warts (PTSG_04961) encodes the Warts/LATS-family (NDR subgroup, AGC group) serine/threonine protein kinase of the colonial choanoflagellate Salpingoeca rosetta. The 833-residue protein has a long, largely disordered N-terminal region, a C-terminal protein kinase domain (residues 456-763) with the conserved ATP-binding lysine and catalytic aspartate and an insert between the two kinase lobes, and an AGC-kinase C-terminal extension. It is the S. rosetta member of the choanoflagellate-plus-animal Warts clade that includes human LATS1/2 and Drosophila Warts. In animals Warts/LATS, activated by Hippo, phosphorylates the coactivator Yorkie/YAP on HXRXXS motifs and keeps it out of the nucleus; the S. rosetta Yorkie homolog carries four such motifs. In S. rosetta, warts knockout cells proliferate more slowly than wild type, the reverse of the animal overgrowth phenotype, yet form rosettes with about twice as many cells as wild type (up to about 60 versus at most 25) around a larger, more branched core of extracellular matrix. Warts and Yorkie both influence expression of extracellular matrix genes, including couscous. Its kinase activity and substrates have not been measured.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000082 G1/S transition of mitotic cell cycle | IEA GO_REF:0000118 | MARK AS OVER ANNOTATED | Summary: TreeGrafter transfer. F2U943 was grafted onto PTN001220369, the Monosiga brevicollis Warts leaf, and inherits the IBD terms of its parent PTN002390470, a speciation node labelled "Metazoa-Choanoflagellida" whose descendants include human LATS1/2, Drosophila wts and Nematostella and M. brevicollis Warts (PANTHER 19 tree). The IBA for this term on human LATS1 cites mouse Lats2 and human LATS2. No cell-cycle phase analysis has been done in S. rosetta; warts knockout cells proliferate more slowly, the opposite of the animal loss-of-function phenotype [DOI:10.1101/2024.07.13.603360]. Reason: G1/S transition is a eukaryote-wide process, so there is no taxon violation. But the donor evidence is a cell-cycle role of vertebrate LATS2 (mouse Lats2, human LATS2), which says little about a choanoflagellate. In S. rosetta, loss of warts slows rather than accelerates proliferation, and no cell-cycle phase was measured, so nothing supports a direct role in the G1/S transition. Kept as an over-annotation rather than removed, because the IBD placement at the Choanozoa node is a phylogenetic judgement I cannot refute with S. rosetta data. Propagation Review Root cause: PROPAGATION BAD Failure modes: CONTEXT OR TISSUE MISMATCH Sources checked: PANTHER:PTN002390470 · Warts/LATS, Metazoa-Choanoflagellida node SUPPORTS SOURCE BUT NOT TARGET IBD supported by vertebrate LATS2 cell-cycle evidence (MGI Lats2, human LATS2); sound for vertebrates, but no evidence the role extends to choanoflagellates. PANTHER:PTN001220369 · M. brevicollis Warts leaf NOT RELEVANT Graft point only; carries no experimental annotation of its own. Supporting Evidence: DOI:10.1101/2024.07.13.603360 On the other hand, hippopac1 and warts pac1 KO clones proliferated markedly slower DOI:10.1101/2024.07.13.603360 down proliferation in S. rosetta, which is the opposite of their animal loss-of-function phenotype |
| GO:0000166 nucleotide binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: UniRule assignment from the protein kinase ATP-binding signature (ATP-binding residue K485). Reason: True but uninformative parent of ATP binding, itself a component of the kinase activity. No binding measurements exist. Supporting Evidence: file:SALRS/warts/warts-uniprot.txt PROSITE; PS00107; PROTEIN_KINASE_ATP; 1. |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO from the Ser/Thr kinase active-site signature (IPR008271); the catalytic aspartate (D579, proton acceptor) is annotated as present. Reason: Correct, but the more specific protein serine/threonine kinase activity term is also present and better describes a Warts/LATS AGC kinase. Kept as a true, less specific parent. Supporting Evidence: file:SALRS/warts/warts-uniprot.txt PROSITE; PS00108; PROTEIN_KINASE_ST; 1. |
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA (InterPro AGC C-terminal domain, EC 2.7.11.1, PANTHER, UniRule). F2U943 is classified in PANTHER PTHR24356:SF418 "SERINE_THREONINE-PROTEIN KINASE WARTS", the subfamily of human LATS1/2, and has an intact kinase domain (ATP-binding K485, catalytic D579) followed by an AGC C-terminal domain. In animals Warts/LATS phosphorylates Yorkie/YAP on HXRXXS motifs [PMID:35659869]. Reason: This is the core molecular activity of a Warts/LATS kinase, and the catalytic residues are retained. Unlike Capsaspora coWts, whose TreeGrafter placement is in the Rho-effector kinase family, the S. rosetta protein sits in the LATS clade, so the family-level inference is sound. Kinase activity has not been assayed for the S. rosetta protein. Supporting Evidence: file:SALRS/warts/warts-uniprot.txt PANTHER; PTHR24356:SF418; SERINE_THREONINE-PROTEIN KINASE WARTS; 1. file:SALRS/warts/warts-uniprot.txt PROSITE; PS00108; PROTEIN_KINASE_ST; 1. PMID:35659869 In mammals and in Drosophila, YAP/TAZ/Yorkie is regulated by cytoplasmic sequestration in response to phosphorylation of HXRXXS motifs by the LATS/Warts kinases (Zheng and Pan, 2019). |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | ACCEPT | Summary: Domain-based assignment from the protein kinase domain and its ATP-binding signature (K485). Reason: Kinase domain with the conserved ATP-binding lysine; ATP binding is required for the predicted catalytic activity. Sound domain-level inference, not measured. Supporting Evidence: file:SALRS/warts/warts-uniprot.txt InterPro; IPR017441; Protein_kinase_ATP_BS. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: ARBA rule and UniProt subcellular-location keyword. Warts/LATS kinases act in the cytoplasm, where they retain phosphorylated Yorkie/YAP. No localization data exist for the S. rosetta protein. Reason: Cytoplasm is the expected compartment for a Warts/LATS kinase, and the term is broad enough to be safe without direct data. Accepted as a homology-based location. Supporting Evidence: file:SALRS/warts/warts-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm PMID:35659869 In mammals and in Drosophila, YAP/TAZ/Yorkie is regulated by cytoplasmic sequestration in response to phosphorylation of HXRXXS motifs by the LATS/Warts kinases (Zheng and Pan, 2019). |
| GO:0016301 kinase activity | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: UniRule assignment of the general kinase activity parent term. Reason: True but redundant with the more specific protein serine/threonine kinase activity annotation. Supporting Evidence: file:SALRS/warts/warts-uniprot.txt PROSITE; PS50011; PROTEIN_KINASE_DOM; 1. |
| GO:0016740 transferase activity | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: UniRule assignment of the very general transferase activity term. Reason: True (a kinase transfers phosphate) but too general to be informative. Kept as a non-core parent of protein serine/threonine kinase activity. Supporting Evidence: file:SALRS/warts/warts-uniprot.txt PROSITE; PS50011; PROTEIN_KINASE_DOM; 1. |
| GO:0035329 hippo signaling | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer of the IBD placed at PTN002390470 (Warts/LATS, "Metazoa-Choanoflagellida"), supported by experimental annotations on Drosophila wts, human LATS1/2 and mouse Lats. In S. rosetta, Warts and Yorkie regulate several extracellular matrix genes [PMID:41037400], the Yorkie homolog carries four HXRXXS Warts motifs [PMID:38729842], and in Capsaspora the Warts homolog restrains nuclear entry of the Yorkie homolog [PMID:38517944]. Reason: Warts/LATS performs a step of the hippo signaling cascade (phosphorylating Yorkie), and the IBD node includes choanoflagellates. That placement is consistent with Capsaspora evidence that the Hippo-Warts-Yorkie cascade predates animals, so the family-level inference is sound and the term describes the core role. Caveats: no S. rosetta experiment shows Warts phosphorylating Yorkie or controlling its localization; hippo knockout does not phenocopy the warts rosette-size phenotype; and the predicted TEAD-binding region of S. rosetta Yorkie is poorly conserved. A NEW IMP is not added: the knockout phenotypes (rosette size, proliferation) are downstream outcomes and do not test participation in the cascade itself. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002390470 · Warts/LATS, Metazoa-Choanoflagellida node SUPPORTS TRANSFER IBD supported by fly wts and mammalian LATS1/2; the cascade is shown to predate animals in Capsaspora, so inheritance at the Choanozoa node is plausible. Supporting Evidence: PMID:41037400 RNA sequencing revealed that Warts and Yorkie regulated several extracellular matrix genes involved in multicellularity (including couscous) PMID:38729842 srYki has 4 putative Warts phosphorylation sites 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. |
| GO:0035556 intracellular signal transduction | IEA GO_REF:0000118 | KEEP AS NON CORE | Summary: TreeGrafter transfer of a general signalling term from the PTHR24356 tree; parent of hippo signaling. Reason: True for a signalling kinase but less informative than hippo signaling, which is also annotated. Kept as a non-core parent. Supporting Evidence: file:SALRS/warts/warts-uniprot.txt PANTHER; PTHR24356:SF418; SERINE_THREONINE-PROTEIN KINASE WARTS; 1. |
| GO:0043065 positive regulation of apoptotic process | IEA GO_REF:0000118 | MARK AS OVER ANNOTATED | Summary: TreeGrafter transfer of an IBD at PTN002390470 whose experimental support is Drosophila wts (FBgn0011739), where Warts promotes apoptosis by inactivating Yorkie and so lowering expression of the caspase inhibitor diap1. No cell death has been examined in S. rosetta warts knockouts; the enlarged rosettes were described with a slower, not faster, proliferation rate [DOI:10.1101/2024.07.13.603360]. Reason: The term is permitted for opisthokonts, so it is not a taxon violation, and a role in cell survival in a choanoflagellate is not impossible. But the donor biology is Yorkie-dependent control of an animal anti-apoptotic programme in growing tissue. There is no evidence that choanoflagellate Warts or Yorkie regulate apoptosis, and the Yorkie homolog's TEAD-binding region is poorly conserved, so transfer of this tissue-growth-control output is unsupported. Not removed, because no data contradict it. Propagation Review Root cause: PROPAGATION BAD Failure modes: CONTEXT OR TISSUE MISMATCH Sources checked: PANTHER:PTN002390470 · Warts/LATS, Metazoa-Choanoflagellida node SUPPORTS SOURCE BUT NOT TARGET Seeded by Drosophila wts tissue-growth and apoptosis phenotypes; no choanoflagellate data on apoptosis. Supporting Evidence: PMID:38729842 The degree to which choanoflagellate Yorkie orthologs interact with Sd/TEAD proteins is therefore currently unclear DOI:10.1101/2024.07.13.603360 down proliferation in S. rosetta, which is the opposite of their animal loss-of-function phenotype |
| GO:0046620 regulation of organ growth | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter transfer of an IBD at PTN002390470 seeded by mouse Lats2 (MGI:1354386). GO defines the term as modulating "growth of an organ of an organism"; it is a child of regulation of developmental growth and regulation of multicellular organismal process. S. rosetta is a choanoflagellate that lives as single cells or as clonal rosettes of a few dozen cells; it has no organs. Its warts knockout does enlarge rosettes [DOI:10.1101/2024.07.13.603360], but a rosette is not an organ. Reason: An organ is a multicellular anatomical structure of an animal or plant body, which choanoflagellates do not have, so the term cannot be true of this protein whatever its function. The source annotation is sound for mice. The fault is that an organ-level outcome term was placed at a node ("Metazoa-Choanoflagellida") that includes unicellular organisms, while the GO term's taxon constraints exclude only bacteria, archaea and two yeasts. The rosette-size phenotype is the closest S. rosetta counterpart, but no GO term fits it (there is no rosette term; see the NTR drafted in the rosetteless review). It is also a downstream outcome of altered extracellular matrix gene expression, not work done by Warts, so no replacement process term is proposed. Propagation Review Root cause: PROPAGATION BAD Failure modes: LINEAGE OR TAXON MISMATCH Sources checked: PANTHER:PTN002390470 · Warts/LATS, Metazoa-Choanoflagellida node SUPPORTS SOURCE BUT NOT TARGET Organ-growth IBD seeded by mouse Lats2; valid for animals, but the node spans choanoflagellates, which have no organs. PANTHER:PTN001220369 · M. brevicollis Warts leaf NOT RELEVANT Graft point only; M. brevicollis also receives this IBA and has no organs either. Supporting Evidence: DOI:10.1101/2024.07.13.603360 warts pac1 cells grew into giant rosettes containing about twice as many cells as wild-type ones (21.1 ± 4.4 versus 10.9 ± 3.8 cells per rosette, respectively) PMID:41037400 suggesting that Hippo signaling regulates multicellular size in choanoflagellates by modulating matrix secretion |
| GO:0071944 cell periphery | IEA GO_REF:0000117 | UNDECIDED | Summary: ARBA machine-learning rule. In animals Warts/LATS is recruited to the plasma membrane by Mats/MOB1 and upstream adaptors during activation. The localization of S. rosetta Warts has not been examined. Reason: A membrane-proximal pool is plausible for a Warts/LATS kinase, but there are no S. rosetta data for or against it, and the rule is not specific to the LATS clade. Supporting Evidence: file:SALRS/warts/warts-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
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Download this section (compressed HTML)Q: Does S. rosetta Warts phosphorylate the Yorkie homolog on its four HXRXXS motifs, and is Yorkie more nuclear in warts knockout cells?
Q: Are the enlarged rosettes of warts knockouts caused by Yorkie? yorkie knockout rosettes are of normal size, so a warts yorkie double knockout is the key epistasis test.
Q: Why does hippo knockout not phenocopy the warts rosette-size phenotype? Which kinases activate Warts in S. rosetta?
Q: Do larger warts knockout rosettes arise from more cell divisions before rosette release, reduced cell loss, or altered matrix secretion (e.g. via couscous)?
Q: Should GO add taxon constraints restricting organ-level growth terms such as regulation of organ growth (GO:0046620) to taxa with organs, or should PAINT place the organ-growth IBD at a metazoan rather than a Metazoa-plus-choanoflagellate node?
Experiment: In vitro kinase assay of immunopurified wild-type versus kinase-dead (K485A or D579A) Warts on recombinant Yorkie and a four-site S-to-A Yorkie mutant, plus Phos-tag immunoblots of Yorkie in wild-type and warts knockout cells.
Hypothesis: S. rosetta Warts is an active kinase that phosphorylates Yorkie at HXRXXS motifs.
Type: biochemical kinase assay
Experiment: Generate warts yorkie double knockouts with the selection-based cassette method and count cells per rosette; express a non-phosphorylatable Yorkie in wild-type cells to test whether it phenocopies warts loss.
Hypothesis: The warts rosette-size phenotype is mediated by Yorkie.
Type: genetic epistasis
Experiment: Complement warts knockout cells with wild-type or kinase-dead Warts and score cells per rosette, ECM core morphology and doubling time.
Hypothesis: Warts kinase activity is required to limit rosette size.
Type: genetic complementation
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