{"gene":"LCP2","run_date":"2026-04-28T18:30:27","timeline":{"discoveries":[{"year":1995,"finding":"SLP-76 (LCP2) was molecularly cloned as a 76 kDa hematopoietic-specific adaptor protein containing a C-terminal SH2 domain; it directly associates with Grb2 via in vitro GST pulldown, and its SH2 domain precipitates tyrosine phosphoproteins from T cell lysates including PLC-γ1-associated proteins.","method":"Molecular cloning, GST fusion protein pulldown, co-immunoprecipitation, in vitro translation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — original cloning paper with direct binding assays and multiple orthogonal methods","pmids":["7706237"],"is_preprint":false},{"year":1996,"finding":"ZAP-70 phosphorylates SLP-76 in vitro and in heterologous cell systems; SLP-76 phosphorylation is diminished when catalytically inactive ZAP-70 is expressed. SLP-76 overexpression hyperactivates the TCR, while a non-phosphorylatable SLP-76 attenuates signaling. The SH2 domain of SLP-76 is independently required for TCR function.","method":"In vitro kinase assay, dominant-negative ZAP-70 expression, overexpression/mutagenesis in T cell lines, reporter gene assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus mutagenesis with functional readout, confirmed across multiple approaches","pmids":["8702662"],"is_preprint":false},{"year":1996,"finding":"SLP-76 directly associates with Vav via the Vav SH2 domain binding to phosphorylated SLP-76; co-overexpression of Vav and SLP-76 synergistically induces NF-AT activation, placing SLP-76 in a complex with Vav during TCR signaling.","method":"Co-immunoprecipitation, overexpression, NF-AT reporter assay in Jurkat cells","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP and functional reporter assay, replicated across labs","pmids":["8673706"],"is_preprint":false},{"year":1996,"finding":"TCR engagement leads to tyrosine phosphorylation of SLP-76 at its N-terminal acidic region; tyrosines Y113 and Y128 (YESP motifs) and Y145 are phosphorylated, with Y145 being the most important for NF-AT promoter activity.","method":"Site-directed mutagenesis, TCR stimulation, NF-AT reporter assay in Jurkat cells","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with functional readout, replicated by multiple labs","pmids":["8892604"],"is_preprint":false},{"year":1996,"finding":"SLP-76 is required for optimal TCR-induced activation of PLC-γ1 and Ras pathways; an SLP-76-deficient T cell line showed that SLP-76 is not required for general tyrosine phosphorylation but is specifically required for PLC-γ1 phosphorylation and TCR-inducible gene expression.","method":"SLP-76-deficient cell line (genetic loss), phosphorylation assays, gene expression reporter","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — genetic loss-of-function with specific biochemical and functional readouts","pmids":["9665884"],"is_preprint":false},{"year":1996,"finding":"Grb2 constitutively associates with unphosphorylated SLP-76; the SH2 domain of SLP-76 associates with 62 kDa and 130 kDa tyrosine-phosphorylated proteins and a serine/threonine kinase after TCR stimulation.","method":"Co-immunoprecipitation, GST-SH2 domain pulldown, in vitro binding","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP and pulldown, single lab","pmids":["8666952"],"is_preprint":false},{"year":1996,"finding":"SHP-1 constitutively associates with SLP-76 in B cells via SHP-1 SH2 domains; BCR ligation induces tyrosine phosphorylation of SLP-76 and SHP-1.","method":"Co-immunoprecipitation, GST-SH2 domain pulldown, Western blot","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 3 — single-lab co-IP with limited mechanistic follow-up","pmids":["8760799"],"is_preprint":false},{"year":1997,"finding":"ZAP-70 phosphorylates two YESP motifs of SLP-76 (Y113 and Y128) to create binding sites for the Vav SH2 domain; a third motif (Y145, pYEPP) fails to bind Vav SH2. Vav-SLP-76 binding is not absolutely required for IL-2 production in all T cells.","method":"In vitro kinase assay, phosphopeptide binding, overexpression in T cell hybridoma","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus phosphopeptide binding mapping specific sites","pmids":["9047237"],"is_preprint":false},{"year":1997,"finding":"SLAP-130 (FYB/ADAP) is a 130 kDa SLP-76-associated phosphoprotein that interacts via the SH2 domain of SLP-76; overexpression of SLAP-130 negatively regulates TCR-induced IL-2 promoter activity and counteracts SLP-76 augmentation of signaling.","method":"Molecular cloning, co-immunoprecipitation with SH2 domain, reporter gene assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — molecular cloning plus functional reporter assay, single lab","pmids":["9115214"],"is_preprint":false},{"year":1997,"finding":"SLP-76 function requires three distinct protein-interaction domains (N-terminal tyrosine-containing acidic region, proline-rich region, C-terminal SH2 domain) for optimal augmentation of TCR-induced NF-AT activity and ERK activation.","method":"Domain deletion mutagenesis, overexpression in Jurkat cells, reporter gene assay, kinase assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — structure-function with multiple domain deletions and functional readouts","pmids":["9257823"],"is_preprint":false},{"year":1998,"finding":"SLP-76 is required for normal T cell development; SLP-76-null mice have a complete block in thymopoiesis at an early stage with no peripheral T cells, establishing SLP-76 as an essential signal transducer for pre-TCR signaling.","method":"Targeted gene knockout in mice, flow cytometry of thymic populations","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with defined developmental phenotype, independently replicated by two labs (PMID 9665885 and 9695951)","pmids":["9665885","9695951"],"is_preprint":false},{"year":1998,"finding":"SLP-76 interacts with Nck (an adaptor protein) and Vav (a GEF for Rho-family GTPases), forming a trimolecular complex that activates PAK1 (p21-activated kinase 1) and facilitates actin polymerization downstream of the TCR.","method":"Co-immunoprecipitation, PAK1 kinase assay, actin polymerization assay","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP plus enzymatic (kinase) assay and actin polymerization readout","pmids":["9846482"],"is_preprint":false},{"year":1998,"finding":"SLP-76 is a direct substrate of SHP-1 in T cells and NK cells; SHP-1, recruited to killer cell inhibitory receptors, dephosphorylates SLP-76, and tyrosine-phosphorylated SLP-76 is required for optimal cytotoxic lymphocyte activation.","method":"Direct binding assay (SHP-1 catalytic domain on SLP-76), functional inhibitory receptor co-engagement assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding and functional assay in relevant cell types","pmids":["9765283"],"is_preprint":false},{"year":1998,"finding":"SLP-76 is required for optimal PLC-γ2 activation in platelets downstream of the collagen receptor; SLP-76-deficient platelets show impaired collagen-induced platelet aggregation, granule release, and PLC-γ2 tyrosine phosphorylation.","method":"SLP-76 KO mouse platelets, platelet aggregation assay, phosphorylation assay, granule release assay","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with defined biochemical and functional phenotype in platelets","pmids":["9884330"],"is_preprint":false},{"year":1999,"finding":"Gads (Grb2-related adaptor) constitutively interacts with SLP-76 via the C-terminal SH3 domain of Gads and a proline-rich region of SLP-76; Gads also binds tyrosine-phosphorylated LAT via its SH2 domain, bridging LAT and SLP-76 and promoting T cell signaling.","method":"Co-immunoprecipitation, domain mapping, NF-AT reporter assay","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP plus domain mapping and functional assay, replicated","pmids":["10021361"],"is_preprint":false},{"year":1999,"finding":"SLP-76 deficiency in mast cells impairs FcεRI-mediated signaling: SLP-76-null BMMCs fail to release β-hexosaminidase and IL-6, and show reduced PLC-γ1 tyrosine phosphorylation and calcium mobilization after FcεRI cross-linking, while Syk phosphorylation is normal.","method":"SLP-76 KO mouse mast cells, degranulation assay, cytokine secretion, phosphorylation assay, calcium flux","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple orthogonal functional and biochemical readouts","pmids":["10377180"],"is_preprint":false},{"year":1999,"finding":"In platelets, tyrosine phosphorylation of SLP-76 by the collagen receptor pathway is downstream of Syk; SLP-76 co-immunoprecipitates with SLAP-130, Vav, Fyn, Lyn, and the FcR γ-chain after collagen stimulation, and SLP-76 deficiency abrogates PLC-γ2 phosphorylation and Ca2+ mobilization.","method":"Syk-deficient platelet analysis, co-immunoprecipitation, in vitro kinase assay, calcium mobilization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis (Syk-deficient platelets) plus co-IP and functional assays","pmids":["10026222"],"is_preprint":false},{"year":1999,"finding":"Nck SH2 domain interacts with tyrosine-phosphorylated SLP-76 in activated T cells; phosphopeptides corresponding to Y113 and Y128 of SLP-76 compete binding of SLP-76 to the Nck SH2 domain.","method":"Co-immunoprecipitation, phosphopeptide competition, in vitro binding","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus peptide competition mapping specific sites","pmids":["10229072"],"is_preprint":false},{"year":1999,"finding":"FYN-T selectively phosphorylates FYB/SLAP, creating binding sites for the SH2 domains of FYN-T and SLP-76; co-expression of FYN-T, FYB, and SLP-76 synergistically up-regulates IL-2 transcription via TCR ligation.","method":"In vitro kinase assay, co-immunoprecipitation, NF-AT/IL-2 reporter assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro kinase assay plus functional reporter, single lab","pmids":["10409671"],"is_preprint":false},{"year":1999,"finding":"SLP-76 SH2 domain binds HPK1 at Tyr379; HPK1 interacts with SLP-76 in T cells and inhibits AP-1 activation in an SLP-76 SH2-domain-dependent manner, integrating HPK1 into antigen receptor signaling cascades.","method":"Yeast two-hybrid, co-immunoprecipitation, reporter gene assay, homology modeling","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — yeast two-hybrid plus co-IP and functional assay","pmids":["11487585"],"is_preprint":false},{"year":1999,"finding":"GrpL (Grb2-related adaptor) co-immunoprecipitates with SLP-76 but not with Sos1/2 in Jurkat cells; GrpL cooperates with SLP-76 to augment NF-AT activation, defining a distinct Grb2-like adaptor complex with SLP-76.","method":"Co-immunoprecipitation, NF-AT reporter assay","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP with functional reporter, single lab","pmids":["10209041"],"is_preprint":false},{"year":2000,"finding":"Fyb/SLAP (FYB/ADAP) links TCR signaling to actin cytoskeleton remodeling by acting as a ligand for Ena/VASP EVH1 domains; upon TCR engagement, Fyb/SLAP localizes to the T cell-APC interface within complexes containing WASP, Nck, and SLP-76.","method":"Co-immunoprecipitation, confocal microscopy, WASP/Arp2/3 inhibition, biochemical fractionation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — co-IP plus localization imaging and functional inhibition with defined actin phenotype","pmids":["10747096"],"is_preprint":false},{"year":2000,"finding":"SLP-76 recruitment to glycolipid-enriched membrane microdomains (GEMs) requires amino acids 224-244 of SLP-76 and is critical for TCR signaling; forced membrane localization of SLP-76 via a LAT/SLP-76 chimera reconstitutes PLC-γ1 phosphorylation, ERK activation, and NF-AT activity in LAT-deficient cells.","method":"Chimeric protein expression, fractionation into GEMs, reporter gene assay, phosphorylation assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — chimeric protein reconstitution plus mutagenesis and multiple functional readouts","pmids":["11015445"],"is_preprint":false},{"year":2000,"finding":"SLP-76 relays signals from platelet integrin αIIbβ3 to the actin cytoskeleton: fibrinogen binding triggers Syk-dependent SLP-76 phosphorylation, which promotes SLP-76 association with Nck and Vav1, lamellipodia formation, and PAK kinase activation.","method":"CHO cell expression system, co-immunoprecipitation, PAK kinase assay, microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reconstitution in CHO cells plus co-IP, kinase assay and genetic KO comparison","pmids":["11113155"],"is_preprint":false},{"year":2001,"finding":"SLP-76 directly interacts with the SH3 domain of PLC-γ1 via a proline-rich 'P-1 domain' (67 amino acids); this interaction is constitutive and required for TCR-mediated ERK, PLC-γ1, and NF-AT activation. The Gads-binding domain of SLP-76 mediates inducible recruitment to a PLC-γ1-LAT complex.","method":"Mutagenesis, co-immunoprecipitation, reporter gene assay, reconstitution in SLP-76-deficient cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — domain mutagenesis identifying direct interaction plus reconstitution and multiple functional readouts","pmids":["11390650"],"is_preprint":false},{"year":2001,"finding":"SLAP-130 (FYB/ADAP) interaction with SLP-76 requires phospho-Y559 of SLAP-130; this interaction is important for the negative regulatory role of SLAP-130, specifically inhibiting TCR-induced ERK activation but not PLC-γ1 phosphorylation.","method":"Mutagenesis, co-immunoprecipitation, ERK activation assay, NF-AT reporter assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — point mutagenesis plus functional assays, single lab","pmids":["10671560"],"is_preprint":false},{"year":2001,"finding":"WASP recruitment to the T cell-APC contact site requires Nck (via WASP proline-rich domain), while WASP activation requires Vav-1 for localized Cdc42 activation; SLP-76 coordinates both events by acting as a scaffold bringing Nck and WASP near Vav-1/Cdc42-GTP.","method":"Vav-1-deficient T cells, microscopy of WASP localization, co-immunoprecipitation, Cdc42 activation assay","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with Vav-1-deficient T cells plus imaging and biochemical assays","pmids":["12874226"],"is_preprint":false},{"year":2001,"finding":"GADS is required for coupling SLP-76 to LAT in T cells; GADS-deficient thymocytes fail to respond to CD3 cross-linking and the association between SLP-76 and LAT is uncoupled, establishing GADS as the physical bridge between SLP-76 and LAT.","method":"GADS KO mice, co-immunoprecipitation, thymocyte proliferation assay, in vivo selection assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — genetic KO plus co-IP demonstrating loss of SLP-76/LAT association","pmids":["11239162"],"is_preprint":false},{"year":2001,"finding":"SLP-76 mediates signaling via Fcγ receptors and integrins in neutrophils; SLP-76-deficient neutrophils show decreased FcγR-induced calcium flux and reactive oxygen intermediate production, and fail to produce ROI, spread, or activate key downstream regulators upon integrin ligation.","method":"SLP-76 KO mouse neutrophils, calcium flux, ROI production assay, spreading assay","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple functional readouts","pmids":["14614862"],"is_preprint":false},{"year":2002,"finding":"The RxxK motif in SLP-76 (R237 and K240) mediates high-affinity binding to the C-terminal SH3 domain of Gads; single point mutations in R237 or K240 abrogate SLP-76/Gads association in vivo and impair SLP-76 function in TCR signaling.","method":"Peptide array, in vitro binding (Kd measurement), site-directed mutagenesis, co-immunoprecipitation, NF-AT reporter assay","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 1 — quantitative affinity measurement plus mutagenesis and in vivo functional confirmation","pmids":["12176364"],"is_preprint":false},{"year":2003,"finding":"Crystal structure (1.7 Å) of the Mona/Gads C-terminal SH3 domain complexed with a SLP-76 peptide reveals a novel non-proline-type II helix binding mode: the SLP-76 RSTK peptide forms a 3(10) helix inserting into a negatively charged double pocket on the SH3 domain.","method":"X-ray crystallography (1.7 Å resolution), mutagenesis, in vitro binding","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with supporting mutagenesis and binding data","pmids":["12773374"],"is_preprint":false},{"year":2003,"finding":"NMR solution structure of the Gads C-terminal SH3 domain in complex with an SLP-76 RSTK-containing peptide shows the peptide adopts a right-handed 3(10) helix at the RSTK locus engaging four distinct binding pockets, confirming a novel mode of SH3 recognition.","method":"NMR structure determination, mutagenesis, peptide binding assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with mutagenesis validation","pmids":["12620234"],"is_preprint":false},{"year":2003,"finding":"SLP-76 deficiency causes failure of blood-lymphatic vascular separation in mice; SLP-76 is required in hematopoietic cells (not endothelial cells) for this separation, as bone marrow reconstitution with SLP-76-deficient cells confers blood-filled lymphatics in wild-type mice.","method":"SLP-76 KO mice, bone marrow reconstitution, vascular histology","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — genetic KO plus bone marrow reconstitution establishing hematopoietic cell-autonomous mechanism","pmids":["12522250"],"is_preprint":false},{"year":2004,"finding":"ADAP binding to SLP-76 differentially regulates peripheral SMAC (pSMAC) formation at the immunological synapse versus T cell-APC conjugation; ADAP-SLP-76 interaction requires specific YDDV sites of ADAP, and loss of this interaction acts as a dominant negative on pSMAC formation and IL-2 production.","method":"Mutagenesis of ADAP, immunological synapse imaging, IL-2 production assay, LFA-1 clustering assay","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis plus imaging and functional readout, single lab","pmids":["15477347"],"is_preprint":false},{"year":2005,"finding":"TCR-activated reactive oxygen species (ROS) induce transient oxidative inactivation of SHP-2 (but not SHP-1); SHP-2 is recruited to the LAT-Gads-SLP-76 complex and directly regulates phosphorylation of Vav1 and ADAP, with the ADAP-SLP-76 association regulated by SHP-2 in a redox-dependent manner.","method":"ROS detection, phosphatase activity assay, co-immunoprecipitation, T cell adhesion assay","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with redox-dependent functional assay, single lab","pmids":["15933714"],"is_preprint":false},{"year":2005,"finding":"SLP-76 forms signaling microclusters at TCR contact sites with APCs containing ZAP-70; microclusters are continuously generated at the periphery, migrate toward the central SMAC, and tyrosine phosphorylation and calcium influx occur as microclusters form. Inhibition of signaling prevents ZAP-70 recruitment into microclusters.","method":"Live-cell imaging (TIRF microscopy), fluorescently-tagged SLP-76 and ZAP-70, signaling inhibitors","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — direct live imaging of protein dynamics with functional correlation, highly cited","pmids":["16273097"],"is_preprint":false},{"year":2005,"finding":"CD6 cytoplasmic tail phospho-Y662 directly binds the SH2 domain of SLP-76 (Kd = 0.5 µM at 37°C); this interaction is required for CD6-mediated T cell costimulation.","method":"Phosphopeptide binding (equilibrium dissociation constant measured), co-immunoprecipitation, mutagenesis, costimulation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — quantitative binding assay plus mutagenesis and functional validation","pmids":["16914752"],"is_preprint":false},{"year":2006,"finding":"SLP-76 forms dynamic membrane clusters upon FcεRI cross-linking in mast cells, colocalizing with FcεRI, Syk, LAT, and phosphotyrosine; disruption of the SLP-76-Gads interaction (by SLP-76 mutation) prevents translocation and clustering, inhibiting calcium flux, degranulation, and cytokine secretion.","method":"Confocal real-time imaging, mutagenesis of SLP-76 Gads-binding region, calcium flux, degranulation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — direct imaging plus mutagenesis and multiple functional readouts","pmids":["16479002"],"is_preprint":false},{"year":2006,"finding":"SLP-76 Tyr145 is the most critical N-terminal tyrosine for T cell development and function; Y145 is required for optimal association of SLP-76 with ITK, while Y112/Y128 associate with Vav and Nck and are required for TCR-induced Ca2+ flux and actin reorganization.","method":"Individual tyrosine mutagenesis in reconstituted SLP-76-deficient cells and primary T cells, calcium flux, actin assay, co-immunoprecipitation","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — systematic mutagenesis with multiple functional readouts in both cell lines and primary cells","pmids":["16456002"],"is_preprint":false},{"year":2006,"finding":"The Gads-binding domain of SLP-76 is required for PLC-γ1 recruitment to GEMs, while the N-terminal tyrosine phosphorylation sites and P-1 region are required for PLC-γ1 phosphorylation at Y783; ITK (associated with SLP-76 via N-terminal tyrosines) efficiently phosphorylates PLC-γ1 at Y783 in vitro.","method":"Lipid raft fractionation, PLC-γ1 phosphorylation assay, in vitro kinase assay with ITK, mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus mutagenesis and biochemical fractionation with functional readout","pmids":["17148460"],"is_preprint":false},{"year":2007,"finding":"HPK1 phosphorylates SLP-76 at serine 376; this serine phosphorylation mediates recruitment of 14-3-3ε and ζ proteins to SLP-76, which attenuates TCR-induced tyrosine phosphorylation of SLP-76 and PLC-γ1 and reduces IL-2 transcription. S376A mutation or HPK-1 knockdown enhances TCR signaling.","method":"RNAi knockdown, in vitro phosphorylation, mutagenesis (S376A), co-immunoprecipitation, IL-2 reporter assay, phosphorylation assay","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus mutagenesis, RNAi, and multiple functional readouts","pmids":["17353368"],"is_preprint":false},{"year":2007,"finding":"SLP-76 is required for TCR-induced activation of ITK; ITK bound to SLP-76 represents most of the catalytically active ITK, and ITK activity is lost upon mild elution from the SLP-76 complex but restored upon reconstitution. SLP-76 N-terminal tyrosines are required for ITK activation but not ZAP-70 activation.","method":"In vitro kinase assay (ITK vs ZAP-70 on PLC-γ1), co-immunoprecipitation, mutagenesis, reconstitution experiments","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus mutagenesis and reconstitution revealing SLP-76 maintains ITK active conformation","pmids":["17420479"],"is_preprint":false},{"year":2007,"finding":"The Gads C-SH3 domain binds the SLP-76 RxxK motif with very high affinity (Kd = 8-20 nM); T cell signaling efficiency declines with decreasing Gads-SLP-76 binding affinity, demonstrating a quantitative affinity requirement for efficient TCR signaling.","method":"Quantitative affinity measurement, SLP-76 mutants with graded affinities, TCR signaling reporter assays in Jurkat cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — quantitative affinity measurements with functional correlates across multiple mutants","pmids":["17235283"],"is_preprint":false},{"year":2008,"finding":"SLP-76 N-terminal tyrosines Y145 and Y112-128 are required for thymocyte selection but can complement one another in trans (on separate SLP-76 molecules); Y145 differentially supports Itk-dependent pathways while Y112-128 supports Vav1 phosphorylation, demonstrating cooperativity between SLP-76 molecules.","method":"Knock-in mice (Y145F and Y112-128F), compound heterozygote analysis, phosphorylation assays, co-immunoprecipitation","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — knock-in genetic approach with biochemical and developmental functional readouts","pmids":["18342008"],"is_preprint":false},{"year":2008,"finding":"VLA-4 integrin costimulation retards actin-driven inward flows and prevents centralization of SLP-76 microclusters, prolonging lateral SLP-76/ZAP-70 interactions and retaining SLP-76 in tyrosine-phosphorylated peripheral structures, thereby sustaining T cell signaling.","method":"Live TIRF microscopy of SLP-76 microclusters, integrin ligand modulation, actin flow measurement","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — direct live imaging correlating cytoskeletal dynamics with SLP-76 microcluster behavior and signaling","pmids":["18549800"],"is_preprint":false},{"year":2009,"finding":"SLP-76 mediates 'outside-in' integrin signaling in T cells; SLP-76 relocalizes to integrin-initiated microclusters by a mechanism requiring ADAP binding (not LAT/Gads), distinct from TCR-initiated microcluster assembly, and is required for T cell adhesion to integrin ligands.","method":"SLP-76 KO T cells, domain mutagenesis (ADAP-binding vs Gads-binding mutants), microcluster imaging, adhesion assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — genetic KO plus structure-function mutagenesis and imaging with defined functional phenotype","pmids":["19667077"],"is_preprint":false},{"year":2010,"finding":"The SLP-76/Nck/VAV1 complex has defined stoichiometry (one SLP-76, two Nck, two VAV1); a direct Nck-VAV1 interaction via the C-terminal SH3 domain of Nck and the VAV1 N-terminal SH3 domain is required for actin polymerization after T cell activation.","method":"Analytical ultracentrifugation, co-immunoprecipitation, mutagenesis of Nck-VAV1 interface, actin polymerization assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — stoichiometry determined by biophysics plus mutagenesis with functional actin polymerization assay","pmids":["20562827"],"is_preprint":false},{"year":2010,"finding":"Platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling; platelet-specific deletion of SLP-76 (via PF4-Cre) is sufficient to cause lymphatic vascular defects, and SLP-76-dependent platelet aggregate formation occurs on lymphatic endothelial cell surfaces.","method":"Conditional KO (PF4-Cre x Slp76 flox), in vivo and ex vivo vascular imaging, platelet-LEC co-culture","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — conditional KO in specific cell type with in vivo and ex vivo mechanistic validation","pmids":["20363774"],"is_preprint":false},{"year":2012,"finding":"HPK1-dependent serine 376 phosphorylation of SLP-76 mediates ubiquitination of SLP-76 at lysine 30, targeting phosphorylated SLP-76 for proteasomal degradation during TCR signaling; K30R mutation enhances anti-CD3-induced ERK and JNK activation.","method":"In vitro ubiquitination assay, mutagenesis (S376A, K30R), proteasome inhibition, ERK/JNK activation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro ubiquitination plus mutagenesis and functional signaling readout","pmids":["22902619"],"is_preprint":false},{"year":2012,"finding":"In NK cells, SLP-76 Y113 and Y128 are phosphorylated by distinct co-activation receptor pairs (e.g., NKG2D and 2B4), enabling SLP-76 binding to Vav1; combined phosphorylation of both tyrosines is required for synergistic Ca2+ mobilization and NK cell activation.","method":"SLP-76 KD and reconstitution with tyrosine mutants, Ca2+ flux assay, cytotoxicity assay, co-immunoprecipitation","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 — reconstitution with point mutants and multiple functional readouts","pmids":["22786724"],"is_preprint":false},{"year":2012,"finding":"SLP-76 and ADAP are required for E-selectin-mediated integrin activation and slow leukocyte rolling in neutrophils; two N-terminal tyrosines and the SH2 domain of SLP-76 are required for downstream signaling, placing BTK and then PI3Kγ/PLCγ2 downstream of SLP-76 in this pathway.","method":"Genetically engineered KO mice, transduced primary leukocytes, intravital microscopy, kinase assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with mutagenesis and in vivo functional readout","pmids":["22291096"],"is_preprint":false},{"year":2012,"finding":"SLP-76, Nck, and Vav1 form a trimolecular complex in live T cells; Nck-Vav1 dimers are constitutively formed independently of T cell activation and SLP-76-Nck association; after TCR stimulation, SLP-76 phosphorylation enables Nck binding and complex assembly. The Nck-Vav1 interaction requires Vav1 proline-rich/SH3 domain and is critical for actin rearrangement.","method":"Triple-color FRET (3FRET) in live T cells, mutagenesis of Vav1 SH3, actin rearrangement assay","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 — live-cell 3FRET plus mutagenesis and functional actin assay","pmids":["22534133"],"is_preprint":false},{"year":2013,"finding":"ADAP contains three binding sites for the SLP-76 SH2 domain; multipoint binding of SLP-76 to ADAP oligomerizes SLP-76 SH2 domain in vitro and is critical for SLP-76 microcluster assembly in stimulated T cells; any combination of two sites partially restores microclusters.","method":"Analytical ultracentrifugation, biophysical binding assays, confocal imaging of microclusters, ADAP mutagenesis, functional T cell assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — biophysical reconstitution plus cellular imaging and mutagenesis with functional correlates","pmids":["23979596"],"is_preprint":false},{"year":2021,"finding":"SLP-76 directly binds the cytosolic tail of RAGE via its SAM (sterile α motif) domain; this interaction mediates downstream p38 MAPK, ERK1/2, and IKKα/β phosphorylation as well as cytokine release in macrophages. TAT-SAM peptide delivery blocks RAGE-SLP-76 interaction and attenuates sepsis in mice.","method":"Co-immunoprecipitation in vitro and in vivo, genetic deficiency of RAGE/SLP-76, TAT-peptide cell delivery, CLP sepsis model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — co-IP plus genetic KO and peptide intervention with in vivo functional validation","pmids":["33436632"],"is_preprint":false}],"current_model":"SLP-76 (LCP2) is a hematopoietic-specific multi-domain scaffold/adaptor protein (N-terminal acidic tyrosine-containing region, central proline-rich region, C-terminal SH2 domain) that is phosphorylated by ZAP-70 (and ITK/Rlk) at Y112, Y128, and Y145 downstream of ITAM-containing receptors (TCR, FcεRI, GPVI, FcγR) and integrins; it is recruited to membrane microdomains via constitutive association with Gads (which bridges it to LAT) and assembles a central signalosome with Nck, Vav1, and ITK to activate PLC-γ1, actin remodeling (via WASP/Arp2/3), Ras/ERK, Ca2+/NFAT, and NF-κB pathways, while being negatively regulated by HPK1-mediated S376 phosphorylation that recruits 14-3-3 proteins and triggers K30 ubiquitination and proteasomal degradation of SLP-76, and by SHP-1-mediated dephosphorylation downstream of inhibitory receptors."},"narrative":{"teleology":[{"year":1995,"claim":"Molecular cloning of SLP-76 established the existence of a novel hematopoietic adaptor protein with an SH2 domain capable of engaging tyrosine-phosphorylated signaling partners including PLC-γ1-associated proteins, opening the question of its receptor context and upstream regulation.","evidence":"Cloning from IL-2-stimulated T cells with GST pulldown and co-immunoprecipitation","pmids":["7706237"],"confidence":"High","gaps":["Identity of the 62 kDa and 130 kDa SH2 domain-associated proteins unknown","Upstream kinase not identified","In vivo function unknown"]},{"year":1996,"claim":"Identification of ZAP-70 as the kinase phosphorylating SLP-76 at Y113, Y128, and Y145, and discovery that phospho-SLP-76 recruits Vav via its SH2 domain to synergistically activate NF-AT, established SLP-76 as a TCR-proximal signaling node linking receptor engagement to transcription factor activation and identified the key phosphotyrosine residues.","evidence":"In vitro kinase assays, site-directed mutagenesis, NF-AT reporter assays, co-immunoprecipitation in Jurkat T cells","pmids":["8702662","8892604","8673706"],"confidence":"High","gaps":["Which tyrosine recruits which effector not yet resolved","In vivo requirement for SLP-76 not tested"]},{"year":1998,"claim":"Genetic knockout of SLP-76 revealed it is essential for T cell development (complete thymopoiesis block), platelet collagen-receptor signaling (PLC-γ2 activation, aggregation), and that SLP-76 organizes a trimolecular complex with Nck and Vav to activate PAK1 and actin polymerization, transforming the view of SLP-76 from a simple adaptor to a multifunctional signalosome scaffold.","evidence":"SLP-76 KO mice (two independent labs), platelet aggregation/degranulation assays, co-IP and PAK1 kinase assays","pmids":["9665885","9695951","9884330","9846482"],"confidence":"High","gaps":["Mechanism of SLP-76 membrane recruitment unknown","Role in other hematopoietic lineages not yet explored"]},{"year":1999,"claim":"Discovery that Gads constitutively bridges SLP-76 to LAT and identification of ADAP (SLAP-130), HPK1, and Nck as SLP-76 SH2 domain or phosphotyrosine-dependent interactors expanded the signalosome model and revealed that SLP-76 serves as a hub integrating both positive (Nck, Vav) and negative (HPK1, ADAP) regulators.","evidence":"Co-IP and domain mapping in T cells and mast cells, NF-AT reporter assays, yeast two-hybrid, SLP-76 KO mast cell functional assays","pmids":["10021361","10377180","10229072","11487585","9115214"],"confidence":"High","gaps":["Structural basis of Gads-SLP-76 interaction unknown","Mechanism of HPK1-mediated negative regulation unclear"]},{"year":2000,"claim":"Demonstrating that SLP-76 recruitment to glycolipid-enriched membrane microdomains (GEMs) is essential for TCR signaling and that SLP-76 coordinates actin remodeling via WASP/ADAP/Ena-VASP at the immunological synapse established the spatial dimension of SLP-76 function.","evidence":"LAT/SLP-76 chimeric protein reconstitution in LAT-deficient cells, GEM fractionation, confocal imaging of T cell-APC contacts","pmids":["11015445","10747096","11113155"],"confidence":"High","gaps":["Dynamic behavior of SLP-76 at the synapse not resolved","How integrin signals feed into SLP-76 not clear"]},{"year":2001,"claim":"Mapping of a direct SLP-76–PLC-γ1 SH3 domain interaction via a proline-rich P-1 region and confirmation that GADS KO uncouples SLP-76 from LAT completed the minimal wiring diagram of the LAT-Gads-SLP-76-PLC-γ1 signalosome.","evidence":"GADS KO mice with co-IP, SLP-76 domain mutagenesis and reconstitution in SLP-76-deficient cells, functional reporter assays","pmids":["11390650","11239162"],"confidence":"High","gaps":["Quantitative affinity requirements for each interaction not defined","Stoichiometry of the complex unknown"]},{"year":2003,"claim":"Crystal and NMR structures of the Gads C-SH3/SLP-76 RSTK peptide complex revealed a novel non-proline-based SH3 recognition mode (3₁₀ helix), providing the first atomic-level understanding of a key SLP-76 interaction; separately, blood-lymphatic separation failure in SLP-76 KO mice disclosed an unexpected developmental role in a hematopoietic cell-autonomous vascular process.","evidence":"X-ray crystallography (1.7 Å), NMR solution structure, bone marrow reconstitution in KO mice, vascular histology","pmids":["12773374","12620234","12522250"],"confidence":"High","gaps":["Identity of the hematopoietic cell type mediating vascular separation unknown","Full-length SLP-76 structure unavailable"]},{"year":2005,"claim":"Live-cell TIRF imaging showed SLP-76 forms dynamic signaling microclusters at TCR contact sites that migrate centripetally; this established that signaling is organized in discrete membrane-associated clusters rather than uniformly distributed, with ZAP-70 co-recruited and signaling correlated with cluster formation.","evidence":"TIRF microscopy with fluorescently tagged SLP-76 and ZAP-70 in T cell-APC conjugates","pmids":["16273097"],"confidence":"High","gaps":["Molecular determinants of microcluster formation not identified","Role of ADAP in cluster assembly not yet explored"]},{"year":2006,"claim":"Systematic tyrosine mutagenesis resolved the division of labor among SLP-76 N-terminal tyrosines: Y145 recruits ITK for PLC-γ1 phosphorylation, while Y112/Y128 recruit Vav1 and Nck for Ca²⁺ flux and actin reorganization, and the Gads-binding domain delivers PLC-γ1 to membrane microdomains for phosphorylation by ITK.","evidence":"Individual tyrosine-to-phenylalanine mutants in SLP-76-deficient cells and primary T cells, lipid raft fractionation, in vitro ITK kinase assay","pmids":["16456002","17148460"],"confidence":"High","gaps":["Whether Y145 directly contacts ITK SH2 domain not structurally confirmed","Redundancy between Y112 and Y128 not fully resolved"]},{"year":2007,"claim":"HPK1 was identified as the kinase phosphorylating SLP-76 S376, which recruits 14-3-3 proteins to attenuate TCR signaling, and SLP-76 was shown to allosterically maintain ITK in its catalytically active conformation, establishing SLP-76 as both a positive allosteric activator and a target of negative feedback.","evidence":"In vitro kinase assays, S376A mutagenesis, RNAi of HPK1, ITK activity reconstitution upon elution/rebinding to SLP-76","pmids":["17353368","17420479"],"confidence":"High","gaps":["E3 ligase mediating SLP-76 ubiquitination not identified","Structural basis for ITK allosteric activation unknown"]},{"year":2010,"claim":"Biophysical determination of a 1:2:2 SLP-76/Nck/Vav1 stoichiometry and demonstration that platelet-specific SLP-76 deletion (via PF4-Cre) suffices to cause lymphatic vascular defects via CLEC-2/PDPN signaling resolved both the molecular architecture of the actin-regulatory complex and the cell type responsible for blood-lymphatic separation.","evidence":"Analytical ultracentrifugation, conditional KO mice, platelet-lymphatic endothelial cell co-culture","pmids":["20562827","20363774"],"confidence":"High","gaps":["Full structural model of the pentameric complex not available","Downstream effectors of CLEC-2/SLP-76 in vascular separation not fully mapped"]},{"year":2012,"claim":"HPK1-dependent S376 phosphorylation was linked to K30 ubiquitination and proteasomal degradation of SLP-76, completing the negative feedback circuit; in parallel, ADAP was shown to oligomerize SLP-76 via multipoint SH2 domain binding, providing a mechanism for microcluster assembly, and SLP-76 tyrosine phosphorylation was shown to integrate co-activation receptor signals in NK cells.","evidence":"In vitro ubiquitination assays with K30R mutant, analytical ultracentrifugation of ADAP-SLP-76, confocal imaging of microclusters, NK cell reconstitution with tyrosine mutants","pmids":["22902619","23979596","22786724"],"confidence":"High","gaps":["Identity of the E3 ubiquitin ligase targeting K30 still unknown","Whether ADAP-mediated oligomerization occurs in non-T cells not tested"]},{"year":2021,"claim":"Discovery that SLP-76 directly binds the RAGE cytoplasmic tail via its SAM domain to drive p38/ERK/IKK signaling and cytokine release in macrophages extended SLP-76 function beyond ITAM/integrin-receptor pathways to a pattern-recognition receptor axis relevant to sepsis.","evidence":"Co-IP in macrophages, RAGE/SLP-76 genetic deficiency, TAT-SAM peptide inhibition, CLP sepsis model in mice","pmids":["33436632"],"confidence":"High","gaps":["Whether the SAM domain interaction is direct or requires post-translational modification not established","Relevance to human sepsis not validated","Structural basis of RAGE-SAM interaction unknown"]},{"year":null,"claim":"A full-length structure of SLP-76 in complex with its signalosome partners, the identity of the E3 ubiquitin ligase for K30 ubiquitination, and the mechanism by which SLP-76 allosterically activates ITK remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length SLP-76 structure available","E3 ligase for K30 ubiquitination unidentified","Structural mechanism of ITK allosteric activation by SLP-76 unknown","Role of SLP-76 SAM-RAGE axis in other inflammatory contexts untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,9,14,24,41,46]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[22,35,37,44,52]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,14]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,4,10,15,28,38,49]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,40,41,53]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[13,16,23,47]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[10,32,47]}],"complexes":["LAT-Gads-SLP-76 signalosome","SLP-76/Nck/Vav1 trimolecular complex","SLP-76/ITK/PLC-γ1 complex"],"partners":["GRAP2","VAV1","NCK1","ITK","PLCG1","FYB1","ZAP70","HPK1"],"other_free_text":[]},"mechanistic_narrative":"SLP-76 (LCP2) is a hematopoietic-specific multi-domain scaffold protein that nucleates signalosome assembly downstream of ITAM-containing receptors (TCR, FcεRI, FcγR, GPVI, CLEC-2) and integrins in T cells, platelets, mast cells, NK cells, and neutrophils. ZAP-70/Syk phosphorylates SLP-76 at N-terminal tyrosines Y112, Y128, and Y145, creating docking sites for Vav1 and Nck (Y112/Y128) and ITK (Y145), thereby coordinating PLC-γ1/γ2 activation, Ca²⁺ mobilization, Ras/ERK signaling, NF-AT transcription, and actin cytoskeleton remodeling via WASP/Arp2/3 [PMID:8892604, PMID:16456002, PMID:17420479, PMID:9846482]. SLP-76 is constitutively bridged to LAT-containing membrane microdomains through Gads binding to a central RxxK motif, and it additionally recruits ADAP via its C-terminal SH2 domain to drive integrin activation and microcluster oligomerization [PMID:11239162, PMID:23979596, PMID:19667077]. SLP-76 is negatively regulated by HPK1-mediated S376 phosphorylation, which recruits 14-3-3 proteins and triggers K30 ubiquitination and proteasomal degradation, and by SHP-1-mediated dephosphorylation downstream of inhibitory receptors; SLP-76 knockout in mice causes a complete block in T cell development and defective blood-lymphatic vascular separation through platelet CLEC-2/PDPN signaling [PMID:17353368, PMID:22902619, PMID:9765283, PMID:9665885, PMID:20363774]."},"prefetch_data":{"uniprot":{"accession":"Q13094","full_name":"Lymphocyte cytosolic protein 2","aliases":["SH2 domain-containing leukocyte protein of 76 kDa","SLP-76 tyrosine phosphoprotein","SLP76"],"length_aa":533,"mass_kda":60.2,"function":"Adapter protein primarily involved in signaling pathways within T-cells, as well as other immune cells such as platelets, mast cells, and natural killer (NK) cells (PubMed:11313406, PubMed:33159816). Plays a crucial role for transducing signal from the T-cell receptor (TCR) after antigen recognition leading to T-cell activation. Mechanistically, once phosphorylated by the kinase ZAP70, mediates interactions with the guanine-nucleotide exchange factor VAV1, the adapter protein NCK and the kinase ITK (PubMed:8673706, PubMed:8702662). In turn, stimulates the activation of PKC-theta/PRKCQ and NF-kappa-B transcriptional activity in response to CD3 and CD28 costimulation (PubMed:11313406). Also plays an essential role in AGER-induced signaling pathways including p38 MAPK and ERK1/2 activation leading to cytokine release and pro-inflammatory responses (PubMed:33436632)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q13094/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/LCP2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/LCP2","total_profiled":1310},"omim":[{"mim_id":"619374","title":"IMMUNODEFICIENCY 81; IMD81","url":"https://www.omim.org/entry/619374"},{"mim_id":"618478","title":"FYN-BINDING PROTEIN 2; FYB2","url":"https://www.omim.org/entry/618478"},{"mim_id":"614406","title":"SLP ADAPTOR- AND CSK-INTERACTING MEMBRANE PROTEIN; SCIMP","url":"https://www.omim.org/entry/614406"},{"mim_id":"611434","title":"CYTOKINE-DEPENDENT HEMATOPOIETIC CELL LINKER; CLNK","url":"https://www.omim.org/entry/611434"},{"mim_id":"609201","title":"UBIQUITIN-ASSOCIATED AND SH3 DOMAIN-CONTAINING PROTEIN B; UBASH3B","url":"https://www.omim.org/entry/609201"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":154.8},{"tissue":"lymphoid tissue","ntpm":107.3}],"url":"https://www.proteinatlas.org/search/LCP2"},"hgnc":{"alias_symbol":["SLP-76"],"prev_symbol":["SLP76"]},"alphafold":{"accession":"Q13094","domains":[{"cath_id":"1.10.150.50","chopping":"10-76","consensus_level":"high","plddt":88.5961,"start":10,"end":76},{"cath_id":"3.30.505.10","chopping":"416-530","consensus_level":"high","plddt":90.023,"start":416,"end":530}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13094","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13094-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13094-F1-predicted_aligned_error_v6.png","plddt_mean":62.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=LCP2","jax_strain_url":"https://www.jax.org/strain/search?query=LCP2"},"sequence":{"accession":"Q13094","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13094.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13094/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13094"}},"corpus_meta":[{"pmid":"16273097","id":"PMC_16273097","title":"Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.","date":"2005","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/16273097","citation_count":566,"is_preprint":false},{"pmid":"9665884","id":"PMC_9665884","title":"Uncoupling of nonreceptor tyrosine kinases from PLC-gamma1 in an SLP-76-deficient T cell.","date":"1998","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/9665884","citation_count":342,"is_preprint":false},{"pmid":"9665885","id":"PMC_9665885","title":"Requirement for the leukocyte-specific adapter protein SLP-76 for normal T cell development.","date":"1998","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/9665885","citation_count":340,"is_preprint":false},{"pmid":"20363774","id":"PMC_20363774","title":"Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.","date":"2010","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/20363774","citation_count":332,"is_preprint":false},{"pmid":"12522250","id":"PMC_12522250","title":"Regulation of blood and lymphatic vascular separation by signaling proteins SLP-76 and Syk.","date":"2003","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/12522250","citation_count":321,"is_preprint":false},{"pmid":"8702662","id":"PMC_8702662","title":"Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8702662","citation_count":311,"is_preprint":false},{"pmid":"9695951","id":"PMC_9695951","title":"Impaired viability and profound block in thymocyte development in mice lacking the adaptor protein SLP-76.","date":"1998","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/9695951","citation_count":302,"is_preprint":false},{"pmid":"7706237","id":"PMC_7706237","title":"Molecular cloning of SLP-76, a 76-kDa tyrosine phosphoprotein associated with Grb2 in T cells.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7706237","citation_count":288,"is_preprint":false},{"pmid":"8673706","id":"PMC_8673706","title":"Vav and SLP-76 interact and functionally cooperate in IL-2 gene activation.","date":"1996","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/8673706","citation_count":286,"is_preprint":false},{"pmid":"10021361","id":"PMC_10021361","title":"The hematopoietic-specific adaptor protein gads functions in T-cell signaling via interactions with the SLP-76 and LAT adaptors.","date":"1999","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/10021361","citation_count":254,"is_preprint":false},{"pmid":"10747096","id":"PMC_10747096","title":"Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton.","date":"2000","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10747096","citation_count":246,"is_preprint":false},{"pmid":"16493428","id":"PMC_16493428","title":"SLP76 and SLP65: complex regulation of signalling in lymphocytes and beyond.","date":"2006","source":"Nature reviews. 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\"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original cloning paper with direct binding assays and multiple orthogonal methods\",\n      \"pmids\": [\"7706237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"ZAP-70 phosphorylates SLP-76 in vitro and in heterologous cell systems; SLP-76 phosphorylation is diminished when catalytically inactive ZAP-70 is expressed. SLP-76 overexpression hyperactivates the TCR, while a non-phosphorylatable SLP-76 attenuates signaling. The SH2 domain of SLP-76 is independently required for TCR function.\",\n      \"method\": \"In vitro kinase assay, dominant-negative ZAP-70 expression, overexpression/mutagenesis in T cell lines, reporter gene assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus mutagenesis with functional readout, confirmed across multiple approaches\",\n      \"pmids\": [\"8702662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"SLP-76 directly associates with Vav via the Vav SH2 domain binding to phosphorylated SLP-76; co-overexpression of Vav and SLP-76 synergistically induces NF-AT activation, placing SLP-76 in a complex with Vav during TCR signaling.\",\n      \"method\": \"Co-immunoprecipitation, overexpression, NF-AT reporter assay in Jurkat cells\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP and functional reporter assay, replicated across labs\",\n      \"pmids\": [\"8673706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"TCR engagement leads to tyrosine phosphorylation of SLP-76 at its N-terminal acidic region; tyrosines Y113 and Y128 (YESP motifs) and Y145 are phosphorylated, with Y145 being the most important for NF-AT promoter activity.\",\n      \"method\": \"Site-directed mutagenesis, TCR stimulation, NF-AT reporter assay in Jurkat cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with functional readout, replicated by multiple labs\",\n      \"pmids\": [\"8892604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"SLP-76 is required for optimal TCR-induced activation of PLC-γ1 and Ras pathways; an SLP-76-deficient T cell line showed that SLP-76 is not required for general tyrosine phosphorylation but is specifically required for PLC-γ1 phosphorylation and TCR-inducible gene expression.\",\n      \"method\": \"SLP-76-deficient cell line (genetic loss), phosphorylation assays, gene expression reporter\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic loss-of-function with specific biochemical and functional readouts\",\n      \"pmids\": [\"9665884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Grb2 constitutively associates with unphosphorylated SLP-76; the SH2 domain of SLP-76 associates with 62 kDa and 130 kDa tyrosine-phosphorylated proteins and a serine/threonine kinase after TCR stimulation.\",\n      \"method\": \"Co-immunoprecipitation, GST-SH2 domain pulldown, in vitro binding\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP and pulldown, single lab\",\n      \"pmids\": [\"8666952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"SHP-1 constitutively associates with SLP-76 in B cells via SHP-1 SH2 domains; BCR ligation induces tyrosine phosphorylation of SLP-76 and SHP-1.\",\n      \"method\": \"Co-immunoprecipitation, GST-SH2 domain pulldown, Western blot\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single-lab co-IP with limited mechanistic follow-up\",\n      \"pmids\": [\"8760799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"ZAP-70 phosphorylates two YESP motifs of SLP-76 (Y113 and Y128) to create binding sites for the Vav SH2 domain; a third motif (Y145, pYEPP) fails to bind Vav SH2. Vav-SLP-76 binding is not absolutely required for IL-2 production in all T cells.\",\n      \"method\": \"In vitro kinase assay, phosphopeptide binding, overexpression in T cell hybridoma\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus phosphopeptide binding mapping specific sites\",\n      \"pmids\": [\"9047237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"SLAP-130 (FYB/ADAP) is a 130 kDa SLP-76-associated phosphoprotein that interacts via the SH2 domain of SLP-76; overexpression of SLAP-130 negatively regulates TCR-induced IL-2 promoter activity and counteracts SLP-76 augmentation of signaling.\",\n      \"method\": \"Molecular cloning, co-immunoprecipitation with SH2 domain, reporter gene assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — molecular cloning plus functional reporter assay, single lab\",\n      \"pmids\": [\"9115214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"SLP-76 function requires three distinct protein-interaction domains (N-terminal tyrosine-containing acidic region, proline-rich region, C-terminal SH2 domain) for optimal augmentation of TCR-induced NF-AT activity and ERK activation.\",\n      \"method\": \"Domain deletion mutagenesis, overexpression in Jurkat cells, reporter gene assay, kinase assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — structure-function with multiple domain deletions and functional readouts\",\n      \"pmids\": [\"9257823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"SLP-76 is required for normal T cell development; SLP-76-null mice have a complete block in thymopoiesis at an early stage with no peripheral T cells, establishing SLP-76 as an essential signal transducer for pre-TCR signaling.\",\n      \"method\": \"Targeted gene knockout in mice, flow cytometry of thymic populations\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined developmental phenotype, independently replicated by two labs (PMID 9665885 and 9695951)\",\n      \"pmids\": [\"9665885\", \"9695951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"SLP-76 interacts with Nck (an adaptor protein) and Vav (a GEF for Rho-family GTPases), forming a trimolecular complex that activates PAK1 (p21-activated kinase 1) and facilitates actin polymerization downstream of the TCR.\",\n      \"method\": \"Co-immunoprecipitation, PAK1 kinase assay, actin polymerization assay\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus enzymatic (kinase) assay and actin polymerization readout\",\n      \"pmids\": [\"9846482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"SLP-76 is a direct substrate of SHP-1 in T cells and NK cells; SHP-1, recruited to killer cell inhibitory receptors, dephosphorylates SLP-76, and tyrosine-phosphorylated SLP-76 is required for optimal cytotoxic lymphocyte activation.\",\n      \"method\": \"Direct binding assay (SHP-1 catalytic domain on SLP-76), functional inhibitory receptor co-engagement assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding and functional assay in relevant cell types\",\n      \"pmids\": [\"9765283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"SLP-76 is required for optimal PLC-γ2 activation in platelets downstream of the collagen receptor; SLP-76-deficient platelets show impaired collagen-induced platelet aggregation, granule release, and PLC-γ2 tyrosine phosphorylation.\",\n      \"method\": \"SLP-76 KO mouse platelets, platelet aggregation assay, phosphorylation assay, granule release assay\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined biochemical and functional phenotype in platelets\",\n      \"pmids\": [\"9884330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Gads (Grb2-related adaptor) constitutively interacts with SLP-76 via the C-terminal SH3 domain of Gads and a proline-rich region of SLP-76; Gads also binds tyrosine-phosphorylated LAT via its SH2 domain, bridging LAT and SLP-76 and promoting T cell signaling.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, NF-AT reporter assay\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus domain mapping and functional assay, replicated\",\n      \"pmids\": [\"10021361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"SLP-76 deficiency in mast cells impairs FcεRI-mediated signaling: SLP-76-null BMMCs fail to release β-hexosaminidase and IL-6, and show reduced PLC-γ1 tyrosine phosphorylation and calcium mobilization after FcεRI cross-linking, while Syk phosphorylation is normal.\",\n      \"method\": \"SLP-76 KO mouse mast cells, degranulation assay, cytokine secretion, phosphorylation assay, calcium flux\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple orthogonal functional and biochemical readouts\",\n      \"pmids\": [\"10377180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"In platelets, tyrosine phosphorylation of SLP-76 by the collagen receptor pathway is downstream of Syk; SLP-76 co-immunoprecipitates with SLAP-130, Vav, Fyn, Lyn, and the FcR γ-chain after collagen stimulation, and SLP-76 deficiency abrogates PLC-γ2 phosphorylation and Ca2+ mobilization.\",\n      \"method\": \"Syk-deficient platelet analysis, co-immunoprecipitation, in vitro kinase assay, calcium mobilization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis (Syk-deficient platelets) plus co-IP and functional assays\",\n      \"pmids\": [\"10026222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Nck SH2 domain interacts with tyrosine-phosphorylated SLP-76 in activated T cells; phosphopeptides corresponding to Y113 and Y128 of SLP-76 compete binding of SLP-76 to the Nck SH2 domain.\",\n      \"method\": \"Co-immunoprecipitation, phosphopeptide competition, in vitro binding\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus peptide competition mapping specific sites\",\n      \"pmids\": [\"10229072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"FYN-T selectively phosphorylates FYB/SLAP, creating binding sites for the SH2 domains of FYN-T and SLP-76; co-expression of FYN-T, FYB, and SLP-76 synergistically up-regulates IL-2 transcription via TCR ligation.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, NF-AT/IL-2 reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro kinase assay plus functional reporter, single lab\",\n      \"pmids\": [\"10409671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"SLP-76 SH2 domain binds HPK1 at Tyr379; HPK1 interacts with SLP-76 in T cells and inhibits AP-1 activation in an SLP-76 SH2-domain-dependent manner, integrating HPK1 into antigen receptor signaling cascades.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, reporter gene assay, homology modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid plus co-IP and functional assay\",\n      \"pmids\": [\"11487585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"GrpL (Grb2-related adaptor) co-immunoprecipitates with SLP-76 but not with Sos1/2 in Jurkat cells; GrpL cooperates with SLP-76 to augment NF-AT activation, defining a distinct Grb2-like adaptor complex with SLP-76.\",\n      \"method\": \"Co-immunoprecipitation, NF-AT reporter assay\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP with functional reporter, single lab\",\n      \"pmids\": [\"10209041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Fyb/SLAP (FYB/ADAP) links TCR signaling to actin cytoskeleton remodeling by acting as a ligand for Ena/VASP EVH1 domains; upon TCR engagement, Fyb/SLAP localizes to the T cell-APC interface within complexes containing WASP, Nck, and SLP-76.\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy, WASP/Arp2/3 inhibition, biochemical fractionation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus localization imaging and functional inhibition with defined actin phenotype\",\n      \"pmids\": [\"10747096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SLP-76 recruitment to glycolipid-enriched membrane microdomains (GEMs) requires amino acids 224-244 of SLP-76 and is critical for TCR signaling; forced membrane localization of SLP-76 via a LAT/SLP-76 chimera reconstitutes PLC-γ1 phosphorylation, ERK activation, and NF-AT activity in LAT-deficient cells.\",\n      \"method\": \"Chimeric protein expression, fractionation into GEMs, reporter gene assay, phosphorylation assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — chimeric protein reconstitution plus mutagenesis and multiple functional readouts\",\n      \"pmids\": [\"11015445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SLP-76 relays signals from platelet integrin αIIbβ3 to the actin cytoskeleton: fibrinogen binding triggers Syk-dependent SLP-76 phosphorylation, which promotes SLP-76 association with Nck and Vav1, lamellipodia formation, and PAK kinase activation.\",\n      \"method\": \"CHO cell expression system, co-immunoprecipitation, PAK kinase assay, microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reconstitution in CHO cells plus co-IP, kinase assay and genetic KO comparison\",\n      \"pmids\": [\"11113155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SLP-76 directly interacts with the SH3 domain of PLC-γ1 via a proline-rich 'P-1 domain' (67 amino acids); this interaction is constitutive and required for TCR-mediated ERK, PLC-γ1, and NF-AT activation. The Gads-binding domain of SLP-76 mediates inducible recruitment to a PLC-γ1-LAT complex.\",\n      \"method\": \"Mutagenesis, co-immunoprecipitation, reporter gene assay, reconstitution in SLP-76-deficient cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — domain mutagenesis identifying direct interaction plus reconstitution and multiple functional readouts\",\n      \"pmids\": [\"11390650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SLAP-130 (FYB/ADAP) interaction with SLP-76 requires phospho-Y559 of SLAP-130; this interaction is important for the negative regulatory role of SLAP-130, specifically inhibiting TCR-induced ERK activation but not PLC-γ1 phosphorylation.\",\n      \"method\": \"Mutagenesis, co-immunoprecipitation, ERK activation assay, NF-AT reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — point mutagenesis plus functional assays, single lab\",\n      \"pmids\": [\"10671560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"WASP recruitment to the T cell-APC contact site requires Nck (via WASP proline-rich domain), while WASP activation requires Vav-1 for localized Cdc42 activation; SLP-76 coordinates both events by acting as a scaffold bringing Nck and WASP near Vav-1/Cdc42-GTP.\",\n      \"method\": \"Vav-1-deficient T cells, microscopy of WASP localization, co-immunoprecipitation, Cdc42 activation assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with Vav-1-deficient T cells plus imaging and biochemical assays\",\n      \"pmids\": [\"12874226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"GADS is required for coupling SLP-76 to LAT in T cells; GADS-deficient thymocytes fail to respond to CD3 cross-linking and the association between SLP-76 and LAT is uncoupled, establishing GADS as the physical bridge between SLP-76 and LAT.\",\n      \"method\": \"GADS KO mice, co-immunoprecipitation, thymocyte proliferation assay, in vivo selection assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO plus co-IP demonstrating loss of SLP-76/LAT association\",\n      \"pmids\": [\"11239162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SLP-76 mediates signaling via Fcγ receptors and integrins in neutrophils; SLP-76-deficient neutrophils show decreased FcγR-induced calcium flux and reactive oxygen intermediate production, and fail to produce ROI, spread, or activate key downstream regulators upon integrin ligation.\",\n      \"method\": \"SLP-76 KO mouse neutrophils, calcium flux, ROI production assay, spreading assay\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple functional readouts\",\n      \"pmids\": [\"14614862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The RxxK motif in SLP-76 (R237 and K240) mediates high-affinity binding to the C-terminal SH3 domain of Gads; single point mutations in R237 or K240 abrogate SLP-76/Gads association in vivo and impair SLP-76 function in TCR signaling.\",\n      \"method\": \"Peptide array, in vitro binding (Kd measurement), site-directed mutagenesis, co-immunoprecipitation, NF-AT reporter assay\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — quantitative affinity measurement plus mutagenesis and in vivo functional confirmation\",\n      \"pmids\": [\"12176364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Crystal structure (1.7 Å) of the Mona/Gads C-terminal SH3 domain complexed with a SLP-76 peptide reveals a novel non-proline-type II helix binding mode: the SLP-76 RSTK peptide forms a 3(10) helix inserting into a negatively charged double pocket on the SH3 domain.\",\n      \"method\": \"X-ray crystallography (1.7 Å resolution), mutagenesis, in vitro binding\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with supporting mutagenesis and binding data\",\n      \"pmids\": [\"12773374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"NMR solution structure of the Gads C-terminal SH3 domain in complex with an SLP-76 RSTK-containing peptide shows the peptide adopts a right-handed 3(10) helix at the RSTK locus engaging four distinct binding pockets, confirming a novel mode of SH3 recognition.\",\n      \"method\": \"NMR structure determination, mutagenesis, peptide binding assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with mutagenesis validation\",\n      \"pmids\": [\"12620234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"SLP-76 deficiency causes failure of blood-lymphatic vascular separation in mice; SLP-76 is required in hematopoietic cells (not endothelial cells) for this separation, as bone marrow reconstitution with SLP-76-deficient cells confers blood-filled lymphatics in wild-type mice.\",\n      \"method\": \"SLP-76 KO mice, bone marrow reconstitution, vascular histology\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO plus bone marrow reconstitution establishing hematopoietic cell-autonomous mechanism\",\n      \"pmids\": [\"12522250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ADAP binding to SLP-76 differentially regulates peripheral SMAC (pSMAC) formation at the immunological synapse versus T cell-APC conjugation; ADAP-SLP-76 interaction requires specific YDDV sites of ADAP, and loss of this interaction acts as a dominant negative on pSMAC formation and IL-2 production.\",\n      \"method\": \"Mutagenesis of ADAP, immunological synapse imaging, IL-2 production assay, LFA-1 clustering assay\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis plus imaging and functional readout, single lab\",\n      \"pmids\": [\"15477347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TCR-activated reactive oxygen species (ROS) induce transient oxidative inactivation of SHP-2 (but not SHP-1); SHP-2 is recruited to the LAT-Gads-SLP-76 complex and directly regulates phosphorylation of Vav1 and ADAP, with the ADAP-SLP-76 association regulated by SHP-2 in a redox-dependent manner.\",\n      \"method\": \"ROS detection, phosphatase activity assay, co-immunoprecipitation, T cell adhesion assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with redox-dependent functional assay, single lab\",\n      \"pmids\": [\"15933714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SLP-76 forms signaling microclusters at TCR contact sites with APCs containing ZAP-70; microclusters are continuously generated at the periphery, migrate toward the central SMAC, and tyrosine phosphorylation and calcium influx occur as microclusters form. Inhibition of signaling prevents ZAP-70 recruitment into microclusters.\",\n      \"method\": \"Live-cell imaging (TIRF microscopy), fluorescently-tagged SLP-76 and ZAP-70, signaling inhibitors\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct live imaging of protein dynamics with functional correlation, highly cited\",\n      \"pmids\": [\"16273097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CD6 cytoplasmic tail phospho-Y662 directly binds the SH2 domain of SLP-76 (Kd = 0.5 µM at 37°C); this interaction is required for CD6-mediated T cell costimulation.\",\n      \"method\": \"Phosphopeptide binding (equilibrium dissociation constant measured), co-immunoprecipitation, mutagenesis, costimulation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — quantitative binding assay plus mutagenesis and functional validation\",\n      \"pmids\": [\"16914752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SLP-76 forms dynamic membrane clusters upon FcεRI cross-linking in mast cells, colocalizing with FcεRI, Syk, LAT, and phosphotyrosine; disruption of the SLP-76-Gads interaction (by SLP-76 mutation) prevents translocation and clustering, inhibiting calcium flux, degranulation, and cytokine secretion.\",\n      \"method\": \"Confocal real-time imaging, mutagenesis of SLP-76 Gads-binding region, calcium flux, degranulation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct imaging plus mutagenesis and multiple functional readouts\",\n      \"pmids\": [\"16479002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SLP-76 Tyr145 is the most critical N-terminal tyrosine for T cell development and function; Y145 is required for optimal association of SLP-76 with ITK, while Y112/Y128 associate with Vav and Nck and are required for TCR-induced Ca2+ flux and actin reorganization.\",\n      \"method\": \"Individual tyrosine mutagenesis in reconstituted SLP-76-deficient cells and primary T cells, calcium flux, actin assay, co-immunoprecipitation\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic mutagenesis with multiple functional readouts in both cell lines and primary cells\",\n      \"pmids\": [\"16456002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The Gads-binding domain of SLP-76 is required for PLC-γ1 recruitment to GEMs, while the N-terminal tyrosine phosphorylation sites and P-1 region are required for PLC-γ1 phosphorylation at Y783; ITK (associated with SLP-76 via N-terminal tyrosines) efficiently phosphorylates PLC-γ1 at Y783 in vitro.\",\n      \"method\": \"Lipid raft fractionation, PLC-γ1 phosphorylation assay, in vitro kinase assay with ITK, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus mutagenesis and biochemical fractionation with functional readout\",\n      \"pmids\": [\"17148460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"HPK1 phosphorylates SLP-76 at serine 376; this serine phosphorylation mediates recruitment of 14-3-3ε and ζ proteins to SLP-76, which attenuates TCR-induced tyrosine phosphorylation of SLP-76 and PLC-γ1 and reduces IL-2 transcription. S376A mutation or HPK-1 knockdown enhances TCR signaling.\",\n      \"method\": \"RNAi knockdown, in vitro phosphorylation, mutagenesis (S376A), co-immunoprecipitation, IL-2 reporter assay, phosphorylation assay\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus mutagenesis, RNAi, and multiple functional readouts\",\n      \"pmids\": [\"17353368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SLP-76 is required for TCR-induced activation of ITK; ITK bound to SLP-76 represents most of the catalytically active ITK, and ITK activity is lost upon mild elution from the SLP-76 complex but restored upon reconstitution. SLP-76 N-terminal tyrosines are required for ITK activation but not ZAP-70 activation.\",\n      \"method\": \"In vitro kinase assay (ITK vs ZAP-70 on PLC-γ1), co-immunoprecipitation, mutagenesis, reconstitution experiments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus mutagenesis and reconstitution revealing SLP-76 maintains ITK active conformation\",\n      \"pmids\": [\"17420479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The Gads C-SH3 domain binds the SLP-76 RxxK motif with very high affinity (Kd = 8-20 nM); T cell signaling efficiency declines with decreasing Gads-SLP-76 binding affinity, demonstrating a quantitative affinity requirement for efficient TCR signaling.\",\n      \"method\": \"Quantitative affinity measurement, SLP-76 mutants with graded affinities, TCR signaling reporter assays in Jurkat cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — quantitative affinity measurements with functional correlates across multiple mutants\",\n      \"pmids\": [\"17235283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"SLP-76 N-terminal tyrosines Y145 and Y112-128 are required for thymocyte selection but can complement one another in trans (on separate SLP-76 molecules); Y145 differentially supports Itk-dependent pathways while Y112-128 supports Vav1 phosphorylation, demonstrating cooperativity between SLP-76 molecules.\",\n      \"method\": \"Knock-in mice (Y145F and Y112-128F), compound heterozygote analysis, phosphorylation assays, co-immunoprecipitation\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knock-in genetic approach with biochemical and developmental functional readouts\",\n      \"pmids\": [\"18342008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"VLA-4 integrin costimulation retards actin-driven inward flows and prevents centralization of SLP-76 microclusters, prolonging lateral SLP-76/ZAP-70 interactions and retaining SLP-76 in tyrosine-phosphorylated peripheral structures, thereby sustaining T cell signaling.\",\n      \"method\": \"Live TIRF microscopy of SLP-76 microclusters, integrin ligand modulation, actin flow measurement\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct live imaging correlating cytoskeletal dynamics with SLP-76 microcluster behavior and signaling\",\n      \"pmids\": [\"18549800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"SLP-76 mediates 'outside-in' integrin signaling in T cells; SLP-76 relocalizes to integrin-initiated microclusters by a mechanism requiring ADAP binding (not LAT/Gads), distinct from TCR-initiated microcluster assembly, and is required for T cell adhesion to integrin ligands.\",\n      \"method\": \"SLP-76 KO T cells, domain mutagenesis (ADAP-binding vs Gads-binding mutants), microcluster imaging, adhesion assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO plus structure-function mutagenesis and imaging with defined functional phenotype\",\n      \"pmids\": [\"19667077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The SLP-76/Nck/VAV1 complex has defined stoichiometry (one SLP-76, two Nck, two VAV1); a direct Nck-VAV1 interaction via the C-terminal SH3 domain of Nck and the VAV1 N-terminal SH3 domain is required for actin polymerization after T cell activation.\",\n      \"method\": \"Analytical ultracentrifugation, co-immunoprecipitation, mutagenesis of Nck-VAV1 interface, actin polymerization assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — stoichiometry determined by biophysics plus mutagenesis with functional actin polymerization assay\",\n      \"pmids\": [\"20562827\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling; platelet-specific deletion of SLP-76 (via PF4-Cre) is sufficient to cause lymphatic vascular defects, and SLP-76-dependent platelet aggregate formation occurs on lymphatic endothelial cell surfaces.\",\n      \"method\": \"Conditional KO (PF4-Cre x Slp76 flox), in vivo and ex vivo vascular imaging, platelet-LEC co-culture\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO in specific cell type with in vivo and ex vivo mechanistic validation\",\n      \"pmids\": [\"20363774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"HPK1-dependent serine 376 phosphorylation of SLP-76 mediates ubiquitination of SLP-76 at lysine 30, targeting phosphorylated SLP-76 for proteasomal degradation during TCR signaling; K30R mutation enhances anti-CD3-induced ERK and JNK activation.\",\n      \"method\": \"In vitro ubiquitination assay, mutagenesis (S376A, K30R), proteasome inhibition, ERK/JNK activation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro ubiquitination plus mutagenesis and functional signaling readout\",\n      \"pmids\": [\"22902619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In NK cells, SLP-76 Y113 and Y128 are phosphorylated by distinct co-activation receptor pairs (e.g., NKG2D and 2B4), enabling SLP-76 binding to Vav1; combined phosphorylation of both tyrosines is required for synergistic Ca2+ mobilization and NK cell activation.\",\n      \"method\": \"SLP-76 KD and reconstitution with tyrosine mutants, Ca2+ flux assay, cytotoxicity assay, co-immunoprecipitation\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reconstitution with point mutants and multiple functional readouts\",\n      \"pmids\": [\"22786724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SLP-76 and ADAP are required for E-selectin-mediated integrin activation and slow leukocyte rolling in neutrophils; two N-terminal tyrosines and the SH2 domain of SLP-76 are required for downstream signaling, placing BTK and then PI3Kγ/PLCγ2 downstream of SLP-76 in this pathway.\",\n      \"method\": \"Genetically engineered KO mice, transduced primary leukocytes, intravital microscopy, kinase assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with mutagenesis and in vivo functional readout\",\n      \"pmids\": [\"22291096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SLP-76, Nck, and Vav1 form a trimolecular complex in live T cells; Nck-Vav1 dimers are constitutively formed independently of T cell activation and SLP-76-Nck association; after TCR stimulation, SLP-76 phosphorylation enables Nck binding and complex assembly. The Nck-Vav1 interaction requires Vav1 proline-rich/SH3 domain and is critical for actin rearrangement.\",\n      \"method\": \"Triple-color FRET (3FRET) in live T cells, mutagenesis of Vav1 SH3, actin rearrangement assay\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live-cell 3FRET plus mutagenesis and functional actin assay\",\n      \"pmids\": [\"22534133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ADAP contains three binding sites for the SLP-76 SH2 domain; multipoint binding of SLP-76 to ADAP oligomerizes SLP-76 SH2 domain in vitro and is critical for SLP-76 microcluster assembly in stimulated T cells; any combination of two sites partially restores microclusters.\",\n      \"method\": \"Analytical ultracentrifugation, biophysical binding assays, confocal imaging of microclusters, ADAP mutagenesis, functional T cell assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biophysical reconstitution plus cellular imaging and mutagenesis with functional correlates\",\n      \"pmids\": [\"23979596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SLP-76 directly binds the cytosolic tail of RAGE via its SAM (sterile α motif) domain; this interaction mediates downstream p38 MAPK, ERK1/2, and IKKα/β phosphorylation as well as cytokine release in macrophages. TAT-SAM peptide delivery blocks RAGE-SLP-76 interaction and attenuates sepsis in mice.\",\n      \"method\": \"Co-immunoprecipitation in vitro and in vivo, genetic deficiency of RAGE/SLP-76, TAT-peptide cell delivery, CLP sepsis model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus genetic KO and peptide intervention with in vivo functional validation\",\n      \"pmids\": [\"33436632\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SLP-76 (LCP2) is a hematopoietic-specific multi-domain scaffold/adaptor protein (N-terminal acidic tyrosine-containing region, central proline-rich region, C-terminal SH2 domain) that is phosphorylated by ZAP-70 (and ITK/Rlk) at Y112, Y128, and Y145 downstream of ITAM-containing receptors (TCR, FcεRI, GPVI, FcγR) and integrins; it is recruited to membrane microdomains via constitutive association with Gads (which bridges it to LAT) and assembles a central signalosome with Nck, Vav1, and ITK to activate PLC-γ1, actin remodeling (via WASP/Arp2/3), Ras/ERK, Ca2+/NFAT, and NF-κB pathways, while being negatively regulated by HPK1-mediated S376 phosphorylation that recruits 14-3-3 proteins and triggers K30 ubiquitination and proteasomal degradation of SLP-76, and by SHP-1-mediated dephosphorylation downstream of inhibitory receptors.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SLP-76 (LCP2) is a hematopoietic-specific multi-domain scaffold protein that nucleates signalosome assembly downstream of ITAM-containing receptors (TCR, FcεRI, FcγR, GPVI, CLEC-2) and integrins in T cells, platelets, mast cells, NK cells, and neutrophils. ZAP-70/Syk phosphorylates SLP-76 at N-terminal tyrosines Y112, Y128, and Y145, creating docking sites for Vav1 and Nck (Y112/Y128) and ITK (Y145), thereby coordinating PLC-γ1/γ2 activation, Ca²⁺ mobilization, Ras/ERK signaling, NF-AT transcription, and actin cytoskeleton remodeling via WASP/Arp2/3 [PMID:8892604, PMID:16456002, PMID:17420479, PMID:9846482]. SLP-76 is constitutively bridged to LAT-containing membrane microdomains through Gads binding to a central RxxK motif, and it additionally recruits ADAP via its C-terminal SH2 domain to drive integrin activation and microcluster oligomerization [PMID:11239162, PMID:23979596, PMID:19667077]. SLP-76 is negatively regulated by HPK1-mediated S376 phosphorylation, which recruits 14-3-3 proteins and triggers K30 ubiquitination and proteasomal degradation, and by SHP-1-mediated dephosphorylation downstream of inhibitory receptors; SLP-76 knockout in mice causes a complete block in T cell development and defective blood-lymphatic vascular separation through platelet CLEC-2/PDPN signaling [PMID:17353368, PMID:22902619, PMID:9765283, PMID:9665885, PMID:20363774].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Molecular cloning of SLP-76 established the existence of a novel hematopoietic adaptor protein with an SH2 domain capable of engaging tyrosine-phosphorylated signaling partners including PLC-γ1-associated proteins, opening the question of its receptor context and upstream regulation.\",\n      \"evidence\": \"Cloning from IL-2-stimulated T cells with GST pulldown and co-immunoprecipitation\",\n      \"pmids\": [\"7706237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the 62 kDa and 130 kDa SH2 domain-associated proteins unknown\", \"Upstream kinase not identified\", \"In vivo function unknown\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Identification of ZAP-70 as the kinase phosphorylating SLP-76 at Y113, Y128, and Y145, and discovery that phospho-SLP-76 recruits Vav via its SH2 domain to synergistically activate NF-AT, established SLP-76 as a TCR-proximal signaling node linking receptor engagement to transcription factor activation and identified the key phosphotyrosine residues.\",\n      \"evidence\": \"In vitro kinase assays, site-directed mutagenesis, NF-AT reporter assays, co-immunoprecipitation in Jurkat T cells\",\n      \"pmids\": [\"8702662\", \"8892604\", \"8673706\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which tyrosine recruits which effector not yet resolved\", \"In vivo requirement for SLP-76 not tested\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Genetic knockout of SLP-76 revealed it is essential for T cell development (complete thymopoiesis block), platelet collagen-receptor signaling (PLC-γ2 activation, aggregation), and that SLP-76 organizes a trimolecular complex with Nck and Vav to activate PAK1 and actin polymerization, transforming the view of SLP-76 from a simple adaptor to a multifunctional signalosome scaffold.\",\n      \"evidence\": \"SLP-76 KO mice (two independent labs), platelet aggregation/degranulation assays, co-IP and PAK1 kinase assays\",\n      \"pmids\": [\"9665885\", \"9695951\", \"9884330\", \"9846482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of SLP-76 membrane recruitment unknown\", \"Role in other hematopoietic lineages not yet explored\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Discovery that Gads constitutively bridges SLP-76 to LAT and identification of ADAP (SLAP-130), HPK1, and Nck as SLP-76 SH2 domain or phosphotyrosine-dependent interactors expanded the signalosome model and revealed that SLP-76 serves as a hub integrating both positive (Nck, Vav) and negative (HPK1, ADAP) regulators.\",\n      \"evidence\": \"Co-IP and domain mapping in T cells and mast cells, NF-AT reporter assays, yeast two-hybrid, SLP-76 KO mast cell functional assays\",\n      \"pmids\": [\"10021361\", \"10377180\", \"10229072\", \"11487585\", \"9115214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Gads-SLP-76 interaction unknown\", \"Mechanism of HPK1-mediated negative regulation unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrating that SLP-76 recruitment to glycolipid-enriched membrane microdomains (GEMs) is essential for TCR signaling and that SLP-76 coordinates actin remodeling via WASP/ADAP/Ena-VASP at the immunological synapse established the spatial dimension of SLP-76 function.\",\n      \"evidence\": \"LAT/SLP-76 chimeric protein reconstitution in LAT-deficient cells, GEM fractionation, confocal imaging of T cell-APC contacts\",\n      \"pmids\": [\"11015445\", \"10747096\", \"11113155\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamic behavior of SLP-76 at the synapse not resolved\", \"How integrin signals feed into SLP-76 not clear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Mapping of a direct SLP-76–PLC-γ1 SH3 domain interaction via a proline-rich P-1 region and confirmation that GADS KO uncouples SLP-76 from LAT completed the minimal wiring diagram of the LAT-Gads-SLP-76-PLC-γ1 signalosome.\",\n      \"evidence\": \"GADS KO mice with co-IP, SLP-76 domain mutagenesis and reconstitution in SLP-76-deficient cells, functional reporter assays\",\n      \"pmids\": [\"11390650\", \"11239162\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative affinity requirements for each interaction not defined\", \"Stoichiometry of the complex unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Crystal and NMR structures of the Gads C-SH3/SLP-76 RSTK peptide complex revealed a novel non-proline-based SH3 recognition mode (3₁₀ helix), providing the first atomic-level understanding of a key SLP-76 interaction; separately, blood-lymphatic separation failure in SLP-76 KO mice disclosed an unexpected developmental role in a hematopoietic cell-autonomous vascular process.\",\n      \"evidence\": \"X-ray crystallography (1.7 Å), NMR solution structure, bone marrow reconstitution in KO mice, vascular histology\",\n      \"pmids\": [\"12773374\", \"12620234\", \"12522250\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the hematopoietic cell type mediating vascular separation unknown\", \"Full-length SLP-76 structure unavailable\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Live-cell TIRF imaging showed SLP-76 forms dynamic signaling microclusters at TCR contact sites that migrate centripetally; this established that signaling is organized in discrete membrane-associated clusters rather than uniformly distributed, with ZAP-70 co-recruited and signaling correlated with cluster formation.\",\n      \"evidence\": \"TIRF microscopy with fluorescently tagged SLP-76 and ZAP-70 in T cell-APC conjugates\",\n      \"pmids\": [\"16273097\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular determinants of microcluster formation not identified\", \"Role of ADAP in cluster assembly not yet explored\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Systematic tyrosine mutagenesis resolved the division of labor among SLP-76 N-terminal tyrosines: Y145 recruits ITK for PLC-γ1 phosphorylation, while Y112/Y128 recruit Vav1 and Nck for Ca²⁺ flux and actin reorganization, and the Gads-binding domain delivers PLC-γ1 to membrane microdomains for phosphorylation by ITK.\",\n      \"evidence\": \"Individual tyrosine-to-phenylalanine mutants in SLP-76-deficient cells and primary T cells, lipid raft fractionation, in vitro ITK kinase assay\",\n      \"pmids\": [\"16456002\", \"17148460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Y145 directly contacts ITK SH2 domain not structurally confirmed\", \"Redundancy between Y112 and Y128 not fully resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"HPK1 was identified as the kinase phosphorylating SLP-76 S376, which recruits 14-3-3 proteins to attenuate TCR signaling, and SLP-76 was shown to allosterically maintain ITK in its catalytically active conformation, establishing SLP-76 as both a positive allosteric activator and a target of negative feedback.\",\n      \"evidence\": \"In vitro kinase assays, S376A mutagenesis, RNAi of HPK1, ITK activity reconstitution upon elution/rebinding to SLP-76\",\n      \"pmids\": [\"17353368\", \"17420479\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase mediating SLP-76 ubiquitination not identified\", \"Structural basis for ITK allosteric activation unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Biophysical determination of a 1:2:2 SLP-76/Nck/Vav1 stoichiometry and demonstration that platelet-specific SLP-76 deletion (via PF4-Cre) suffices to cause lymphatic vascular defects via CLEC-2/PDPN signaling resolved both the molecular architecture of the actin-regulatory complex and the cell type responsible for blood-lymphatic separation.\",\n      \"evidence\": \"Analytical ultracentrifugation, conditional KO mice, platelet-lymphatic endothelial cell co-culture\",\n      \"pmids\": [\"20562827\", \"20363774\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full structural model of the pentameric complex not available\", \"Downstream effectors of CLEC-2/SLP-76 in vascular separation not fully mapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"HPK1-dependent S376 phosphorylation was linked to K30 ubiquitination and proteasomal degradation of SLP-76, completing the negative feedback circuit; in parallel, ADAP was shown to oligomerize SLP-76 via multipoint SH2 domain binding, providing a mechanism for microcluster assembly, and SLP-76 tyrosine phosphorylation was shown to integrate co-activation receptor signals in NK cells.\",\n      \"evidence\": \"In vitro ubiquitination assays with K30R mutant, analytical ultracentrifugation of ADAP-SLP-76, confocal imaging of microclusters, NK cell reconstitution with tyrosine mutants\",\n      \"pmids\": [\"22902619\", \"23979596\", \"22786724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the E3 ubiquitin ligase targeting K30 still unknown\", \"Whether ADAP-mediated oligomerization occurs in non-T cells not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovery that SLP-76 directly binds the RAGE cytoplasmic tail via its SAM domain to drive p38/ERK/IKK signaling and cytokine release in macrophages extended SLP-76 function beyond ITAM/integrin-receptor pathways to a pattern-recognition receptor axis relevant to sepsis.\",\n      \"evidence\": \"Co-IP in macrophages, RAGE/SLP-76 genetic deficiency, TAT-SAM peptide inhibition, CLP sepsis model in mice\",\n      \"pmids\": [\"33436632\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the SAM domain interaction is direct or requires post-translational modification not established\", \"Relevance to human sepsis not validated\", \"Structural basis of RAGE-SAM interaction unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length structure of SLP-76 in complex with its signalosome partners, the identity of the E3 ubiquitin ligase for K30 ubiquitination, and the mechanism by which SLP-76 allosterically activates ITK remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length SLP-76 structure available\", \"E3 ligase for K30 ubiquitination unidentified\", \"Structural mechanism of ITK allosteric activation by SLP-76 unknown\", \"Role of SLP-76 SAM-RAGE axis in other inflammatory contexts untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 9, 14, 24, 41, 46]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [22, 35, 37, 44, 52]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 4, 10, 15, 28, 38, 49]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 40, 41, 53]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [13, 16, 23, 47]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [10, 32, 47]}\n    ],\n    \"complexes\": [\n      \"LAT-Gads-SLP-76 signalosome\",\n      \"SLP-76/Nck/Vav1 trimolecular complex\",\n      \"SLP-76/ITK/PLC-γ1 complex\"\n    ],\n    \"partners\": [\n      \"GRAP2\",\n      \"VAV1\",\n      \"NCK1\",\n      \"ITK\",\n      \"PLCG1\",\n      \"FYB1\",\n      \"ZAP70\",\n      \"HPK1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}