STK36 (Fused homolog) is a 1315-residue serine/threonine protein kinase (EC 2.7.11.1) and the vertebrate orthologue of Drosophila Fused, with which it shares PANTHER subfamily PTHR22983:SF6. Its N-terminal kinase domain is activated by trans-autophosphorylation of the activation loop on Thr158 and Ser159 in response to Hedgehog stimulation, and is followed by a long C-terminal region of unknown structure. STK36 has two distinct roles. In Hedgehog signalling it is one of two vertebrate Fused-family kinases, with ULK3, that convert the latent GLI transcription factors into their activator form: at the tip of the primary cilium, in a complex with the pseudokinase ULK4 that bridges kinase to substrate, STK36 phosphorylates GLI2 on Ser230 and Ser232 - sites conserved with the Fused sites on Cubitus interruptus - which weakens the GLI-SUFU interaction and permits GLI nuclear entry and target-gene transcription. STK36 also phosphorylates ULK4 itself on Thr1021 and Thr1023, and the two proteins depend on each other to accumulate at the ciliary tip. Independently of Hedgehog signalling, STK36 is required to build the central pair apparatus of motile 9+2 cilia, where it sits between the radial spokes and the central pair and works with the kinesin KIF27 and central-pair structural proteins. Because ULK3 can substitute for it in Hedgehog signalling, mouse knockouts develop normally and respond normally to Hedgehog, but die postnatally of progressive hydrocephalus from defective ependymal ciliary beating and disrupted cerebrospinal fluid flow. In humans, biallelic loss of function causes primary ciliary dyskinesia 46, with a central-pair axonemal defect, disordered ciliary orientation and no situs abnormality. STK36 is cytoplasmic and axonemal, with a low-level nuclear pool, and is expressed most highly in adult testis germ cells and in respiratory epithelium.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0001222 transcription corepressor binding | IDA PMID:10806483 Gli regulation by the opposing activities of fused and suppr... | KEEP AS NON CORE | Summary: The STK36-SUFU interaction, reported by Murone et al. and annotated using a term that names SUFU as a transcription corepressor. The interaction is well replicated - IntAct records three experiments, and an independent in situ proximity ligation assay detected it on endogenous protein in HeLa cells (PMID:25241761). Reason: Retained because the binding event is real and the term at least says something specific about the partner, but not promoted to core, for two reasons. First, STK36 does not act on SUFU: its catalytic target is GLI, and the effect on SUFU is indirect - phosphorylation of Ci/GLI alters its affinity for SUFU rather than STK36 modifying SUFU itself (PMID:31279575). Second, the premise that SUFU is a transcription corepressor rests on TAS-only rows in SUFU's own GOA record, and the review of SUFU in this repository keeps its corepressor activity as a non-core annotation. Making the STK36 side core would place more weight on that premise than the evidence behind it supports. The cached record for PMID:10806483 holds citation metadata only, with no abstract text, so the paper itself could not be read. It was adjudicated instead against the structured UniProt annotations that cite it with experimental evidence codes, and against later experimental rows that reach the same conclusion. Supporting Evidence: file:human/STK36/STK36-uniprot.txt Interacts with GLI1, GLI2 and GLI3 (PubMed:10806483). file:human/STK36/STK36-uniprot.txt Q9NRP7; Q9UMX1: SUFU; NbExp=3; IntAct=EBI-863797, EBI-740595; PMID:31279575 These phosphorylation events alter Ci binding to the pathway inhibitor Suppressor of fused (Sufu) and facilitate the recruitment of Transportion and the transcriptional coactivator CBP. PMID:25241761 Herein, we collected ∼ 700 primary antibodies and employed a highly sensitive and specific technique, an in situ proximity ligation assay, to investigate 1204 endogenous PPIs in HeLa cells, and 557 PPIs of them tested positive. |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | MODIFY | Summary: Generic protein kinase activity inferred from the InterPro protein-kinase active-site signature IPR008271. STK36 is a kinase and the domain call is right (UniProt DOMAIN 4..254 "Protein kinase", proton acceptor Asp-125), but its specific activity has been determined directly. Reason: This is the "term is too general" case rather than a wrong call. Serine/threonine specificity is established by direct assay - immunopurified STK36 phosphorylates GST-Gli2N in vitro (PMID:38096226) - and by UniProt, which records both the seryl (Rhea:RHEA:17989) and the threonyl (Rhea:RHEA:46608) half-reactions with experimental evidence codes and assigns EC 2.7.11.1. GO:0004674 is already present on this record from IDA, IMP, IEA and TAS rows, so replacing the generic parent loses nothing and removes an uninformative level. Proposed replacements: protein serine/threonine kinase activity Supporting Evidence: file:human/STK36/STK36-uniprot.txt /note="Protein kinase" PMID:38096226 In an in vitro kinase assay, we found that immunopurified Stk36 phosphorylated GST-Gli2N equally well regardless of whether it interacted with Ulk4 or not, suggesting that Ulk4 does not regulate Stk36 kinase activity. PMID:31279575 Furthermore, we provide evidence that Sonic hedgehog (Shh) activates Gli2 by stimulating its phosphorylation on conserved sites through the Fu-family kinases ULK3 and mFu/STK36 in a manner depending on Gli2 ciliary localization. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:38096226 Ulk4 promotes Shh signaling by regulating Stk36 ciliary loca... | ACCEPT | Summary: Direct demonstration of serine/threonine kinase activity: immunopurified STK36 phosphorylates GST-Gli2N in vitro, and a second in vitro assay shows it phosphorylates a ULK4 peptide spanning Thr1021. Activation-loop autophosphorylation on Thr158/Ser159 was used as the readout of activation, with the kinase-dead form as control (PMID:38096226). Reason: The strongest molecular-function row on this record, and core. The assay is on purified protein with defined substrates, the specificity is confirmed by the kinase-dead and phospho-mutant controls, and the activation mechanism is the same trans-autophosphorylation of the activation loop described for Drosophila Fu. UniProt records the resulting modified residues (Thr-158 phosphothreonine, Ser-159 phosphoserine, both by autocatalysis). This is the term the pan-metazoan Hedgehog module assigns to its Fused-family kinase annoton, and the term the GO-CAM model 696022cd00001645 gives STK36. Supporting Evidence: PMID:38096226 In an in vitro kinase assay, we found that immunopurified Stk36 phosphorylated GST-Gli2N equally well regardless of whether it interacted with Ulk4 or not, suggesting that Ulk4 does not regulate Stk36 kinase activity. PMID:38096226 As shown in Figure 3D, Shh induced phosphorylation of CFP-Stk36WT but not CFP-Stk36KR on T158/S159, suggesting that Shh promotes the phosphorylation of Stk36 AL via its kinase activity, likely through trans-autophosphorylation as are the cases for other kinases. PMID:38096226 We found that the phospho-mimetic mutation (Stk36EE) increased, whereas phospho-deficient mutation (Stk36AA) abolished Stk36 kinase activity as determined by both Stk36 T158/S159 phosphorylation and Gli2 S230/S232 phosphorylation PMID:38096226 To confirm T1021 phosphorylation by Stk36, we carried out an in vitro kinase assay using immunopurified Fg-Stk36 as kinase and GST fusion protein containing hUlk4 sequence from aa1017 to aa1036 (GST-hUlk4C) as substrate. file:human/STK36/STK36-uniprot.txt Autophosphorylated at Thr-158 and Ser-159, activating the protein |
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automated assignment combining an ARBA model, orthology to mouse Stk36 (UniProtKB:Q69ZM6) and the EC mapping 2.7.11.1. All three routes converge on the activity that is directly demonstrated in the IDA row. Reason: An electronic row that is correct at the right level of specificity. The EC number it uses is not a guess: UniProt assigns EC 2.7.11.1 to STK36 with ECO:0000269 experimental evidence from PMID:31279575 and PMID:38096226. Redundant with the experimental rows rather than adding anything, but nothing here needs changing. Supporting Evidence: PMID:38096226 In an in vitro kinase assay, we found that immunopurified Stk36 phosphorylated GST-Gli2N equally well regardless of whether it interacted with Ulk4 or not, suggesting that Ulk4 does not regulate Stk36 kinase activity. file:human/STK36/STK36-uniprot.txt EC=2.7.11.1 {ECO:0000269|PubMed:31279575, ECO:0000269|PubMed:38096226}; |
| GO:0004674 protein serine/threonine kinase activity | IMP PMID:31279575 Phosphorylation of Ci/Gli by Fused Family Kinases Promotes H... | ACCEPT | Summary: Han et al. showed that Hedgehog signalling activates Gli2 through phosphorylation on sites conserved with the Fused sites on Ci, carried out by the two Fu-family kinases ULK3 and mFu/STK36. The annotation was made on the human gene by FlyBase curators from a paper that does the Drosophila and the mammalian experiments side by side. Reason: Core. This is the paper that established Fu-family phosphorylation of Ci/GLI as the conserved activation mechanism, working out the fly sites (Ser218 and Ser1230 on Ci, primed for CK1) and then showing the same logic operates on Gli2 through ULK3 and STK36. Accepting a molecular function from an IMP is appropriate here because the kinase requirement is read out as loss of a specific phosphorylation event on a named substrate, and the same activity is independently confirmed in vitro on purified protein (PMID:38096226). Supporting Evidence: PMID:31279575 Here, we demonstrate that Hh stimulates the phosphorylation of Ci by the Ser/Thr kinase Fused (Fu) and that Fu-mediated phosphorylation of Ci promotes its activation. PMID:31279575 Furthermore, we provide evidence that Sonic hedgehog (Shh) activates Gli2 by stimulating its phosphorylation on conserved sites through the Fu-family kinases ULK3 and mFu/STK36 in a manner depending on Gli2 ciliary localization. PMID:31279575 Hence, Fu-family kinase-mediated phosphorylation of Ci/Gli serves as a conserved mechanism that activates the Hh pathway transcription factor. PMID:38096226 In an in vitro kinase assay, we found that immunopurified Stk36 phosphorylated GST-Gli2N equally well regardless of whether it interacted with Ulk4 or not, suggesting that Ulk4 does not regulate Stk36 kinase activity. |
| GO:0004674 protein serine/threonine kinase activity | TAS PMID:10806483 Gli regulation by the opposing activities of fused and suppr... | ACCEPT | Summary: Author statement from Murone et al., the paper that first characterised human Fused homolog as a kinase acting on GLI transcription factors. UniProt cites the same paper for the functional description and for the ATP-binding Lys-33 mutagenesis. Reason: A TAS row that later direct assays have fully vindicated, so there is nothing to correct even though the statement itself is not an experiment record. The cached record for PMID:10806483 holds citation metadata only, with no abstract text, so the paper itself could not be read. It was adjudicated instead against the structured UniProt annotations that cite it with experimental evidence codes, and against later experimental rows that reach the same conclusion. UniProt's FUNCTION line attributes the GLI-regulating kinase description to this paper together with three later ones, and the specific activity is established independently by IDA and IMP rows. Supporting Evidence: file:human/STK36/STK36-uniprot.txt GLI2; GLI3 AND SUFU, TISSUE SPECIFICITY, SUBCELLULAR LOCATION, AND file:human/STK36/STK36-uniprot.txt Interacts with GLI1, GLI2 and GLI3 (PubMed:10806483). PMID:38096226 In an in vitro kinase assay, we found that immunopurified Stk36 phosphorylated GST-Gli2N equally well regardless of whether it interacted with Ulk4 or not, suggesting that Ulk4 does not regulate Stk36 kinase activity. |
| GO:0005515 protein binding | IPI PMID:10806483 Gli regulation by the opposing activities of fused and suppr... | MODIFY | Summary: STK36-SUFU co-immunoprecipitation from Murone et al., the founding observation that vertebrate Fused and Suppressor of fused act on GLI in opposite directions. IntAct records three experiments for the pair. Reason: A bare protein-binding row carries no functional information, and here a more informative term for the same contact is already on this record from the same 2000 paper: GO:0001222 transcription corepressor binding. Replacing the generic row with the specific one is therefore the MODIFY case rather than the removal case, and it does not require inventing anything. What should not be read into these rows is a catalytic relationship: STK36 does not phosphorylate SUFU. The mechanism is the other way round - STK36 phosphorylates Ci/GLI, and that phosphorylation weakens the Ci/GLI-SUFU interface (PMID:31279575). The cached record for PMID:10806483 holds citation metadata only, with no abstract text, so the paper itself could not be read. It was adjudicated instead against the structured UniProt annotations that cite it with experimental evidence codes, and against later experimental rows that reach the same conclusion. Proposed replacements: transcription corepressor binding Supporting Evidence: file:human/STK36/STK36-uniprot.txt Q9NRP7; Q9UMX1: SUFU; NbExp=3; IntAct=EBI-863797, EBI-740595; file:human/STK36/STK36-uniprot.txt GLI2; GLI3 AND SUFU, TISSUE SPECIFICITY, SUBCELLULAR LOCATION, AND PMID:31279575 These phosphorylation events alter Ci binding to the pathway inhibitor Suppressor of fused (Sufu) and facilitate the recruitment of Transportion and the transcriptional coactivator CBP. |
| GO:0005515 protein binding | IPI PMID:25241761 Using an in situ proximity ligation assay to systematically ... | MODIFY | Summary: STK36-SUFU interaction detected by in situ proximity ligation assay on endogenous proteins in HeLa cells, as part of a systematic survey of 1204 candidate pathway interactions (PMID:25241761). Reason: A bare protein-binding row carries no functional information, and here a more informative term for the same contact is already on this record from the same 2000 paper: GO:0001222 transcription corepressor binding. Replacing the generic row with the specific one is therefore the MODIFY case rather than the removal case, and it does not require inventing anything. What should not be read into these rows is a catalytic relationship: STK36 does not phosphorylate SUFU. The mechanism is the other way round - STK36 phosphorylates Ci/GLI, and that phosphorylation weakens the Ci/GLI-SUFU interface (PMID:31279575). This row is worth keeping in preferred form rather than deleting because it is methodologically the strongest evidence for the pair: proximity ligation reports on endogenous, untagged protein at native abundance, which is a different and stronger claim than a co-immunoprecipitation of overexpressed constructs. Proposed replacements: transcription corepressor binding Supporting Evidence: PMID:25241761 Herein, we collected ∼ 700 primary antibodies and employed a highly sensitive and specific technique, an in situ proximity ligation assay, to investigate 1204 endogenous PPIs in HeLa cells, and 557 PPIs of them tested positive. file:human/STK36/STK36-uniprot.txt Q9NRP7; Q9UMX1: SUFU; NbExp=3; IntAct=EBI-863797, EBI-740595; PMID:31279575 These phosphorylation events alter Ci binding to the pathway inhibitor Suppressor of fused (Sufu) and facilitate the recruitment of Transportion and the transcriptional coactivator CBP. |
| GO:0005515 protein binding | IPI PMID:32707033 Kinase Interaction Network Expands Functional and Disease Ro... | MODIFY | Summary: STK36-ULK4 interaction from a mass-spectrometry kinase interaction network covering more than 300 human kinases (PMID:32707033). The pair has since been validated by yeast two-hybrid, co-immunoprecipitation and in vitro phosphorylation (PMID:37584603, PMID:38096226). Reason: This is not a generic contact and the bare row understates what is known. ULK4 is a pseudokinase that acts as the adaptor bridging STK36 to its GLI2 substrate, and it is itself an STK36 substrate: an in vitro kinase assay with immunopurified STK36 and a GST-ULK4 peptide identified phosphorylation of Thr1021/Thr1023, and that phosphorylation is what drives ULK4 to the ciliary tip (PMID:38096226). The informative molecular function behind these rows is therefore the serine/threonine kinase activity STK36 exerts on ULK4, not an unqualified binding statement. UniProt records the functional reading directly: "Interacts with ULK4; promoting association with GLI2 substrate" and "Also catalyzes phosphorylation of ULK4 adapter". Proposed replacements: protein serine/threonine kinase activity Supporting Evidence: PMID:32707033 Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. PMID:38096226 To confirm T1021 phosphorylation by Stk36, we carried out an in vitro kinase assay using immunopurified Fg-Stk36 as kinase and GST fusion protein containing hUlk4 sequence from aa1017 to aa1036 (GST-hUlk4C) as substrate. PMID:38096226 We further show that ciliary tip localization of Ulk4 depends on Stk36-mediated phosphorylation of Ulk4 on Thr1023, and that ciliary tip localization of Ulk4 is essential for its function in Hh signal transduction. file:human/STK36/STK36-uniprot.txt Interacts with ULK4; promoting association with GLI2 substrate file:human/STK36/STK36-uniprot.txt ULK4 adapter (PubMed:38096226). GLI2 requires an additional function of |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: STK36-DMWD interaction reported by a systematic yeast two-hybrid screen of about 500 neurodegeneration-related proteins, which generated roughly 30,000 candidate interactions across some 5,000 proteins (PMID:32814053). No functional follow-up connects DMWD to STK36. Reason: Removal is not a claim that the two-hybrid result is false, and IntAct records three experiments for the pair. It is a statement that the row carries no functional information and that nothing supports a more informative molecular function. DMWD has no established role in Hedgehog signalling, in motile or primary ciliogenesis, or as a kinase substrate of STK36, so neither an adaptor term nor a kinase-activity term can be justified from this evidence, and inventing one would be exactly the error the protein-binding policy warns against. The five rows this screen contributed to STK36 all fall the same way. Supporting Evidence: PMID:32814053 Here, we report on an interactome map that focuses on neurodegenerative disease (ND), connects ∼5,000 human proteins via ∼30,000 candidate interactions and is generated by systematic yeast two-hybrid interaction screening of ∼500 ND-related proteins and integration of literature interactions. file:human/STK36/STK36-uniprot.txt Q9NRP7; G5E9A7: DMWD; NbExp= |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: STK36-KLF11 interaction reported by a systematic yeast two-hybrid screen of about 500 neurodegeneration-related proteins, which generated roughly 30,000 candidate interactions across some 5,000 proteins (PMID:32814053). No functional follow-up connects KLF11 to STK36. Reason: Removal is not a claim that the two-hybrid result is false, and IntAct records three experiments for the pair. It is a statement that the row carries no functional information and that nothing supports a more informative molecular function. KLF11 has no established role in Hedgehog signalling, in motile or primary ciliogenesis, or as a kinase substrate of STK36, so neither an adaptor term nor a kinase-activity term can be justified from this evidence, and inventing one would be exactly the error the protein-binding policy warns against. The five rows this screen contributed to STK36 all fall the same way. Supporting Evidence: PMID:32814053 Here, we report on an interactome map that focuses on neurodegenerative disease (ND), connects ∼5,000 human proteins via ∼30,000 candidate interactions and is generated by systematic yeast two-hybrid interaction screening of ∼500 ND-related proteins and integration of literature interactions. file:human/STK36/STK36-uniprot.txt Q9NRP7; O14901: KLF11; NbExp= |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: STK36-GFAP interaction reported by a systematic yeast two-hybrid screen of about 500 neurodegeneration-related proteins, which generated roughly 30,000 candidate interactions across some 5,000 proteins (PMID:32814053). No functional follow-up connects GFAP to STK36. Reason: Removal is not a claim that the two-hybrid result is false, and IntAct records three experiments for the pair. It is a statement that the row carries no functional information and that nothing supports a more informative molecular function. GFAP has no established role in Hedgehog signalling, in motile or primary ciliogenesis, or as a kinase substrate of STK36, so neither an adaptor term nor a kinase-activity term can be justified from this evidence, and inventing one would be exactly the error the protein-binding policy warns against. The five rows this screen contributed to STK36 all fall the same way. Supporting Evidence: PMID:32814053 Here, we report on an interactome map that focuses on neurodegenerative disease (ND), connects ∼5,000 human proteins via ∼30,000 candidate interactions and is generated by systematic yeast two-hybrid interaction screening of ∼500 ND-related proteins and integration of literature interactions. file:human/STK36/STK36-uniprot.txt Q9NRP7; P14136: GFAP; NbExp= |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: STK36-ATXN3 interaction reported by a systematic yeast two-hybrid screen of about 500 neurodegeneration-related proteins, which generated roughly 30,000 candidate interactions across some 5,000 proteins (PMID:32814053). No functional follow-up connects ATXN3 to STK36. Reason: Removal is not a claim that the two-hybrid result is false, and IntAct records three experiments for the pair. It is a statement that the row carries no functional information and that nothing supports a more informative molecular function. ATXN3 has no established role in Hedgehog signalling, in motile or primary ciliogenesis, or as a kinase substrate of STK36, so neither an adaptor term nor a kinase-activity term can be justified from this evidence, and inventing one would be exactly the error the protein-binding policy warns against. The five rows this screen contributed to STK36 all fall the same way. Supporting Evidence: PMID:32814053 Here, we report on an interactome map that focuses on neurodegenerative disease (ND), connects ∼5,000 human proteins via ∼30,000 candidate interactions and is generated by systematic yeast two-hybrid interaction screening of ∼500 ND-related proteins and integration of literature interactions. file:human/STK36/STK36-uniprot.txt Q9NRP7; P54252: ATXN3; NbExp= |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: STK36-SPRED1 interaction reported by a systematic yeast two-hybrid screen of about 500 neurodegeneration-related proteins, which generated roughly 30,000 candidate interactions across some 5,000 proteins (PMID:32814053). No functional follow-up connects SPRED1 to STK36. Reason: Removal is not a claim that the two-hybrid result is false, and IntAct records three experiments for the pair. It is a statement that the row carries no functional information and that nothing supports a more informative molecular function. SPRED1 has no established role in Hedgehog signalling, in motile or primary ciliogenesis, or as a kinase substrate of STK36, so neither an adaptor term nor a kinase-activity term can be justified from this evidence, and inventing one would be exactly the error the protein-binding policy warns against. The five rows this screen contributed to STK36 all fall the same way. Supporting Evidence: PMID:32814053 Here, we report on an interactome map that focuses on neurodegenerative disease (ND), connects ∼5,000 human proteins via ∼30,000 candidate interactions and is generated by systematic yeast two-hybrid interaction screening of ∼500 ND-related proteins and integration of literature interactions. file:human/STK36/STK36-uniprot.txt Q9NRP7; Q7Z699: SPRED1; NbExp= |
| GO:0005515 protein binding | IPI PMID:37584603 The putative protein kinase Stk36 is essential for ciliogene... | MODIFY | Summary: STK36-ULK4 interaction demonstrated by yeast two-hybrid and confirmed by co-immunoprecipitation in HEK293T cells, with the interface mapped to the N-terminal kinase domains of both proteins (PMID:37584603). The same study showed that Stk36 and Ulk4 hypomorphs share a phenotype. Reason: This is not a generic contact and the bare row understates what is known. ULK4 is a pseudokinase that acts as the adaptor bridging STK36 to its GLI2 substrate, and it is itself an STK36 substrate: an in vitro kinase assay with immunopurified STK36 and a GST-ULK4 peptide identified phosphorylation of Thr1021/Thr1023, and that phosphorylation is what drives ULK4 to the ciliary tip (PMID:38096226). The informative molecular function behind these rows is therefore the serine/threonine kinase activity STK36 exerts on ULK4, not an unqualified binding statement. UniProt records the functional reading directly: "Interacts with ULK4; promoting association with GLI2 substrate" and "Also catalyzes phosphorylation of ULK4 adapter". The domain mapping in this paper adds something the other rows do not: the contact is kinase-domain to pseudokinase-domain, which is consistent with ULK4 both engaging the catalytic face of STK36 and being phosphorylated by it. Proposed replacements: protein serine/threonine kinase activity Supporting Evidence: PMID:37584603 Here, we report that serine/threonine kinase 36 (STK36), a paralog of ULK4, directly interacts with ULK4 and this was demonstrated by yeast two-hybrid (Y2H) in yeast and coimmunoprecipitation (co-IP) assays in HEK293T cells, respectively. PMID:37584603 The interaction region was confined to their respective N-terminal kinase domain. PMID:38096226 To confirm T1021 phosphorylation by Stk36, we carried out an in vitro kinase assay using immunopurified Fg-Stk36 as kinase and GST fusion protein containing hUlk4 sequence from aa1017 to aa1036 (GST-hUlk4C) as substrate. file:human/STK36/STK36-uniprot.txt Q9NRP7; Q96C45: ULK4; NbExp=4; IntAct=EBI-863797, EBI-3922541; |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: ATP binding inferred from the protein-kinase domain signatures IPR000719 and IPR017441. UniProt records two ATP-binding features, the glycine-rich loop at residues 10-18 and Lys-33, the latter with evidence from PMID:10806483 and PMID:31279575. Reason: Correct and core - an active kinase must bind ATP, and STK36 demonstrably turns ATP over on GLI2 and ULK4 substrates in vitro. Lys-33 is the residue Murone et al. mutated to make the catalytically inactive form, which is the functional confirmation that this site is the ATP site rather than a pattern match. Supporting Evidence: file:human/STK36/STK36-uniprot.txt /note="Protein kinase" file:human/STK36/STK36-uniprot.txt ECO:0000305|PubMed:10806483, PMID:38096226 In an in vitro kinase assay, we found that immunopurified Stk36 phosphorylated GST-Gli2N equally well regardless of whether it interacted with Ulk4 or not, suggesting that Ulk4 does not regulate Stk36 kinase activity. |
| GO:0005524 ATP binding | TAS PMID:10806483 Gli regulation by the opposing activities of fused and suppr... | ACCEPT | Summary: ATP binding as an author statement from Murone et al., who made the Lys-33 substitution to generate a kinase-inactive STK36 and used it to separate the catalytic from the non-catalytic requirements of GLI2 activation. Reason: The statement is correct and is backed by the experiment the same paper performed: UniProt records BINDING 33 for ATP citing this paper, and the reference block lists MUTAGENESIS OF LYS-33 among its contributions. The cached record for PMID:10806483 holds citation metadata only, with no abstract text, so the paper itself could not be read. It was adjudicated instead against the structured UniProt annotations that cite it with experimental evidence codes, and against later experimental rows that reach the same conclusion. Duplication with the InterPro row is not a problem. Supporting Evidence: file:human/STK36/STK36-uniprot.txt GLI2; GLI3 AND SUFU, TISSUE SPECIFICITY, SUBCELLULAR LOCATION, AND file:human/STK36/STK36-uniprot.txt ECO:0000305|PubMed:10806483, |
| GO:0005634 nucleus | IDA PMID:10806483 Gli regulation by the opposing activities of fused and suppr... | KEEP AS NON CORE | Summary: Nuclear localisation observed by Murone et al. UniProt qualifies it explicitly: "Low levels also present in the nucleus (PubMed:10806483)". The functional pool of STK36 is at the ciliary tip, where it phosphorylates GLI2 (PMID:38096226). Reason: Not doubted as an observation - the same paper reported that STK36 promotes nuclear localisation of GLI1, GLI2 and GLI3, so a nuclear pool is expected - but kept off the core list because it is a low-level pool with no activity assigned to it. Everything mechanistic about STK36 happens elsewhere: GLI2 phosphorylation at the ciliary tip, and central-pair assembly in the axoneme. GLI nuclear entry is a consequence of the phosphorylation, not evidence that STK36 acts in the nucleus. The cached record for PMID:10806483 holds citation metadata only, with no abstract text, so the paper itself could not be read. It was adjudicated instead against the structured UniProt annotations that cite it with experimental evidence codes, and against later experimental rows that reach the same conclusion. Supporting Evidence: file:human/STK36/STK36-uniprot.txt Nucleus {ECO:0000269|PubMed:10806483}. Note=STK36 and ULK4 are mutually PMID:38096226 In this study, we found that Stk36 accumulated to the tip of primary cilium and colocalized with Gli2 in response to Shh stimulation, consistent with Gli2 being converted into Gli2A at the ciliary tip. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Nucleus derived from the UniProt subcellular-location keyword SL-0191, which itself comes from the low-level nuclear pool reported in PMID:10806483. Reason: A faithful mapping of the UniProt location, so there is nothing mechanically wrong with it; but the keyword mapping necessarily drops the "low levels" qualifier that UniProt attaches to the free-text annotation, which makes the row look more definite than the underlying observation. Kept for the same reasons as the IDA row and for the same reason not promoted to core. Supporting Evidence: file:human/STK36/STK36-uniprot.txt Nucleus {ECO:0000269|PubMed:10806483}. Note=STK36 and ULK4 are mutually |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Cytoplasm as the active compartment, from PAINT node PTN000541535, whose donors include Drosophila fu, mouse Stk36, human STK36, an Arabidopsis and a Dictyostelium kinase. Reason: True, but not where the function is. STK36 acts at the tip of the primary cilium in Hedgehog signalling and in the axoneme of motile cilia in central-pair assembly; both are cytoplasmic in the ontology sense, so the term is not wrong, merely uninformative at this level. It is kept rather than removed for a specific reason: the Drosophila arm of this pathway genuinely runs the Fused step on a cytoplasmic complex with no cilium involved, so cytoplasm is the location that makes the pan-metazoan statement, and the PAINT node behind the IBA row includes Drosophila fu among its donors. Supporting Evidence: file:human/STK36/STK36-uniprot.txt {ECO:0000269|PubMed:38096226}. Cytoplasm, cytoskeleton, cilium axoneme file:human/STK36/STK36-uniprot.txt Cytoplasm {ECO:0000269|PubMed:10806483}. |
| GO:0005737 cytoplasm | IDA PMID:10806483 Gli regulation by the opposing activities of fused and suppr... | KEEP AS NON CORE | Summary: Cytoplasmic localisation observed by Murone et al.; UniProt records "Cytoplasm {ECO:0000269|PubMed:10806483}" as one of four locations for STK36. Reason: True, but not where the function is. STK36 acts at the tip of the primary cilium in Hedgehog signalling and in the axoneme of motile cilia in central-pair assembly; both are cytoplasmic in the ontology sense, so the term is not wrong, merely uninformative at this level. It is kept rather than removed for a specific reason: the Drosophila arm of this pathway genuinely runs the Fused step on a cytoplasmic complex with no cilium involved, so cytoplasm is the location that makes the pan-metazoan statement, and the PAINT node behind the IBA row includes Drosophila fu among its donors. The cached record for PMID:10806483 holds citation metadata only, with no abstract text, so the paper itself could not be read. It was adjudicated instead against the structured UniProt annotations that cite it with experimental evidence codes, and against later experimental rows that reach the same conclusion. Supporting Evidence: file:human/STK36/STK36-uniprot.txt Cytoplasm {ECO:0000269|PubMed:10806483}. file:human/STK36/STK36-uniprot.txt GLI2; GLI3 AND SUFU, TISSUE SPECIFICITY, SUBCELLULAR LOCATION, AND |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Cytoplasm from combined automated methods: an ARBA model, orthology to mouse Stk36, and the UniProt subcellular-location keyword SL-0086. Reason: Agrees with the experimental rows and with UniProt, so no correction is needed; kept as non-core for the same reason. True, but not where the function is. STK36 acts at the tip of the primary cilium in Hedgehog signalling and in the axoneme of motile cilia in central-pair assembly; both are cytoplasmic in the ontology sense, so the term is not wrong, merely uninformative at this level. It is kept rather than removed for a specific reason: the Drosophila arm of this pathway genuinely runs the Fused step on a cytoplasmic complex with no cilium involved, so cytoplasm is the location that makes the pan-metazoan statement, and the PAINT node behind the IBA row includes Drosophila fu among its donors. Supporting Evidence: file:human/STK36/STK36-uniprot.txt Cytoplasm {ECO:0000269|PubMed:10806483}. |
| GO:0005829 cytosol | IDA GO_REF:0000052 | KEEP AS NON CORE | Summary: Cytosolic staining recorded from curated immunofluorescence (HPA, GO_REF:0000052). UniProt carries the corresponding IDA:HPA cross-reference for GO:0005829. Reason: An antibody-staining observation in a cell line, kept because it is a real and independently curated localisation but not promoted. Cytosol is more restrictive than cytoplasm - it excludes organelles and the cytoskeleton - and so it sits awkwardly with the two compartments where STK36 function has been localised, the ciliary tip and the axoneme, neither of which is cytosol. In a non-ciliated cultured cell a diffuse pool is what one would expect to see, so the row is best read as describing the unengaged protein. Supporting Evidence: file:human/STK36/STK36-uniprot.txt GO:0005829; C:cytosol; IDA:HPA. PMID:38096226 In this study, we found that Stk36 accumulated to the tip of primary cilium and colocalized with Gli2 in response to Shh stimulation, consistent with Gli2 being converted into Gli2A at the ciliary tip. |
| GO:0005929 cilium | IEA GO_REF:0000044 | ACCEPT | Summary: Cilium from the UniProt subcellular-location keyword SL-0066, which traces to the experimental ciliary localisation in PMID:38096226 ("Cell projection, cilium {ECO:0000269|PubMed:38096226}"). Reason: Correct and core. STK36 is a ciliary protein in both of its roles - at the tip of the primary cilium with ULK4 and GLI2 in Hedgehog signalling, and in the axoneme of motile cilia - and the keyword mapping faithfully reflects an experimentally evidenced UniProt annotation rather than a prediction. The more specific sibling rows (GO:0097542 ciliary tip, GO:0005930 axoneme) give the sub-compartments; the parent term is the right umbrella because the two are different organelle types. Supporting Evidence: file:human/STK36/STK36-uniprot.txt {ECO:0000269|PubMed:38096226}. Cytoplasm, cytoskeleton, cilium axoneme PMID:38096226 In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. PMID:28543983 Transmission electron microscopy analysis demonstrates that STK36 is required for cilia orientation in human respiratory epithelial cells, with a probable localization of STK36 between the RS and CP. |
| GO:0005930 axoneme | IEA GO_REF:0000044 | ACCEPT | Summary: Axoneme from the UniProt subcellular-location keyword SL-0304, tracing to PMID:28543983 ("Cytoplasm, cytoskeleton, cilium axoneme {ECO:0000269|PubMed:28543983}"). Electron microscopy of respiratory cilia from a patient with biallelic STK36 loss placed the protein between the radial spokes and the central pair. Reason: Correct and core, and this is the location that belongs to the Hedgehog-independent half of STK36 function. Mouse work independently places Fu/Stk36 in the central pair apparatus of motile 9+2 cilia and links it to known structural components of that apparatus (PMID:19305393). Accepting the axoneme is what keeps the record from reading as though STK36 were only a signalling protein. Supporting Evidence: file:human/STK36/STK36-uniprot.txt {ECO:0000269|PubMed:38096226}. Cytoplasm, cytoskeleton, cilium axoneme PMID:28543983 Transmission electron microscopy analysis demonstrates that STK36 is required for cilia orientation in human respiratory epithelial cells, with a probable localization of STK36 between the RS and CP. PMID:19305393 Here we show that mouse Fu is essential for construction of the central pair apparatus of motile, 9+2 cilia and offers a new model of human primary ciliary dyskinesia. PMID:19305393 We found that mouse Fu physically interacts with Kif27, a mammalian Cos2 orthologue, and linked Fu to known structural components of the central pair apparatus, providing evidence for the first regulatory component involved in central pair construction. |
| GO:0007017 microtubule-based process | IEA GO_REF:0000117 | MODIFY | Summary: Microtubule-based process assigned by an ARBA machine-learning model. The specific microtubule process STK36 participates in is axonemal: it is required to build the central pair apparatus of motile cilia (PMID:19305393, PMID:28543983). Reason: The parent term is true - axoneme assembly is a descendant of GO:0007017 - but uninformative, and the specific child is already established for this gene by IMP. Replacing the generic machine-learned row with GO:0035082 axoneme assembly states what the microtubule involvement actually is. This is preferable to removal because the ARBA call is not wrong, only vague, and it is preferable to acceptance because nothing in the record would otherwise connect the generic term to the central-pair phenotype. Proposed replacements: axoneme assembly Supporting Evidence: PMID:19305393 Here we show that mouse Fu is essential for construction of the central pair apparatus of motile, 9+2 cilia and offers a new model of human primary ciliary dyskinesia. PMID:28543983 Transmission electron microscopy analysis demonstrates that STK36 is required for cilia orientation in human respiratory epithelial cells, with a probable localization of STK36 between the RS and CP. file:human/STK36/STK36-uniprot.txt Essential for construction of the central pair |
| GO:0007224 smoothened signaling pathway | IBA GO_REF:0000033 | ACCEPT | Summary: Smoothened signaling pathway from PAINT node PTN001122621. The donor set is exactly the one that makes this node interpretable: Drosophila fu (FB:FBgn0001079), mouse Stk36 (MGI:MGI:1920831), human STK36 itself and a zebrafish gene - the Fused-family clade, with experimental evidence in protostome and deuterostome members. Reason: The node placement is sound and the target is squarely inside the clade that inherited the function: human STK36 and Drosophila fu share PANTHER subfamily PTHR22983:SF6, which is direct classification support for the orthology rather than a pathway analogy. Human STK36 appearing in its own WITH/FROM list is expected here, not circular - it has its own experimental annotations for this term, which are among the descendant evidences a PAINT curator would have used, and the IBA then adds the claim that the role is inherited rather than lineage-specific. One apparent conflict has to be addressed before accepting any Hedgehog annotation for STK36: mouse knockouts respond normally to Hedgehog. Merchant et al. found normal neural-tube cell-fate specification and unaffected target-gene induction, and Wilson et al. state flatly that mouse Fu mutants seem to respond normally to Hh signalling. The resolution is paralogue redundancy, and it is now demonstrated rather than assumed: two Fu-family kinases phosphorylate Gli2 in mammals, and knocking down ULK3 and ULK4 together reduces Gli2 phosphorylation further while knocking down STK36 and ULK4 together does not, placing STK36 and ULK4 in one pathway and ULK3 in parallel (PMID:38096226). A single knockout of one of two redundant kinases is expected to be phenotypically silent, so the mouse result is not evidence against the activity. Two further observations confirm the activity is retained in mammals: mouse Fu rescues the Hedgehog-dependent defects of zebrafish fu mutants, where the requirement is not redundant (PMID:19305393), and mouse Fu knockdown severely impaired Hedgehog signalling in a cell line (PMID:18600476). Supporting Evidence: PMID:31279575 Hence, Fu-family kinase-mediated phosphorylation of Ci/Gli serves as a conserved mechanism that activates the Hh pathway transcription factor. PMID:31279575 Here, we demonstrate that Hh stimulates the phosphorylation of Ci by the Ser/Thr kinase Fused (Fu) and that Fu-mediated phosphorylation of Ci promotes its activation. PMID:38096226 Consistent with Ulk4 acting in a liner pathway with Stk36 but in parallel with Ulk3, double KD of Ulk4 and Stk36 decreased S230/232 phosphorylation similarly to single KD of either Ulk4 or Stk36, whereas double KD of Ulk3 and Ulk4 further decreased Shh-induced S230/232 phosphorylation compared with single KD of Ulk3 or Ulk4 PMID:19305393 Mouse Fu rescued both Hh-dependent and -independent defects in zebrafish. file:human/STK36/STK36-uniprot.txt PTHR22983:SF6; SERINE_THREONINE-PROTEIN KINASE 36 |
| GO:0007224 smoothened signaling pathway | IEA GO_REF:0000120 | ACCEPT | Summary: Smoothened signaling pathway from combined automated methods (ARBA model plus orthology to mouse Stk36). Agrees with the IBA, ISS and IMP rows for the same term. Reason: Redundant with better-evidenced rows rather than wrong, and pitched at the right level: the pathway term rather than a regulatory child, which is appropriate for an automated call. No change needed. Supporting Evidence: PMID:31279575 Furthermore, we provide evidence that Sonic hedgehog (Shh) activates Gli2 by stimulating its phosphorylation on conserved sites through the Fu-family kinases ULK3 and mFu/STK36 in a manner depending on Gli2 ciliary localization. |
| GO:0007224 smoothened signaling pathway | IMP PMID:31279575 Phosphorylation of Ci/Gli by Fused Family Kinases Promotes H... | ACCEPT | Summary: Han et al. showed that depleting the Fu-family kinases blocks Shh-induced Gli2 phosphorylation and pathway activation, in a manner depending on Gli2 ciliary localisation. Annotated on the human gene by FlyBase curators from the same paper that establishes the fly mechanism. Reason: Core, and the primary experimental grounding for every other smoothened-pathway row on this record. The paper is unusual and useful in doing the fly and mammalian halves together: it maps the Fused phosphorylation sites on Ci, shows they prime CK1 and alter Ci binding to Sufu, and then shows Shh activates Gli2 through phosphorylation of the conserved sites by ULK3 and mFu/STK36. One apparent conflict has to be addressed before accepting any Hedgehog annotation for STK36: mouse knockouts respond normally to Hedgehog. Merchant et al. found normal neural-tube cell-fate specification and unaffected target-gene induction, and Wilson et al. state flatly that mouse Fu mutants seem to respond normally to Hh signalling. The resolution is paralogue redundancy, and it is now demonstrated rather than assumed: two Fu-family kinases phosphorylate Gli2 in mammals, and knocking down ULK3 and ULK4 together reduces Gli2 phosphorylation further while knocking down STK36 and ULK4 together does not, placing STK36 and ULK4 in one pathway and ULK3 in parallel (PMID:38096226). A single knockout of one of two redundant kinases is expected to be phenotypically silent, so the mouse result is not evidence against the activity. Two further observations confirm the activity is retained in mammals: mouse Fu rescues the Hedgehog-dependent defects of zebrafish fu mutants, where the requirement is not redundant (PMID:19305393), and mouse Fu knockdown severely impaired Hedgehog signalling in a cell line (PMID:18600476). Supporting Evidence: PMID:31279575 Furthermore, we provide evidence that Sonic hedgehog (Shh) activates Gli2 by stimulating its phosphorylation on conserved sites through the Fu-family kinases ULK3 and mFu/STK36 in a manner depending on Gli2 ciliary localization. PMID:31279575 These phosphorylation events alter Ci binding to the pathway inhibitor Suppressor of fused (Sufu) and facilitate the recruitment of Transportion and the transcriptional coactivator CBP. PMID:38096226 Consistent with Ulk4 acting in a liner pathway with Stk36 but in parallel with Ulk3, double KD of Ulk4 and Stk36 decreased S230/232 phosphorylation similarly to single KD of either Ulk4 or Stk36, whereas double KD of Ulk3 and Ulk4 further decreased Shh-induced S230/232 phosphorylation compared with single KD of Ulk3 or Ulk4 PMID:19305393 Mouse Fu rescued both Hh-dependent and -independent defects in zebrafish. |
| GO:0007224 smoothened signaling pathway | ISS GO_REF:0000024 | ACCEPT | Summary: Smoothened signaling pathway transferred by curator judgment from mouse Stk36 (UniProtKB:Q69ZM6). The mouse record grounds the term on an IMP from PMID:18600476, where RNAi against mouse Fu severely impaired Hedgehog signalling in one cell line while overexpression enhanced Gli-induced transcription as human FU does. Reason: A safe one-to-one orthologue transfer - mouse and human STK36 are unambiguous orthologues in PANTHER subfamily PTHR22983:SF6 - and the mouse grounding is a genuine experiment rather than another inference. The mouse source is cell-context dependent, which the source paper says explicitly, and that context dependence is exactly what the later redundancy work explains. One apparent conflict has to be addressed before accepting any Hedgehog annotation for STK36: mouse knockouts respond normally to Hedgehog. Merchant et al. found normal neural-tube cell-fate specification and unaffected target-gene induction, and Wilson et al. state flatly that mouse Fu mutants seem to respond normally to Hh signalling. The resolution is paralogue redundancy, and it is now demonstrated rather than assumed: two Fu-family kinases phosphorylate Gli2 in mammals, and knocking down ULK3 and ULK4 together reduces Gli2 phosphorylation further while knocking down STK36 and ULK4 together does not, placing STK36 and ULK4 in one pathway and ULK3 in parallel (PMID:38096226). A single knockout of one of two redundant kinases is expected to be phenotypically silent, so the mouse result is not evidence against the activity. Two further observations confirm the activity is retained in mammals: mouse Fu rescues the Hedgehog-dependent defects of zebrafish fu mutants, where the requirement is not redundant (PMID:19305393), and mouse Fu knockdown severely impaired Hedgehog signalling in a cell line (PMID:18600476). Supporting Evidence: PMID:18600476 In one cell line with reduced mFu expression the Hedgehog signaling was severely hampered, indicating that mFu may have a role in Hedgehog signaling and Gli regulation in some cellular situations. PMID:18600476 However, in transient overexpression analyses mFu was able to enhance Gli induced transcription in a manner similar to hFU. PMID:18600476 Human FU (hFU) has been shown to enhance the activity of Gli transcription factors, targets of the signaling pathway. PMID:38096226 Consistent with Ulk4 acting in a liner pathway with Stk36 but in parallel with Ulk3, double KD of Ulk4 and Stk36 decreased S230/232 phosphorylation similarly to single KD of either Ulk4 or Stk36, whereas double KD of Ulk3 and Ulk4 further decreased Shh-induced S230/232 phosphorylation compared with single KD of Ulk3 or Ulk4 |
| GO:0009791 post-embryonic development | IEA GO_REF:0000107 | MARK AS OVER ANNOTATED | Summary: Post-embryonic development transferred automatically from mouse Stk36 through Ensembl Compara. The mouse annotation rests on PMID:16055717, where fused knockout mice develop normally and then fail to thrive postnatally from progressive hydrocephalus. Reason: Not wrong, but an over-annotation. The mouse evidence behind this term is that knockouts are born in normal ratios and develop normally, then fail to thrive within two weeks with communicating hydrocephalus and early mortality. That is a whole-organism outcome at the end of a long causal chain: STK36 phosphorylates its substrates, which is needed to build the central pair apparatus of motile cilia, which is needed for ependymal ciliary beating, which is needed for cerebrospinal fluid circulation, the failure of which causes hydrocephalus and hence the failure to thrive. The source paper itself frames the connection as conjecture - "possibly by regulating the CSF homeostasis or ciliary function". STK36 does not execute post-embryonic development in any sense the term intends, and the specific ciliary terms on this record already capture what it does do. The row is marked rather than removed because the genetic dependency is real and the phenotype is the reason STK36 was pursued as a disease gene at all. Supporting Evidence: PMID:16055717 fused knockouts develop normally, being born in Mendelian ratios, but fail to thrive within 2 weeks, displaying profound growth retardation with communicating hydrocephalus and early mortality. PMID:16055717 These results show that the mFu homologue is not required for Hh signaling during embryonic development but is required for proper postnatal development, possibly by regulating the CSF homeostasis or ciliary function. PMID:37584603 The hypomorph mutant of Stk36 (Stk36tmE4-/- ) also developed progressive hydrocephalus postnatally and dysfunctional CSF flow, with multiple defects of motile cilia, including reduced ciliary number, disorganized ciliary orientation, defected axonemal structure and inconsistent base body (BB) orientation. |
| GO:0009791 post-embryonic development | ISS GO_REF:0000024 | MARK AS OVER ANNOTATED | Summary: The same post-embryonic development claim, transferred a second time by curator judgment of sequence similarity to mouse Stk36 rather than by Compara. Same mouse source, same phenotype chain. Reason: Not wrong, but an over-annotation. The mouse evidence behind this term is that knockouts are born in normal ratios and develop normally, then fail to thrive within two weeks with communicating hydrocephalus and early mortality. That is a whole-organism outcome at the end of a long causal chain: STK36 phosphorylates its substrates, which is needed to build the central pair apparatus of motile cilia, which is needed for ependymal ciliary beating, which is needed for cerebrospinal fluid circulation, the failure of which causes hydrocephalus and hence the failure to thrive. The source paper itself frames the connection as conjecture - "possibly by regulating the CSF homeostasis or ciliary function". STK36 does not execute post-embryonic development in any sense the term intends, and the specific ciliary terms on this record already capture what it does do. The row is marked rather than removed because the genetic dependency is real and the phenotype is the reason STK36 was pursued as a disease gene at all. The duplication is not itself a problem - the orthology is not in doubt and duplicate rows from different pipelines are normal - but both rows inherit the same distance between the activity and the term. Supporting Evidence: PMID:16055717 fused knockouts develop normally, being born in Mendelian ratios, but fail to thrive within 2 weeks, displaying profound growth retardation with communicating hydrocephalus and early mortality. PMID:16055717 Specification of neuronal cell fates by Hh in the neural tube was normal in fused knockouts, and induction of Hh target genes in numerous tissues is not affected by the loss of mFu. PMID:16055717 These results show that the mFu homologue is not required for Hh signaling during embryonic development but is required for proper postnatal development, possibly by regulating the CSF homeostasis or ciliary function. |
| GO:0035082 axoneme assembly | IMP PMID:28543983 Mutation of serine/threonine protein kinase 36 (STK36) cause... | ACCEPT | Summary: Biallelic loss-of-function STK36 variants in an individual with primary ciliary dyskinesia and a central-pair axonemal defect; transmission electron microscopy showed disordered cilia orientation in respiratory epithelium and placed STK36 between the radial spokes and the central pair (PMID:28543983). Mouse Fu is essential for construction of the same structure (PMID:19305393). Reason: Core, and the second of STK36's two distinct functions. This is genuine participation rather than a downstream requirement: mouse Fu physically interacts with KIF27 and with known structural components of the central pair apparatus, which makes it the first identified regulatory component of that assembly step rather than something the step merely needs. The human and mouse evidence agree on the structure affected, and UniProt states the conclusion as "Essential for construction of the central pair apparatus of motile cilia". Note the symmetry with the Hedgehog role: in both, STK36 works with a Costal-2-family kinesin - KIF7 in the ciliary-tip GLI complex, KIF27 in the central pair. Supporting Evidence: PMID:28543983 We identified homozygous loss-of-function mutations in STK36 in one PCD-affected individual with situs solitus. PMID:28543983 Transmission electron microscopy analysis demonstrates that STK36 is required for cilia orientation in human respiratory epithelial cells, with a probable localization of STK36 between the RS and CP. PMID:28543983 A minor PCD subgroup including defects of the radial spokes (RS) and central pair (CP) is hallmarked by the absence of laterality defects, subtle beating abnormalities, and unequivocally apparent ultrastructural defects of the ciliary axoneme, making their diagnosis challenging. PMID:19305393 Here we show that mouse Fu is essential for construction of the central pair apparatus of motile, 9+2 cilia and offers a new model of human primary ciliary dyskinesia. PMID:19305393 We found that mouse Fu physically interacts with Kif27, a mammalian Cos2 orthologue, and linked Fu to known structural components of the central pair apparatus, providing evidence for the first regulatory component involved in central pair construction. file:human/STK36/STK36-uniprot.txt Essential for construction of the central pair |
| GO:0045880 positive regulation of smoothened signaling pathway | IDA PMID:38096226 Ulk4 promotes Shh signaling by regulating Stk36 ciliary loca... | ACCEPT | Summary: Zhou et al. showed that STK36, with ULK4, promotes Gli2 phosphorylation on Ser230/Ser232 and Hedgehog pathway activation, that both accumulate with Gli2 at the ciliary tip in response to Shh, and that kinase-inactive STK36 cannot support the step (PMID:38096226). Reason: Core, and the best-evidenced process row on this record. The direction of regulation is established by the readouts used - Gli2 Ser230/232 phosphorylation and Ptch1 and Gli1 target-gene expression both fall when the STK36-ULK4 module is disrupted and are restored by wild-type but not kinase-dead STK36 - so the positive sign is not inferred from a correlation. This is the pairing the Hedgehog module records for its Fused-family kinase annoton (GO:0004674 with GO:0045880) and the pairing GO-CAM model 696022cd00001645 gives STK36 at the ciliary tip. One apparent conflict has to be addressed before accepting any Hedgehog annotation for STK36: mouse knockouts respond normally to Hedgehog. Merchant et al. found normal neural-tube cell-fate specification and unaffected target-gene induction, and Wilson et al. state flatly that mouse Fu mutants seem to respond normally to Hh signalling. The resolution is paralogue redundancy, and it is now demonstrated rather than assumed: two Fu-family kinases phosphorylate Gli2 in mammals, and knocking down ULK3 and ULK4 together reduces Gli2 phosphorylation further while knocking down STK36 and ULK4 together does not, placing STK36 and ULK4 in one pathway and ULK3 in parallel (PMID:38096226). A single knockout of one of two redundant kinases is expected to be phenotypically silent, so the mouse result is not evidence against the activity. Two further observations confirm the activity is retained in mammals: mouse Fu rescues the Hedgehog-dependent defects of zebrafish fu mutants, where the requirement is not redundant (PMID:19305393), and mouse Fu knockdown severely impaired Hedgehog signalling in a cell line (PMID:18600476). Supporting Evidence: PMID:38096226 Here, we show that Unc-51-like kinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. PMID:38096226 In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. PMID:38096226 Adding back wild type Stk36 (Stk36WT) but not its kinase inactive form (Stk36AA) restored Shh-induced Ulk4-HA ciliary tip accumulation in DKO cells PMID:38096226 Consistent with Ulk4 acting in a liner pathway with Stk36 but in parallel with Ulk3, double KD of Ulk4 and Stk36 decreased S230/232 phosphorylation similarly to single KD of either Ulk4 or Stk36, whereas double KD of Ulk3 and Ulk4 further decreased Shh-induced S230/232 phosphorylation compared with single KD of Ulk3 or Ulk4 PMID:31279575 Furthermore, we provide evidence that Sonic hedgehog (Shh) activates Gli2 by stimulating its phosphorylation on conserved sites through the Fu-family kinases ULK3 and mFu/STK36 in a manner depending on Gli2 ciliary localization. |
| GO:0045880 positive regulation of smoothened signaling pathway | IEA GO_REF:0000120 | ACCEPT | Summary: Positive regulation of the smoothened signaling pathway from combined automated methods - an ARBA model plus orthology to mouse Stk36, which carries the term from both IMP and IDA rows. Reason: An electronic row that agrees with the human IDA and the mouse experimental rows, and that gets the regulatory sign right, which is the thing most easily lost in automated transfer for a family containing both activators and inhibitors. No change needed. Supporting Evidence: PMID:38096226 Here, we show that Unc-51-like kinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. |
| GO:0045880 positive regulation of smoothened signaling pathway | ISS GO_REF:0000024 | ACCEPT | Summary: Positive regulation of the smoothened signaling pathway transferred by curator judgment from mouse Stk36, whose record carries the term from an IMP (PMID:18600476) and an IDA (PMID:38096226). Reason: The transfer is between unambiguous one-to-one orthologues in PANTHER subfamily PTHR22983:SF6, the mouse grounding is experimental rather than inferred, and the direction of regulation is the same in both species: in mouse cells, reducing Fu expression impaired Hedgehog signalling and overexpression enhanced Gli-induced transcription. Accepted as core alongside the human IDA. Supporting Evidence: PMID:18600476 In one cell line with reduced mFu expression the Hedgehog signaling was severely hampered, indicating that mFu may have a role in Hedgehog signaling and Gli regulation in some cellular situations. PMID:18600476 However, in transient overexpression analyses mFu was able to enhance Gli induced transcription in a manner similar to hFU. PMID:38096226 Here, we show that Unc-51-like kinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. |
| GO:0060271 cilium assembly | IEA GO_REF:0000120 | MODIFY | Summary: Cilium assembly from combined automated methods (ARBA model plus orthology to mouse Stk36). The ciliary requirement is real but is specific to motile cilia (PMID:19305393, PMID:28543983, PMID:37584603). Reason: The essence is right and the term is one level too broad in a way that matters biologically. GO:0060271 covers primary cilia as well as motile ones, and STK36-null cells build primary cilia perfectly well - that is precisely why Hedgehog signalling still runs in mouse fused knockouts and why the mouse phenotype is postnatal rather than embryonic. Every ciliary defect reported for STK36 loss is in motile 9+2 cilia: respiratory epithelium in the human patient, ependymal cilia in the mouse hypomorph, and Kupffer's vesicle in zebrafish, with reduced ciliary number, disorganised orientation and defective axonemal structure. GO:0044458 motile cilium assembly says this precisely and remains a direct child of GO:0060271, so no information is lost and the misleading implication about primary cilia is removed. Proposed replacements: motile cilium assembly Supporting Evidence: PMID:19305393 Here we show that mouse Fu is essential for construction of the central pair apparatus of motile, 9+2 cilia and offers a new model of human primary ciliary dyskinesia. PMID:19305393 Mouse Fu (also known as Stk36) mutants are viable and seem to respond normally to Hh signalling. PMID:37584603 The hypomorph mutant of Stk36 (Stk36tmE4-/- ) also developed progressive hydrocephalus postnatally and dysfunctional CSF flow, with multiple defects of motile cilia, including reduced ciliary number, disorganized ciliary orientation, defected axonemal structure and inconsistent base body (BB) orientation. PMID:16055717 Specification of neuronal cell fates by Hh in the neural tube was normal in fused knockouts, and induction of Hh target genes in numerous tissues is not affected by the loss of mFu. |
| GO:0060271 cilium assembly | IMP PMID:28543983 Mutation of serine/threonine protein kinase 36 (STK36) cause... | MODIFY | Summary: Cilium assembly inferred from the primary ciliary dyskinesia phenotype of biallelic STK36 loss: a central-pair and radial-spoke defect in respiratory cilia with disordered ciliary orientation and no laterality abnormality (PMID:28543983). Reason: The essence is right and the term is one level too broad in a way that matters biologically. GO:0060271 covers primary cilia as well as motile ones, and STK36-null cells build primary cilia perfectly well - that is precisely why Hedgehog signalling still runs in mouse fused knockouts and why the mouse phenotype is postnatal rather than embryonic. Every ciliary defect reported for STK36 loss is in motile 9+2 cilia: respiratory epithelium in the human patient, ependymal cilia in the mouse hypomorph, and Kupffer's vesicle in zebrafish, with reduced ciliary number, disorganised orientation and defective axonemal structure. GO:0044458 motile cilium assembly says this precisely and remains a direct child of GO:0060271, so no information is lost and the misleading implication about primary cilia is removed. The source evidence here is specifically respiratory epithelial cilia, which are motile, so the more precise term is not an extrapolation but a closer reading of this paper's own data. The sibling axoneme-assembly row from the same paper is accepted unchanged; together they say that STK36 builds the motile cilium and, within it, the central pair of the axoneme. Proposed replacements: motile cilium assembly Supporting Evidence: PMID:28543983 We identified homozygous loss-of-function mutations in STK36 in one PCD-affected individual with situs solitus. PMID:28543983 A minor PCD subgroup including defects of the radial spokes (RS) and central pair (CP) is hallmarked by the absence of laterality defects, subtle beating abnormalities, and unequivocally apparent ultrastructural defects of the ciliary axoneme, making their diagnosis challenging. PMID:37584603 The hypomorph mutant of Stk36 (Stk36tmE4-/- ) also developed progressive hydrocephalus postnatally and dysfunctional CSF flow, with multiple defects of motile cilia, including reduced ciliary number, disorganized ciliary orientation, defected axonemal structure and inconsistent base body (BB) orientation. PMID:19305393 Here we show that mouse Fu is essential for construction of the central pair apparatus of motile, 9+2 cilia and offers a new model of human primary ciliary dyskinesia. |
| GO:0060271 cilium assembly | ISS GO_REF:0000024 | MODIFY | Summary: Cilium assembly transferred by curator judgment from mouse Stk36, whose record grounds the term on PMID:19305393 - the study showing mouse Fu is essential for construction of the central pair apparatus of motile 9+2 cilia. Reason: The essence is right and the term is one level too broad in a way that matters biologically. GO:0060271 covers primary cilia as well as motile ones, and STK36-null cells build primary cilia perfectly well - that is precisely why Hedgehog signalling still runs in mouse fused knockouts and why the mouse phenotype is postnatal rather than embryonic. Every ciliary defect reported for STK36 loss is in motile 9+2 cilia: respiratory epithelium in the human patient, ependymal cilia in the mouse hypomorph, and Kupffer's vesicle in zebrafish, with reduced ciliary number, disorganised orientation and defective axonemal structure. GO:0044458 motile cilium assembly says this precisely and remains a direct child of GO:0060271, so no information is lost and the misleading implication about primary cilia is removed. The orthologue transfer itself is sound; what needs correcting is the term, and the mouse source is if anything more explicit about motility than the human one, since it reports the 9+2 central pair directly and also separates the Hedgehog-dependent from the Hedgehog-independent defects by cross-species rescue. Proposed replacements: motile cilium assembly Supporting Evidence: PMID:19305393 Here we show that mouse Fu is essential for construction of the central pair apparatus of motile, 9+2 cilia and offers a new model of human primary ciliary dyskinesia. PMID:19305393 We also demonstrated that zebrafish Fu is required both for Hh signalling and cilia biogenesis in Kupffer's vesicle. PMID:37584603 The hypomorph mutant of Stk36 (Stk36tmE4-/- ) also developed progressive hydrocephalus postnatally and dysfunctional CSF flow, with multiple defects of motile cilia, including reduced ciliary number, disorganized ciliary orientation, defected axonemal structure and inconsistent base body (BB) orientation. PMID:37584603 we conclude that both Stk36 and Ulk4 are crucial for CSF flow, they cooperate by direct binding with their kinase domain to regulate the Foxj1 transcription factor pathways for ciliogenesis and cilia function, not limited to CSF flow |
| GO:0097542 ciliary tip | IDA PMID:38096226 Ulk4 promotes Shh signaling by regulating Stk36 ciliary loca... | ACCEPT | Summary: STK36 accumulates at the tip of the primary cilium in response to Shh and colocalises there with ULK4 and Gli2; the annotation is is_active_in, which is the right relation because this is where Gli2 phosphorylation happens (PMID:38096226). Reason: Core, and the most informative component annotation STK36 has. Three things make the is_active_in reading rather than mere colocalisation defensible: accumulation is Shh-dependent, it is mutual with ULK4 and requires STK36 kinase activity in the reciprocal direction, and blocking ULK4 ciliary-tip accumulation abolishes Gli2 phosphorylation and target-gene induction without affecting STK36 kinase activation - so the tip is where the activity is exercised, not merely where the protein is found. UniProt states the mutual dependence explicitly, and GO-CAM model 696022cd00001645 places both STK36 and ULK4 at the ciliary tip. Supporting Evidence: PMID:38096226 In this study, we found that Stk36 accumulated to the tip of primary cilium and colocalized with Gli2 in response to Shh stimulation, consistent with Gli2 being converted into Gli2A at the ciliary tip. PMID:38096226 In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. PMID:38096226 Although dispensable for Hh-induced Stk36 kinase activation, Ulk4 is essential for Stk36 ciliary tip localization, Gli2 phosphorylation, and activation. PMID:38096226 We further show that ciliary tip localization of Ulk4 depends on Stk36-mediated phosphorylation of Ulk4 on Thr1023, and that ciliary tip localization of Ulk4 is essential for its function in Hh signal transduction. file:human/STK36/STK36-uniprot.txt dependent for localization to ciliary tip (PubMed:38096226). Low levels |
| GO:0097542 ciliary tip | IEA GO_REF:0000120 | ACCEPT | Summary: Ciliary tip, is_active_in, from combined automated methods (ARBA model plus orthology to mouse Stk36, which carries the same term from an IDA in PMID:38096226). Reason: Agrees with the human IDA row and with the mouse experimental row, and preserves the is_active_in relation rather than degrading it to located_in. Redundant but correct. Supporting Evidence: PMID:38096226 In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | ACCEPT | Summary: Protein serine kinase activity mapped from Rhea reaction RHEA:17989, the seryl half-reaction UniProt records for STK36 with experimental evidence from PMID:31279575 and PMID:38096226. Reason: A Rhea-based row resting on an experimentally evidenced UniProt catalytic-activity annotation, not on a prediction, and the serine specificity is exactly what the substrate work shows: the characterised STK36 sites are Gli2 Ser230/Ser232 and the activation-loop Ser159, alongside Thr158 and Ulk4 Thr1021/Thr1023. UniProt records the threonyl reaction (RHEA:46608) separately, so STK36 is both a serine and a threonine kinase and the parent GO:0004674 is the summary level; this child row is accurate for the half of the activity it names. Supporting Evidence: file:human/STK36/STK36-uniprot.txt Xref=Rhea:RHEA:17989, Rhea:RHEA-COMP:9863, Rhea:RHEA- PMID:38096226 We found that the phospho-mimetic mutation (Stk36EE) increased, whereas phospho-deficient mutation (Stk36AA) abolished Stk36 kinase activity as determined by both Stk36 T158/S159 phosphorylation and Gli2 S230/S232 phosphorylation file:human/STK36/STK36-uniprot.txt Autophosphorylated at Thr-158 and Ser-159, activating the protein |
| GO:0106310 protein serine kinase activity | ISS GO_REF:0000024 | ACCEPT | Summary: Protein serine kinase activity transferred by curator judgment from Drosophila Fused (UniProtKB:P23647). STK36 and fu share PANTHER subfamily PTHR22983:SF6, and UniProt uses fu as the similarity anchor for the STK36 catalytic residue itself (ACT_SITE 125 "Proton acceptor", ECO:0000250|UniProtKB:P23647). Reason: An unusually well-grounded sequence-similarity transfer. The two proteins are in the same PANTHER subfamily rather than merely the same family, the donor activity is experimentally characterised in Drosophila, and the conservation has been demonstrated functionally rather than just inferred: Han et al. showed Fused phosphorylates Ci on Ser218 and Ser1230 and that Shh activates Gli2 through the corresponding conserved sites via STK36, and Zhou et al. showed STK36 activation-loop autophosphorylation follows the same mechanism as Fu. The transfer is also independently confirmed on the target itself by the human IDA and the Rhea mapping, so it is corroborative rather than load-bearing. Supporting Evidence: file:human/STK36/STK36-uniprot.txt PTHR22983:SF6; SERINE_THREONINE-PROTEIN KINASE 36 file:human/STK36/STK36-uniprot.txt /evidence="ECO:0000250|UniProtKB:P23647, PMID:31279575 Hence, Fu-family kinase-mediated phosphorylation of Ci/Gli serves as a conserved mechanism that activates the Hh pathway transcription factor. PMID:38096226 Like Fu, Stk36 is phosphorylated on multiple sites in the activation loop of its kinase domain in response to Shh likely through trans-autophosphorylation, and activation loop phosphorylation is essential for its kinase activation. |
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Download this section (compressed HTML)Q: UniProt records, from the original Murone paper, that GLI2 requires a function of STK36 that does not need an active catalytic site. What is that non-catalytic activity? If it is a scaffolding or adaptor role it would deserve a molecular function of its own, but no mechanism has been proposed in twenty-five years and the claim currently rests on a kinase-dead rescue in overexpression.
Q: Do STK36 and ULK3 phosphorylate GLI2 in the same cells, or does each dominate in a particular tissue? Epistasis places them in parallel pathways, and the difference decides whether the absence of a Hedgehog phenotype in Stk36-null mice reflects true redundancy or a division of labour that the assayed tissues happened not to reveal.
Q: Is the central-pair function of STK36 catalytic? The PCD alleles are loss-of-function rather than kinase-dead point mutants, and no axonemal substrate has been identified, so it remains open whether STK36 phosphorylates a central-pair component or contributes structurally through its interactions with KIF27 and SPAG16.
Q: STK36 partners with a Costal-2-family kinesin in both of its roles - KIF7 in the ciliary tip GLI complex and KIF27 in central-pair construction. Is that a conserved ancestral Fused-kinesin module that the two vertebrate paralogue pairs inherited and specialised, or convergence? A reconstruction across the PTHR22983 and PTHR24115 trees would answer it.
Q: Human STK36 has two isoforms differing by a 21-residue deletion (VSP_051983, residues 838-858) in the uncharacterised C-terminal region, and isoform 2 was cloned from brain. No GOA row for STK36 is isoform-qualified. Do the isoforms differ in ULK4 binding, ciliary targeting, or between motile and primary cilia?
Experiment: Generate knock-in mice and human airway-epithelial lines carrying the kinase-dead (Lys33 or activation-loop Ala) STK36 allele alongside the null, and score both functions in parallel: Gli2 Ser230/232 phosphorylation and Ptch1/Gli1 induction for the Hedgehog arm, and transmission electron microscopy of the central pair plus ciliary beat analysis for the motile arm. If the kinase-dead allele reproduces the null ciliary phenotype, the axonemal role is catalytic and a substrate should be sought; if it rescues ciliary structure while failing in Hedgehog signalling, STK36 contributes structurally to the central pair and the two functions are cleanly separable.
Hypothesis: The Hedgehog and motile-ciliogenesis functions of STK36 are genetically separable, and only the former requires catalytic activity.
Type: kinase-dead knock-in with paired signalling and ultrastructural readouts
Experiment: Perform quantitative phosphoproteomics on isolated axonemes from wild-type and STK36-null multiciliated cultures, differentiated in air-liquid interface so that central-pair assembly occurs in situ, and restrict the analysis to the known central-pair and radial-spoke proteome. Validate the strongest STK36-dependent sites by in vitro kinase assay with immunopurified STK36, and test phospho-null substitutions for the central-pair defect. A named substrate would convert the current IMP-based axoneme-assembly annotation into a mechanistic one.
Hypothesis: The axonemal role of STK36 involves phosphorylation of a central-pair or radial-spoke component.
Type: axoneme-restricted quantitative phosphoproteomics with in vitro validation
Experiment: Build single and compound conditional alleles of Stk36 and Ulk3 in mouse and compare Hedgehog-dependent patterning across several tissues - neural tube, limb bud, growth plate - quantifying Gli2 Ser230/232 phosphorylation and target-gene output in each. Redundancy predicts that neither single mutant is affected anywhere while the double mutant phenocopies loss of GLI activation; specialisation predicts tissue-restricted single-mutant phenotypes. The result determines whether the smoothened-pathway annotations for STK36 should carry a tissue qualifier.
Hypothesis: STK36 and ULK3 are redundant rather than tissue-specialised in vertebrate Hedgehog signalling.
Type: compound conditional mouse genetics with quantitative pathway readouts
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