id: A0A8M6YYZ5 gene_symbol: magi3b product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:7955 label: Danio rerio description: 'magi3b (NCBI LOC564220 in older builds; ZFIN ZDB-GENE-060503-301, chromosome 6) is one of two zebrafish co-orthologs of vertebrate MAGI3; PANTHER places the duplication at the teleost genome duplication. Its paralog is wu:fi36a10 (magi3a in NCBI RefSeq). It encodes a cytoplasmic membrane-associated guanylate kinase with inverted domain order (MAGI): an N-terminal PDZ domain, a guanylate kinase-like (GK) domain that lacks the nucleotide-binding P-loop and is therefore a protein-interaction module rather than an enzyme, two WW domains, five further PDZ domains and a long disordered C-terminal tail. MAGI proteins act as scaffolds at the cytoplasmic face of cell-cell junctions, where their PDZ domains bind C-terminal motifs of receptors and signalling proteins. In zebrafish, magi3b is expressed in endothelial cells, and F0 crispants for magi3b, like those for wu:fi36a10, have reduced brain vascularization that phenocopies loss of the C-terminal ETTV PDZ-binding motif of the Wnt7 co-receptor Gpr124. A morpholino screen reported shorter kidney motile cilia after knockdown of a magi3 gene attributed to this locus. Public RNA-Seq calls are most prominent in retina, early embryo and ovary, and are narrower than those of wu:fi36a10.' existing_annotations: - term: id: GO:0000166 label: nucleotide binding evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: enables supporting_entities: - UniRule:UR000400325 review: summary: Nucleotide binding from UniRule UR000400325 (MAGUK guanylate kinase-like domain). action: REMOVE reason: 'UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in magi3b (as in wu:fi36a10, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain.' supported_by: - &id001 reference_id: PMID:37163606 supporting_text: Unlike other MAGUK GKs, which are composed of three subdomains including guanosine monophosphate (GMP)–binding, LID, and CORE subdomains, MAGI GKs lack the last two subdomains - &id002 reference_id: file:DANRE/wu_fi36a10/wu_fi36a10-bioinformatics/RESULTS.md supporting_text: Neither zebrafish copy, nor gar or human MAGI3, has a Walker A (P-loop) motif at the position UniProt annotates as the ATP-binding site - term: id: GO:0005524 label: ATP binding evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: enables supporting_entities: - UniRule:UR000400325 review: summary: ATP binding from UniRule UR000400325, based on the predicted P-loop of the GK-like domain. action: REMOVE reason: 'UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in magi3b (as in wu:fi36a10, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain.' supported_by: - *id001 - *id002 - term: id: GO:0005737 label: cytoplasm evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in supporting_entities: - MGI:MGI:1203522 - MGI:MGI:1354953 - PANTHER:PTN000817101 - RGD:621855 - UniProtKB:Q86UL8 - UniProtKB:Q96QZ7 - ZFIN:ZDB-GENE-060503-301 review: summary: Cytoplasm (IBA, PANTHER PTN000817101 MAGI node). action: ACCEPT reason: MAGI proteins are cytoplasmic scaffolds at the inner face of the plasma membrane. magi3b is inside the MAGI clade and keeps all its domains. The target appearing in WITH/FROM (ZFIN:ZDB-GENE-060503-301) reflects its own IDA from PMID:25139857, which is expected for a PAINT node, not circular. supported_by: - &id003 reference_id: PMID:25139857 supporting_text: whereas the remainder appeared to localize to a combination of the cytoplasm, nucleus and cell membrane - term: id: GO:0005737 label: cytoplasm evidence_type: IDA original_reference_id: PMID:25139857 qualifier: located_in review: summary: GFP-tagged zebrafish magi3 localized to cytoplasm (plus nucleus and membrane) in Kupffer's vesicle cells; not ciliary. action: ACCEPT reason: Direct localization of the tagged zebrafish protein in the embryo. It is a coarse location, but it is correct and consistent with the IBA. supported_by: - *id003 - term: id: GO:0005886 label: plasma membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0039 review: summary: Plasma membrane (UniProt subcellular location, peripheral membrane protein). action: ACCEPT reason: MAGI3 is a peripheral protein on the cytoplasmic face of the plasma membrane at cell-cell junctions. In zebrafish it binds the C terminus of the transmembrane receptor Gpr124. supported_by: - &id004 reference_id: PMID:10748157 supporting_text: localizes to epithelial cell tight junctions - &id005 reference_id: PMID:35649360 supporting_text: This observation is compatible with a role for Dlg4 and Magi3 as Gpr124 ETTV adaptor proteins, acting in parallel to Dvl. - term: id: GO:0005911 label: cell-cell junction evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in supporting_entities: - MGI:MGI:1203522 - MGI:MGI:1923484 - PANTHER:PTN002569180 - RGD:621855 - UniProtKB:Q86UL8 - UniProtKB:Q96QZ7 review: summary: Cell-cell junction (IBA, PANTHER PTN002569180). action: ACCEPT reason: Junctional localization is the conserved property of MAGI1-3. magi3b keeps the full MAGI domain set, and nothing argues for loss of the function. This is the correct level of specificity for a family-level inference. supported_by: - *id004 - term: id: GO:0005923 label: bicellular tight junction evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0265 review: summary: Bicellular tight junction (UniProt subcellular location). action: ACCEPT reason: Human MAGI3 localizes to epithelial tight junctions. There is no zebrafish test of this, but the domain architecture is conserved. It is a reasonable transfer, more specific than the IBA cell-cell junction. supported_by: - *id004 - term: id: GO:0007165 label: signal transduction evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in supporting_entities: - MGI:MGI:1354953 - PANTHER:PTN000817101 - RGD:621855 review: summary: Signal transduction (IBA, PANTHER PTN000817101). action: ACCEPT reason: 'Very general, but correct. MAGI3 scaffolds signalling complexes: PTEN and AKT in mammals, and in zebrafish the Gpr124 ETTV motif in the Wnt7-dependent brain angiogenesis pathway, where magi3b crispants phenocopy loss of that motif. A more specific process term would need evidence beyond F0 crispants.' supported_by: - reference_id: PMID:10748157 supporting_text: cooperate to modulate the kinase activity of AKT/PKB - *id005 - &id007 reference_id: PMID:35649360 supporting_text: Targeting magi3a, magi3b, or dlg4b, but not dlg4a, partially impaired brain vascularization (Figures 4H and 4I), in agreement with the expression data. - term: id: GO:0016301 label: kinase activity evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: enables supporting_entities: - UniRule:UR000400325 review: summary: Kinase activity from UniRule UR000400325. action: REMOVE reason: 'UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in magi3b (as in wu:fi36a10, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain.' supported_by: - *id001 - *id002 - term: id: GO:0016740 label: transferase activity evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: enables supporting_entities: - UniRule:UR000400325 review: summary: Transferase activity from UniRule UR000400325 (parent of kinase activity). action: REMOVE reason: 'UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in magi3b (as in wu:fi36a10, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain.' supported_by: - *id001 - *id002 - term: id: GO:0044782 label: cilium organization evidence_type: IMP original_reference_id: PMID:25139857 qualifier: acts_upstream_of_or_within supporting_entities: - ZFIN:ZDB-MRPHLNO-141110-18 review: summary: Morpholino knockdown of a magi3 gene shortened kidney motile cilia in a Foxj1-target screen. action: KEEP_AS_NON_CORE reason: The evidence is a single morpholino in a 50-gene screen, with no mutant, rescue or second morpholino in the main text. The locus assignment is ZFIN's. MAGI3 is not a known ciliary protein in other species, and the tagged protein was not ciliary. Kept as a curator-made, uncorroborated non-core annotation. cilium organization is the parent of the co-annotated cilium assembly. supported_by: - &id006 reference_id: PMID:25139857 supporting_text: We found that the loss of six genes – BX470211.1, Dr.81747 (GLB1L2 in human), ect2l, magi3, si:ch211-71m22.1 and si:dkey-26i13.8 (KIF18B in human) – showed a significant (P<5.0×10−7, Student's t-test, two-tailed) reduction in the length of motile cilia in the kidney tubules - reference_id: PMID:25139857 supporting_text: We tested the role of the 50 randomly selected FIGs in cilia formation and function by injecting antisense morpholinos into zebrafish embryos - *id003 - term: id: GO:0060271 label: cilium assembly evidence_type: IMP original_reference_id: PMID:25139857 qualifier: acts_upstream_of_or_within supporting_entities: - ZFIN:ZDB-MRPHLNO-141110-18 review: summary: 'Same morphant: shorter motile cilia in kidney tubules.' action: KEEP_AS_NON_CORE reason: Same single-morpholino evidence as the cilium organization row. There is no mutant confirmation, and the mechanism may be indirect (the protein is cytoplasmic, not ciliary). Not a core function. Do not propagate it to wu:fi36a10 or to human MAGI3 without mutant data. supported_by: - *id006 - *id003 references: - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000104 title: Electronic Gene Ontology annotations created by transferring manual GO annotations between related proteins based on shared sequence features findings: [] - id: PMID:25139857 title: Systematic discovery of novel ciliary genes through functional genomics in the zebrafish. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text. The screen gene is called 'magi3'; ZFIN assigned the morpholino to magi3b (ZDB-GENE-060503-301). Single-morpholino screen evidence. - id: PMID:35649360 title: An integrated model for Gpr124 function in Wnt7a/b signaling among vertebrates. findings: - statement: F0 crispants of magi3b (and of magi3a/wu:fi36a10) partially impair brain vascularization, non-additively, phenocopying Gpr124 dETTV. supporting_text: Targeting magi3a, magi3b, or dlg4b, but not dlg4a, partially impaired brain vascularization (Figures 4H and 4I), in agreement with the expression data. reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text. The paper's magi3a/magi3b names follow NCBI; its probe primers map to wu:fi36a10 and magi3b respectively (see RESULTS.md). - id: PMID:37163606 title: Phosphorylation-dependent recognition of diverse protein targets by the cryptic GK domain of MAGI MAGUKs. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Structural work on MAGI2 GK; states that MAGI GKs lack the LID and CORE subdomains. Family-level background. - id: PMID:10748157 title: Interaction of the tumor suppressor PTEN/MMAC with a PDZ domain of MAGI3, a novel membrane-associated guanylate kinase. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Abstract only; human MAGI3 at tight junctions, binds PTEN. - id: file:DANRE/wu_fi36a10/wu_fi36a10-bioinformatics/RESULTS.md title: magi3b / wu:fi36a10 protein, expression, synteny and primer analyses reference_review: relevance: HIGH correctness: VERIFIED review_notes: Manual audit verified the sidecar maps PMID:35649360 primers to wu:fi36a10 and LOC564220, confirms both MAGI3 copies retain the PDZ/WW/GK-like domain set, and argues against kinase, ATP-binding and nucleotide binding annotations. findings: - statement: Primers of PMID:35649360 identify its magi3b as ZFIN magi3b (LOC564220). supporting_text: the magi3b primers match magi3b (LOC564220) with 0 mismatches core_functions: - description: Cytoplasmic PDZ/WW scaffold at cell-cell junctions. In endothelial cells it acts, with its paralog, as an adaptor for the C-terminal ETTV motif of the Wnt7 co-receptor Gpr124 during brain angiogenesis. Inferred from crispant epistasis and family biochemistry. directly_involved_in: - id: GO:0007165 label: signal transduction locations: - id: GO:0005911 label: cell-cell junction - id: GO:0005886 label: plasma membrane supported_by: - *id005 - *id007 - reference_id: PMID:35649360 supporting_text: dlg4b, magi3a, and magi3b transcripts could readily be amplified, while dlg4a transcripts were not (Figure S2B). suggested_questions: - question: Do stable magi3b mutants (ideally RNA-less, to avoid transcriptional adaptation of wu:fi36a10) reproduce the brain vascularization defect seen in F0 crispants? - question: Does Magi3b bind the Gpr124 ETTV motif directly, as shown for Magi3a by yeast two-hybrid? - question: Is the kidney cilia phenotype of the magi3 morphant reproduced by a magi3b mutant, and which paralog did the morpholino actually target? suggested_experiments: - hypothesis: magi3b and wu:fi36a10 are both required, non-redundantly and dose-dependently, for Gpr124-dependent brain angiogenesis. description: Generate stable single, double and compound-heterozygous mutants and count hindbrain central arteries; test cross-rescue by endothelial (kdrl promoter) expression of each coding sequence. experiment_type: genetic interaction and cross-rescue - hypothesis: The restricted expression of magi3b relative to wu:fi36a10 reflects expression partition, not reduced function. description: Single-cell or FACS-sorted RNA-seq and in situ hybridization for both paralogs in endothelium, retina and kidney, compared with gar magi3. experiment_type: expression profiling