coYki (CAOG_07866) is the single Yorkie/YAP/TAZ ortholog of the amoeboid filasterean Capsaspora owczarzaki, a close unicellular relative of animals that can form calcium-dependent multicellular aggregates. The protein carries the hallmarks of the animal Hippo-pathway transcriptional coactivators: an N-terminal TEAD-binding region, four HXRXXS Warts/LATS phosphorylation motifs (three aligned with sites in YAP and Yorkie) and two WW domains. Expressed in Drosophila together with the Capsaspora Scalloped/TEAD ortholog (coSd), coYki co-immunoprecipitates with coSd, activates a Hippo-responsive diap1 enhancer reporter and endogenous Diap1 and Expanded, and drives eye overgrowth, and the Capsaspora Hippo kinase (coHpo) promotes its phosphorylation and inhibits its activity. In Capsaspora cells, tagged coYki is mostly cytoplasmic; it becomes nuclear when its phosphorylation motifs are mutated or when the Hippo (coHpo) or Warts (coWts) kinase is deleted, so the kinase cascade regulates it by cytoplasmic sequestration as in animals. Unlike its animal counterparts, coYki is not needed for cell proliferation. Instead, cells lacking it show excessive actin-depleted membrane blebbing, increased adhesion to the substrate, and flattened, less circular aggregates, and about 1,200 genes change expression, with genes for actin-binding proteins enriched among those up-regulated. A non-phosphorylatable, hyperactive coYki increases cell packing in aggregates and elongates adherent cells. This effect requires a conserved residue of the TEAD-binding interface, so coYki shapes cell and aggregate morphology by acting as a transcriptional coactivator.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003713 transcription coactivator activity | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer from the PANTHER YAP1/Yorkie family node (PTHR17616:SF8, "TRANSCRIPTIONAL COACTIVATOR YORKIE"). Supported directly for the Capsaspora protein by heterologous assays: coYki and coSd co-immunoprecipitate and together activate a Hippo-responsive diap1 enhancer (HRE) luciferase reporter in Drosophila S2R+ cells and the endogenous targets Diap1 and Ex in the fly eye [PMID:22832104]. In Capsaspora, the phenotypes of hyperactive coYki 4SA are abolished by mutating the residue aligned with YAP1 F95, which YAP needs to bind TEAD, so they depend on transcriptional activity [PMID:38517944]. Reason: The GO definition (activating transcription of specific genes by binding a DNA-binding transcription factor) matches what was shown: coYki binds the DNA-binding transcription factor coSd and the pair activates a target enhancer, and coYki alone does not activate transcription in the fly eye. The cross-species reporter result shows the activity of the Capsaspora protein. The TEAD-interface mutant then shows that the activity is used in Capsaspora itself. This is the core molecular function of coYki. It is a biochemical activity, not an animal-specific process, so placing it at the holozoan-level PANTHER node is phylogenetically sound. Supporting Evidence: PMID:22832104 co-expression of Co-Sd and Co-Yki stimulated the transcription of the HRE-luciferase reporter in Drosophila S2R+ cells PMID:22832104 these data demonstrate the ability of Co-Sd and Co-Yki to form a functional transcription factor complex with striking specificity to activate similar target genes as its Drosophila counterpart PMID:38517944 This result indicates that these phenotypes are mediated by the transcriptional activity of coYki. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Combined ARBA, subcellular-location and TreeGrafter inference. In Capsaspora, mScarlet-coYki is depleted from the nucleus in wild-type cells but is nuclear at or above cytoplasmic levels in most cells lacking coHpo or coWts, and when its four HXRXXS motifs are mutated (4SA) [PMID:35659869, PMID:38517944]. Reason: A coactivator must enter the nucleus to act, and the data show that coYki does so, under kinase control. The nucleus is where coYki carries out its function, even though at steady state in wild-type adherent cells most of the protein is cytoplasmic. Accepted. The supporting observations are of transiently expressed fusion proteins, not endogenous protein. Supporting Evidence: PMID:38517944 the majority of coHpo-/- and coWts-/- cells showed nuclear levels of mScarlet-coYki greater than or equal to that seen in the cytoplasm PMID:35659869 the majority of cells transfected with mScarlet-coYki 4SA showed uniform mScarlet signal throughout the cell or enriched mScarlet signal in the nucleus |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Subcellular-location mapping and TreeGrafter inference. Directly observed in Capsaspora: mScarlet-coYki is enriched in the cytoplasm relative to the nucleus in wild-type cells, and this cytoplasmic retention depends on the HXRXXS motifs and on the coHpo and coWts kinases [PMID:35659869, PMID:38517944]. Reason: The predominant location of coYki in wild-type cells. Keeping it in the cytoplasm is how the Hippo kinase cascade inhibits it, so the term reflects regulated sequestration, not a site of activity. Retained alongside nucleus. The evidence comes from tagged, overexpressed protein. The protein's location under different cell densities or in aggregates has not been reported. Supporting Evidence: PMID:35659869 Cells transfected with mScarlet-coYki showed mScarlet enriched in the cytoplasm relative to the nucleus PMID:38517944 In WT cells, mScarlet-coYki was predominantly cytoplasmic |
| GO:0035329 hippo signaling | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer from the Yorkie family node. GO defines hippo signaling as the cascade in which Hippo phosphorylates LATS/Warts, which in turn phosphorylates YAP1/Yki, causing its cytosolic retention. In Capsaspora this cascade has been shown genetically. Deleting coHpo or coWts makes coYki nuclear. The 4SA mutant also becomes nuclear, and a stable 4SA transgene reproduces the cell-packing and cell-elongation phenotypes of the kinase mutants [PMID:38517944]. In Drosophila cells, coHpo induces coYki phosphorylation, which coexpressed Wts enhances [PMID:22832104]. Reason: Here the IEA is borne out experimentally in the organism itself. The term names the Yorkie/YAP effector in its definition, and it is not restricted to animals or tied to growth control. Its parents are signal-transduction terms, not multicellular or developmental processes. It is therefore not a TAXON_CONSTRAINT_VIOLATION of the kind the propagation audit looks for. Comparator check: human YAP1 (P46937) and Drosophila yki (Q45VV3) both carry GO:0035329 by experimental evidence (IDA/IMP), so annotating the terminal effector is established practice. coYki takes part in the cascade: it is the phosphorylated target whose localization and activity carry the signal, and the phenotypes of the kinase mutants are reproduced by hyperactive coYki. What the pathway regulates in Capsaspora differs from animals (cytoskeleton and aggregate cell packing, not proliferation). That does not affect this signaling-cascade term. Supporting Evidence: PMID:38517944 Together, these results show that the Capsaspora Hippo kinase cascade regulates coYki by cytoplasmic sequestration, indicating a premetazoan origin of this regulatory activity of the Hippo pathway. PMID:38517944 These results suggest that the phenotypes observed in coHpo and coWts mutants are due to coYki activation resulting from a loss of upstream kinase signaling. PMID:22832104 expression of Co-Hpo induced phosphorylation of Co-Yki in S2R+ cells, and this phosphorylation was further enhanced by co-expression of Dm-Wts |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer. Paired with coSd, coYki activates transcription of an RNA polymerase II-transcribed Hippo-responsive reporter and of the endogenous genes Diap1 and Ex in Drosophila [PMID:22832104]. In Capsaspora, coYki loss changes the expression of 1205 genes (397 down, 808 up) [PMID:35659869], and the phenotypes of hyperactive coYki depend on its transcriptional activity [PMID:38517944]. Reason: This process follows directly from the coactivator activity. coYki does part of the work by bringing activation capacity to the TEAD-family factor coSd: coSd alone does not drive the targets, and the complex does. The Capsaspora RNA-seq shows coYki-dependent gene expression in vivo, but cannot separate direct from indirect targets, and more genes go up than down in the mutant. The endogenous genes that coYki activates directly in Capsaspora are not yet known (no ChIP or CUT&RUN). The annotation stands on the heterologous reporter data and the transcription-dependence of the hyperactive allele. Supporting Evidence: PMID:22832104 Third instar eye imaginal discs of GMR>Co-Sd+Co-Yki animals showed a marked upregulation of Diap1 and Ex staining posterior to the morphogenetic furrow PMID:35659869 This analysis revealed 1205 differentially expressed genes, including 397 downregulated PMID:38517944 in contrast to coYki 4SA, the expression of a coYki 4SA F123A mutant did not affect cell or aggregate morphology |
| GO:0140297 DNA-binding transcription factor binding | IPI PMID:22832104 Premetazoan origin of the hippo signaling pathway. | NEW | Summary: Epitope-tagged coYki (HA-CoYki) and coSd (FLAG-CoSd), the Capsaspora Scalloped/TEAD ortholog, co-immunoprecipitate when expressed in Drosophila S2R+ cells [PMID:22832104]. The two proteins also act together in vivo: coYki drives eye overgrowth with coSd but not with Drosophila Sd. In Capsaspora, mutating the coYki residue aligned with the TEAD-binding residue F95 of YAP1 abolishes the activity of hyperactive coYki [PMID:38517944]. Reason: GOA has no experimental annotation for this protein. The co-IP is a direct interaction assay between two proteins from the same species, so IPI with coSd is the right evidence type. Binding to a DNA-binding transcription factor is also how coYki carries out its coactivator activity, and the residue-level mutant data in Capsaspora support the same interface. The more specific functional term is already captured by transcription coactivator activity. This binding term records the evidence, and is used instead of the uninformative "protein binding". The assay used heterologous (fly) cells and overexpressed tagged proteins, and the complex has not yet been isolated from Capsaspora. Supporting Evidence: PMID:22832104 epitope-tagged Co-Sd and Co-Yki immunoprecipitated with each other in Drosophila S2R+ cells (Figure 4A), demonstrating their ability to form a protein complex PMID:22832104 For immunoprecipitation, HA-CoYki and FLAG-CoSd were constructed in the pAc5.1/V5-HisB vector PMID:38729842 coYki and the single Capsaspora Sd/TEAD ortholog (coSd) can bind each other, showing deep conservation of this heterodimer |
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Download this section (compressed HTML)Q: Does coYki bind coSd in Capsaspora cells, and which genomic sites does the complex occupy? Which of the 1205 coYki-dependent genes are direct targets, given that more genes are up-regulated than down-regulated in the knockout?
Q: Which coYki-regulated genes produce the blebbing and cell-substrate adhesion phenotypes? The candidates include the up-regulated actin-binding genes and the integrin-beta genes CAOG_05058 and CAOG_01283.
Q: Is coYki localization regulated by cell density, substrate attachment, or the transition into aggregates, as YAP/TAZ localization is regulated by contact and mechanics in animal cells?
Q: The papers number the TEAD-interface phenylalanine of coYki as F123 (aligned with YAP1 F95), but in the UniProt model A0A0D2WY30 the YAP1-like LPxSFF motif lies at residues 54-59. Is the UniProt gene model N-terminally truncated relative to the RACE-checked cDNA used experimentally?
Q: Does coYki also act at the cell cortex independently of transcription, as reported for Drosophila Yorkie? The F123A result argues against this for the 4SA phenotypes, but it was not tested for the loss-of-function blebbing phenotype.
Experiment: Endogenously tag coYki (and coSd) by homologous recombination and perform co-IP from Capsaspora lysates, plus CUT&RUN or ChIP-seq. Intersect the binding sites with the coYki, coHpo and coWts RNA-seq gene sets to define direct targets.
Hypothesis: coYki and coSd form a complex at target genes in Capsaspora.
Type: protein interaction / chromatin profiling
Experiment: Complement coYki -/- cells with wild-type coYki versus the TEAD-interface mutant (F123A, paper numbering) and score bleb frequency, cell-substrate adhesion and aggregate circularity.
Hypothesis: coYki suppresses blebbing through TEAD-dependent transcription.
Type: genetic complementation
Experiment: Image a stably expressed, low-level tagged coYki (or use an antibody against the endogenous protein) in sparse versus confluent adherent cells and in aggregates, and quantify the nuclear/cytoplasmic ratio. Compare with coHpo and coWts mutants.
Hypothesis: coYki nuclear localization responds to cell density or aggregation.
Type: localization / imaging
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