and dynamic basis of RALB effector binding, revealing that even GTP-loaded RALB samples a non-effector-competent state-1 conformation, so effector engagement is conformationally gated rather than a simple GTP-on switch.\",\n      \"evidence\": \"NMR solution structure, 31P NMR conformational analysis, and backbone dynamics of RALB-GMPPNP with effector titration\",\n      \"pmids\": [\"19166349\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How cellular modifications bias the state-1/state-2 equilibrium unknown\", \"Conformational selection by individual effectors not quantified in cells\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapped the competitive architecture of RALB effector interfaces, showing RLIP76 binds the switch regions via a coiled-coil mode distinct from Sec5/Exo84 and that the three effectors compete for the active GTPase with similar affinities.\",\n      \"evidence\": \"X-ray crystallography of the RalB-RLIP76 complex, interface mutagenesis, and competitive binding assays\",\n      \"pmids\": [\"20696399\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How effector choice is biased in vivo beyond affinity competition not addressed\", \"Did not resolve regulatory inputs that partition effectors spatially\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified PKC phosphorylation of RALB Ser198 in the membrane-targeting HVR as a switch that drives plasma-membrane-to-perinuclear relocalization and is required for actin organization, anchorage-independent growth, migration, and lung metastasis.\",\n      \"evidence\": \"In vitro kinase assay, MS phospho-site mapping, phosphosite mutagenesis with siRNA rescue, and in vivo metastasis assay\",\n      \"pmids\": [\"20940393\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Counteracting phosphatase not identified here\", \"Link between relocalization and metastatic output mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved the downstream consequences of Ser198 phosphorylation and a RLIP76-dependent invasion route, showing PKCα phosphorylation reduces Sec5 binding and reroutes integrin trafficking, while invadopodium formation requires RLIP76 ATPase activity rather than its RhoGAP function.\",\n      \"evidence\": \"Phospho-site mutagenesis, Co-IP, vesicle trafficking and adhesion assays; effector-selective and ATPase-deficient RLIP76 mutants in invadopodium assays\",\n      \"pmids\": [\"22393054\", \"22331470\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate of RLIP76 ATPase activity in invadopodia unknown\", \"How phosphorylation-driven trafficking integrates with effector competition unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established ubiquitylation at Lys47 as the molecular switch governing effector choice, sterically blocking Exo84 (autophagy) while favoring Sec5 (immunity), with USP33 acting as the starvation-induced eraser that licenses autophagy.\",\n      \"evidence\": \"Ubiquitylation site mapping, Lys47 mutagenesis, reciprocal Co-IP, and USP33 perturbation with localization studies\",\n      \"pmids\": [\"24056301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase that writes the Lys47 ubiquitin mark not identified\", \"Signals controlling USP33 relocalization to RalB vesicles incomplete\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placed RALB within an integrin αvβ3-KRAS module at the membrane that activates TBK1-NF-κB to confer tumor initiation, self-renewal, and erlotinib resistance, linking RALB effector signaling to drug-tolerant cancer states.\",\n      \"evidence\": \"Reciprocal Co-IP, plasma-membrane fractionation, gain/loss-of-function, and patient-derived xenografts with pharmacologic inhibition\",\n      \"pmids\": [\"24747441\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How unliganded integrin recruits the KRAS-RALB complex structurally unresolved\", \"Relative contribution of Sec5 versus other effectors not dissected\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended the invasion and effector-interface picture, showing RALB drives TGFβ-induced EMT dissemination via a GEF-H1-exocyst-RhoA traction axis, and that RALB-RLIP76 versus RALB-Sec5 interfaces have distinct energetic landscapes enabling effector-selective mutations.\",\n      \"evidence\": \"siRNA with traction-force microscopy and 3D invasion assays plus Sec5-GEF-H1 Co-IP; ITC/SPR thermodynamic mutagenesis panels\",\n      \"pmids\": [\"26152517\", \"25621740\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GEF-H1-exocyst pathway tested in a single lab\", \"Thermodynamic mapping lacked cellular functional validation\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated that RALB-TBK1-BCL2 signaling is a survival dependency in AML downstream of RAS, and that RAS-independent CDK5-mediated RALB activation drives relapse, nominating the CDK5-RALB axis as a therapeutic target.\",\n      \"evidence\": \"shRNA knockdown with phospho-TBK1/BCL2 and apoptosis readouts in NRAS-inducible AML and PDX models; CDK5 inhibition with dinaciclib\",\n      \"pmids\": [\"27556501\", \"27991934\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which CDK5 activates RALB not biochemically defined\", \"Single-lab in vivo models\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Directly linked plasma-membrane RALB activity to the actin nucleation machinery, showing optogenetically activated RALB recruits the WAVE Regulatory Complex via the exocyst to induce protrusions and invasion, with RGL1/RGL2 relaying RAS input.\",\n      \"evidence\": \"Optogenetic RalB activation, Co-IP, siRNA knockdown, and invasion assays\",\n      \"pmids\": [\"30320548\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct WRC-exocyst contact interface not mapped\", \"How WRC recruitment is coordinated with other effector outputs unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Refined the localization and modification logic of RALB, mapping active RALB and RGL2 to early/recycling endosomes and autophagosomes, identifying SIRT2-reversible Lys200 fatty acylation as a plasma-membrane targeting determinant, and implicating Ral proteins in TBK1 control at depolarized mitochondria.\",\n      \"evidence\": \"FRET biosensor and quantitative imaging with RGL2 knockdown; biochemical acylation assays and Lys200 mutagenesis with SIRT2 knockdown; live-cell relocalization and TBK1 activity assays under mitochondrial depolarization\",\n      \"pmids\": [\"31222145\", \"31433161\", \"30995277\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Acyltransferase adding the Lys200 modification not identified\", \"Mitochondrial TBK1 model rests on single-lab localization/activity correlation\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Uncovered a pro-survival function in which RALB restrains apoptosis by preventing lysosomal degradation of the death receptor DR5, so RALB loss triggers Caspase-8-dependent death and RALB associates with the TRAIL DISC.\",\n      \"evidence\": \"siRNA/shRNA knockdown, proteomics, DR5 KO epistasis, lysosomal degradation assays, and RALB-DISC Co-IP in KRAS-mutant colorectal cells\",\n      \"pmids\": [\"33122623\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which RALB routes DR5 to lysosomes undefined\", \"Effector mediating DR5 trafficking not identified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a non-canonical exocyst engagement in endothelial Weibel-Palade body exocytosis, where RALB binds exocyst in the GDP state at rest and uncouples upon activation, with PKC phosphorylation of the HVR promoting exocyst binding and dephosphorylation augmenting exocytosis.\",\n      \"evidence\": \"siRNA knockdown, nucleotide-state and phospho-site mutants, GDP/GTP-state exocyst binding assays, PKC inhibition, and live-cell WPB exocytosis assays\",\n      \"pmids\": [\"40172988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with canonical GTP-dependent effector engagement incomplete\", \"Phosphatase reversing HVR phosphorylation not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full regulatory circuit that integrates ubiquitylation, phosphorylation, and acylation of the RALB HVR with conformational state-switching to select among competing exocyst effectors in a given compartment remains to be unified, and the writer enzymes (Lys47 E3 ligase, Lys200 acyltransferase) and HVR phosphatase are unidentified.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No E3 ligase identified for Lys47 ubiquitylation\", \"Acyltransferase for Lys200 acylation unknown\", \"Quantitative model coupling PTMs to conformational state and effector partitioning absent\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [13, 14, 16, 22]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 6, 1]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 6, 7, 8, 12]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [16, 18, 3]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2, 23, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [1, 3, 18]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 6, 19]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 6, 11]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [16, 8, 22]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5, 6, 20, 21]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 24, 20]}\n    ],\n    \"complexes\": [\"exocyst\"],\n    \"partners\": [\"EXOC2/Sec5\", \"EXOC8/Exo84\", \"RALBP1/RLIP76\", \"TBK1\", \"USP33\", \"SIRT2\", \"ARHGEF2/GEF-H1\", \"NF2/Merlin\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}