hippo (PTSG_10780) encodes the Hippo/MST1-2-type STE20 (germinal-centre kinase II) serine/threonine protein kinase of the colonial choanoflagellate Salpingoeca rosetta. The 459-residue protein has an N-terminal protein kinase domain (residues 24-275) with the conserved ATP-binding lysine, a disordered linker, and a C-terminal SARAH domain (406-453) of the MST1/2/Hippo type, the dimerization and adaptor-binding module through which animal Hippo kinases bind Salvador and RASSF proteins. It is the only S. rosetta protein combining a kinase domain with an MST-type SARAH domain. In animals Hippo phosphorylates and activates the Warts/LATS kinases, which in turn phosphorylate and inactivate the transcriptional coactivator Yorkie/YAP. S. rosetta encodes Warts, Salvador and Yorkie homologs, and the Capsaspora Hippo homolog activates this cascade, but the S. rosetta protein has not been characterized biochemically. Knockout of hippo in S. rosetta slows cell proliferation (longer doubling time) but, unlike knockout of warts, does not change rosette size, and knockout cells still develop rosettes.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: UniRule assignment from the protein kinase ATP-binding signature. The kinase domain (24-275) carries the PROSITE ATP-binding region and the ATP-binding lysine (K53). Reason: True but uninformative: nucleotide binding is the parent of the ATP binding annotation also present, and ATP binding is only a component of the kinase activity. No nucleotide-binding measurements exist for this protein. Supporting Evidence: file:SALRS/hippo/hippo-uniprot.txt PROSITE; PS00107; PROTEIN_KINASE_ATP; 1. |
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA (InterPro Mst1/2 SARAH domain, EC 2.7.11.1, PANTHER). The protein has a STE20-type kinase domain classified by CDD as the MST1/2 kinase subfamily (cd06612 STKc_MST1_2) and a Hippo-type SARAH domain (cd21884). Homologs of Hippo are defined by exactly this combination of a Ste20-like kinase domain and a SARAH domain [PMID:22832104]. Reason: This is the core activity of an MST/Hippo kinase, and the catalytic domain and its ATP-binding residue are intact. The PANTHER part of the row comes from PTN004698602, a deep duplication node in PTHR48012 spanning several germinal-centre kinase groups (SLK, SPAK/OSR1, MAP4K, GCKIII); serine/threonine kinase activity is shared by all of them, so the broad placement does not affect this term. Activity has not been assayed for the S. rosetta protein; for the Capsaspora homolog, expression in Drosophila S2R+ cells induced phosphorylation of Warts and Yorkie [PMID:22832104]. Supporting Evidence: file:SALRS/hippo/hippo-uniprot.txt CDD; cd06612; STKc_MST1_2; 1. file:SALRS/hippo/hippo-uniprot.txt CDD; cd21884; SARAH_MST_Hpo; 1. PMID:22832104 Our searches further identified homologues of Hpo, defined by the presence of a Ste20-like kinase domain and a SARAH domain |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | ACCEPT | Summary: Domain-based assignment from the protein kinase domain and its ATP-binding signature; UniProt annotates K53 as the ATP-binding residue. Reason: The kinase domain retains the glycine-rich loop and ATP-binding lysine, and ATP binding is required for the predicted catalytic activity. Sound domain-level inference, although binding has not been measured. Supporting Evidence: file:SALRS/hippo/hippo-uniprot.txt InterPro; IPR017441; Protein_kinase_ATP_BS. |
| GO:0005737 cytoplasm | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer from PANTHER node PTN004698602, a duplication node in PTHR48012 whose human members include SLK, STK10, STK24-26, STK39, OXSR1 and MAP4K1-5 (PANTHER 19 tree). Animal Hippo/MST kinases act mainly in the cytoplasm. No localization data exist for the S. rosetta protein. Reason: Cytoplasm is the expected compartment for a soluble STE20 kinase without transmembrane or targeting signals, and it is true of every germinal-centre kinase group under the source node. The term is broad enough to be safe from the deep node placement. Accepted as a homology-based location that should be confirmed by tagging the protein. Supporting Evidence: file:SALRS/hippo/hippo-uniprot.txt STE20 subfamily. |
| GO:0007165 signal transduction | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the SARAH domain (IPR011524). In animals, SARAH domains mediate the signalling interactions of Hippo/MST kinases with Salvador and RASSF proteins. In S. rosetta, hippo knockout slows cell proliferation, as warts knockout does, but does not change rosette size, whereas warts knockout doubles it [DOI:10.1101/2024.07.13.603360]. Reason: A STE20/MST kinase with a SARAH adaptor-binding domain is a signalling kinase, and the broad term is a safe description of its role. The more specific term hippo signaling is not proposed as NEW. That a Hippo-Warts-Yorkie cascade operates in S. rosetta has not been shown, and the hippo and warts knockout phenotypes differ: only warts knockout enlarges rosettes, so Warts may be activated largely independently of Hippo, as the stronger coWts than coHpo phenotype suggests in Capsaspora. The shared slow growth of hippo and warts knockouts fits a common pathway but does not establish one. Supporting Evidence: file:SALRS/hippo/hippo-uniprot.txt InterPro; IPR011524; SARAH_dom. 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 The size of hippopac1 and yorkiepac1 rosettes did not significantly differ from wild type (11.6 ± 1.7 cells and 9.8 ± 3.1 cells |
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Download this section (compressed HTML)Q: Does S. rosetta Hippo phosphorylate S. rosetta Warts on its activation-loop and hydrophobic-motif sites, and is Warts activity reduced in hippo knockout cells?
Q: Why does hippo knockout slow proliferation without changing rosette size, while warts knockout both slows proliferation and doubles rosette size? Are other STE20/MAP4K-type kinases able to activate Warts without Hippo?
Q: Does the S. rosetta SARAH domain bind the Salvador homolog and RASSF-type SARAH proteins encoded in the genome, as in animals?
Q: Is the slower proliferation of hippo knockout cells mediated by Yorkie? yorkie knockout cells proliferate at a wild-type rate, so a double-knockout epistasis test is informative.
Experiment: In vitro kinase assay of immunopurified wild-type versus kinase-dead (K53A) Hippo on recombinant Warts, and phospho-specific or Phos-tag immunoblots of Warts in wild-type and hippo knockout cells.
Hypothesis: S. rosetta Hippo is an active kinase that phosphorylates Warts.
Type: biochemical kinase assay
Experiment: Build hippo yorkie and warts yorkie double knockouts with the selection-based cassette method and test whether loss of Yorkie suppresses the slow-growth phenotype of hippo and warts knockouts; compare Yorkie localization in each background.
Hypothesis: Hippo acts upstream of Warts and Yorkie in S. rosetta.
Type: genetic epistasis
Experiment: Complement hippo knockout cells with wild-type or kinase-dead Hippo and measure doubling time.
Hypothesis: Hippo kinase activity, not only the protein, is required for normal proliferation.
Type: genetic complementation
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