coHpo (CAOG_01932) is the single Hippo/MST-family kinase of the filasterean amoeba Capsaspora owczarzaki, a close unicellular relative of animals. It is a STE20-group serine/threonine protein kinase with an N-terminal kinase domain and a C-terminal SARAH coiled-coil domain, the architecture that defines Hippo/MST1/2 kinases. The locus has three predicted protein models (UniProt A0A0D2WLF3, 430 aa; A0A0D2VKT0, 466 aa; A0A0D2X1E3, 367 aa), of which only A0A0D2WLF3 contains the SARAH domain; this is the form used to rescue the knockout. Homozygous knockout of the kinase domain in Capsaspora shifts the Yorkie/YAP/TAZ ortholog coYki from the cytoplasm into the nucleus, indicating that coHpo acts at the head of a Hippo-Warts kinase cascade that restrains coYki by cytoplasmic sequestration. Loss of coHpo does not alter cell proliferation, but increases the frequency of an elongated, myosin-dependent contractile cell state (rescued by a coHpo transgene) and increases the packing density of cells within multicellular aggregates; these phenotypes are copied by a non-phosphorylatable, transcriptionally active coYki and are therefore attributed to unrestrained coYki activity. When overexpressed in Drosophila, coHpo stimulates phosphorylation of Warts at its Hippo-responsive site and of Yorkie, and restricts eye growth, showing that it can engage the animal kinase cascade. Its catalytic activity has not been measured with purified protein, and its subcellular localization has not been examined directly.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: UniRule-based transfer of nucleotide binding to a protein kinase. coHpo has an intact protein kinase domain with the PROSITE ATP-binding signature (PS00107); nucleotide (ATP) binding is implicit in kinase activity but has not been measured for the Capsaspora protein. Reason: Correct but uninformative; it is a parent of the more specific ATP binding annotation that the same entry carries. Retained as a non-core, redundant statement of the cofactor requirement of the kinase domain rather than removed, because it is not wrong. Supporting Evidence: PMID:22832104 Our searches further identified homologues of Hpo, defined by the presence of a Ste20-like kinase domain and a SARAH domain |
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA (InterPro2GO for the Mst1/2 SARAH domain IPR024205, EC 2.7.11.1 via ARBA, and PANTHER PTN008481874). coHpo is the single Hippo/MST (STE20 subfamily, CDD STKc_MST1_2) kinase of Capsaspora. No in vitro kinase assay has been performed on the Capsaspora protein. Indirect support: overexpressed coHpo induces phosphorylation of coYki and of Drosophila Warts (T1077) and Yorkie (S168) in Drosophila S2R+ cells [PMID:22832104]; in Capsaspora, deletion of the kinase domain causes nuclear accumulation of coYki, the expected outcome of loss of an upstream kinase [PMID:38517944]. Reason: This is the core molecular activity of a Hippo/MST kinase. The domain architecture is complete (kinase domain plus Mst1/2-type SARAH), and the heterologous phosphorylation of the Hippo-responsive Warts site and the kinase-domain knockout phenotype are both consistent with an active Ser/Thr kinase. The activity itself remains an inference: it has not been measured biochemically with purified coHpo, and the Drosophila experiments are overexpression readouts in cells that contain endogenous kinases. Accepted as a well-supported homology-based annotation. Supporting Evidence: PMID:22832104 Interestingly, Co-Hpo also stimulated the phosphorylation of Dm-Wts and Dm-Yki, as revealed by phospho-specific antibodies against P-Dm-Wts-T1077 and P-Dm-Yki-S168, respectively 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 PMID:38517944 homologous recombination was used to replace the kinase domain of both WT alleles with selectable markers |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | ACCEPT | Summary: InterPro/UniRule inference from the protein kinase domain (IPR000719) and its ATP-binding site signature (IPR017441, PROSITE PS00107), both present in A0A0D2WLF3. Not measured experimentally. Reason: ATP is the phosphate donor for a protein kinase and the ATP-binding signature is conserved; this is a standard, correct companion annotation to the kinase activity. Supporting Evidence: PMID:22832104 Our searches further identified homologues of Hpo, defined by the presence of a Ste20-like kinase domain and a SARAH domain |
| GO:0005737 cytoplasm | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer from the PANTHER Hippo/MST family node. Localization of coHpo protein has not been examined in Capsaspora; the only localization data in the primary study are for its target coYki, which is cytoplasmic in wild-type cells and shifts to the nucleus in coHpo-/- cells [PMID:38517944]. Reason: Hippo/MST kinases act in the cytoplasm to phosphorylate Warts/LATS, which in turn retains Yorkie in the cytoplasm. A cytoplasmic site of action for coHpo is consistent with its role in cytoplasmic sequestration of coYki in Capsaspora, and the term is broad enough not to over-claim. It remains a family-level prediction until coHpo is localized directly. 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. |
| GO:0007165 signal transduction | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the SARAH domain (IPR011524). In Capsaspora, homozygous coHpo knockout causes nuclear accumulation of coYki, the same regulatory output as loss of coWts, showing that coHpo acts in a Hippo kinase cascade upstream of coYki [PMID:38517944]. Heterologously, coHpo phosphorylates/activates Drosophila Warts and leads to Yorkie phosphorylation [PMID:22832104]. Reason: Correct but far too general. GO:0035329 hippo signaling is defined as an intracellular cascade that starts with activation of Hippo, which phosphorylates LATS, which phosphorylates YAP/Yorkie leading to its cytosolic retention. That is what has been shown for coHpo: its loss releases coYki from cytoplasmic sequestration in Capsaspora (IMP-type evidence), and the protein activates the Warts-Yorkie cascade in Drosophila. coHpo is the initiating kinase of the cascade, so it performs a step of the process (participation test passes), and the orthologous kinases Drosophila hpo and human STK3/STK4 carry this term. The term has no taxon constraint. Caveats: no epistasis by double mutant was possible, and the coHpo-/- effect on coYki is weaker than that of coWts-/-, consistent with Hpo-independent Wts activity; coHpo phosphorylation of coWts has not been shown in Capsaspora. Proposed replacements: hippo signaling Supporting Evidence: PMID:38517944 In contrast, the majority of coHpo-/- and coWts-/- cells showed nuclear levels of mScarlet-coYki greater than or equal to that seen in the cytoplasm 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 Interestingly, we found that coWts-/- cells were significantly more likely to show nuclear mScarlet-coYki localization than coHpo-/- cells PMID:22832104 Thus, Co-Hpo not only possesses growth-suppressing activity but also functionally activates a signaling cascade leading to the phosphorylation of endogenous Yki in Drosophila. |
| GO:0051262 protein tetramerization | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping from IPR036674 "p53-like tetramerisation domain superfamily", a structural (Gene3D 4.10.170.10 / SUPERFAMILY) fold class that the helical SARAH segment of coHpo matches. GO:0051262 is defined as formation of a protein tetramer. The SARAH domain of Hippo/MST kinases forms antiparallel homodimers and 1:1 heterodimers with RASSF and Salvador SARAH domains, not tetramers [PMID:17517604, PMID:25004971]. No oligomerization data exist for coHpo. Reason: The annotation is a fold-level InterPro2GO artefact: membership of a p53-tetramerisation-like structural superfamily does not imply tetramer formation. For MST1 the SARAH domain forms a homodimer, and when MST1 SARAH meets the tetramer-prone free Rassf5 SARAH domain it converts it into dimers, the opposite of tetramerization. The SARAH-specific InterPro entries for this protein (IPR011524, IPR024205) do not map to tetramerization. If SARAH-mediated oligomerization of coHpo is to be represented, it should be as dimerization activity once tested for the Capsaspora protein. Supporting Evidence: PMID:17517604 The Mst1 SARAH structure forms a homodimer containing two helices per monomer. PMID:17517604 In cross-linking experiments, the major population of the free Rassf5 SARAH domain formed tetramers in solution. However, when the Mst1 SARAH domain was added, dimers were formed PMID:17517604 identified an elongated, tight-binding interface with the Rassf5 SARAH domain and a 1:1 heterodimer formation. |
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Download this section (compressed HTML)Q: Which of the three predicted CAOG_01932 protein models (A0A0D2WLF3 with SARAH, 430 aa; A0A0D2VKT0, 466 aa; A0A0D2X1E3, 367 aa, both lacking SARAH) are expressed in Capsaspora, and are the SARAH-less forms genuine isoforms or gene-model artefacts? The knockout removed the shared kinase domain and so eliminated all of them; rescue used the SARAH-containing form only.
Q: Does purified coHpo phosphorylate coWts directly (at the site corresponding to Drosophila Warts T1077) and is coHpo itself activated by autophosphorylation of its activation loop, as for Hpo/MST1/2?
Q: Does the coHpo SARAH domain homodimerize and heterodimerize with Capsaspora Salvador or RASSF SARAH proteins, and is the SARAH domain required for rescue of the coHpo-/- phenotypes?
Q: Why is the effect of coHpo loss on coYki localization weaker than that of coWts loss - which kinases (e.g. other STE20/MAP4K-family kinases) activate coWts independently of coHpo in Capsaspora?
Q: What upstream signals (cell density, cell contact, mechanical cues in aggregates) regulate coHpo activity in Capsaspora?
Experiment: Express and purify wild-type and kinase-dead (catalytic lysine mutant) coHpo and perform in vitro kinase assays with coWts and the MOB ortholog as substrates, mapping phosphosites by mass spectrometry.
Hypothesis: coHpo is an active Ser/Thr kinase that directly phosphorylates coWts.
Type: biochemical kinase assay
Experiment: Complement coHpo-/- cells with kinase-dead and SARAH-deleted transgenes and score coYki localization, elongated-cell frequency and aggregate cell density.
Hypothesis: Catalytic activity and the SARAH domain are both required for coHpo function.
Type: genetic complementation
Experiment: Stably express a fluorescently tagged coHpo (or endogenously tag the locus) and image its localization in adherent cells and aggregates relative to F-actin foci and the plasma membrane.
Hypothesis: coHpo acts in the cytoplasm/cell cortex.
Type: localization / imaging
Experiment: Once double-mutant or conditional knockdown methods are available, test whether coYki loss suppresses the elongated-cell and dense-packing phenotypes of coHpo-/- cells.
Hypothesis: coHpo phenotypes require coYki.
Type: genetic epistasis
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