ENSA (alpha-endosulfine) is a small (121 residue), intrinsically disordered phosphoprotein of the endosulfine family, the paralog of ARPP19. Its defining activity is phosphorylation-gated inhibition of the PP2A-B55 serine/threonine phosphatase: when Greatwall kinase (MASTL) phosphorylates Ser67 in the conserved central region, phospho-ENSA binds the B55/catalytic interface of PP2A-B55 with very high affinity and is dephosphorylated only slowly, so that it occupies the enzyme and excludes its normal substrates ("inhibition by unfair competition"). This allows cyclin B-CDK1-driven phosphorylations to accumulate and persist at mitotic entry and through mitosis; as MASTL is inactivated at anaphase, phospho-ENSA is turned over (by PP2A-B55 itself and by Fcp1) and PP2A-B55 reactivation lets substrates such as PRC1 be dephosphorylated in an ordered way that couples cytokinesis to chromosome separation. In human cells ENSA is the predominant MASTL substrate in several lines, and it also acts in S phase, where nuclear phospho-ENSA restrains PP2A-mediated dephosphorylation and proteasomal turnover of the origin firing factor Treslin so that replication origins fire at a normal rate. A second, C-terminal site (Ser109) is phosphorylated by PKA/PKG and tunes but does not by itself confer PP2A inhibition. The protein is cytoplasmic and nuclear (nuclear accumulation increases in S phase), widely expressed with high levels in brain and skeletal muscle, and present in anucleate platelets. Historically, recombinant alpha-endosulfine was characterised as an endogenous sulfonylurea-like ligand that displaces glibenclamide from beta-cell membranes, inhibits Kir6.2/SUR1 K-ATP channel currents at micromolar concentrations and stimulates insulin release from a beta-cell line; a related ENSA-K-ATP-neprilysin axis has been described in mouse brain, but the physiological basis of these channel effects remains unresolved.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000086 G2/M transition of mitotic cell cycle | ISS GO_REF:0000024 | ACCEPT | Summary: Transferred from Xenopus Ensa, which when phosphorylated by Greatwall becomes a potent PP2A-B55 inhibitor required for mitotic entry in egg extracts; the same mechanism operates in human cells, where ENSA is the major MASTL substrate in HeLa cells. Reason: The Xenopus source annotation is sound (Mochida et al. 2010 showed that Gwl-phosphorylated Ensa is the PP2A-B55 inhibitor essential for mitosis), and the function is directly demonstrated for the human protein - ENSA Ser67 is phosphorylated by MASTL in mitotic HeLa cells, phospho-ENSA inhibits PP2A-B55, and ENSA rather than ARPP19 is the major MASTL target in those cells (Cundell et al. 2013). In Xenopus egg extracts endogenous Arpp19 rather than Ensa is rate-limiting for mitotic entry, so the relative contribution of the two paralogs is cell-type dependent, but the G2/M role of the phospho-ENSA/PP2A-B55 switch is the core function of the gene and the term is appropriate. Supporting Evidence: PMID:21164013 We report that inhibition of PP2A-B55Ξ΄ results from a small protein, known as Ξ±-endosulfine (Ensa), that is phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of PP2A-B55Ξ΄. PMID:21164014 We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and Ξ±-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. PMID:24120663 Given that ARPP19 was not detected (Figure S3E) and that ARPP19 siRNA had little effect (Figure 4B), ENSA is the major target of Greatwall/MASTL in these cells. |
| GO:0000278 mitotic cell cycle | ISS GO_REF:0000024 | ACCEPT | Summary: Broad parent term transferred from Xenopus Ensa; in human cells ENSA acts at more than one point of the cycle - the G2/M transition, the timing of mitotic exit, and S-phase progression via Treslin stabilisation. Reason: Although general, the term is a fair summary rather than an over-generalisation, because human ENSA has documented roles in three phases of the mitotic cell cycle - mitotic entry and maintenance (Cundell et al. 2013), the ENSA/Greatwall timer that delays PP2A-B55 reactivation and PRC1 dephosphorylation until after chromosome separation (Cundell et al. 2013), and S-phase length control through Treslin (Charrasse et al. 2017). The more specific G2/M term is carried separately. Supporting Evidence: PMID:24120663 Removal of the ENSA/Greatwall (EG) timer module eliminates this second threshold, as well as associated delay in PRC1 dephosphorylation and initiation of cytokinesis, by uncoupling PP2A-B55 from Cdk1-cyclin B activity. PMID:28785014 Unexpectedly, Ensa knockdown promotes a dramatic extension of S phase associated with a lowered density of replication forks. Notably, Ensa depletion results in a decrease of Treslin levels, a pivotal protein for the firing of replication origins. |
| GO:0004864 protein phosphatase inhibitor activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT propagation across the endosulfine family (Drosophila Endos, fission yeast Igo1, budding yeast Igo, human ARPP19); phosphorylation-dependent inhibition of PP2A-B55 is the conserved, defining activity of the family and is directly shown for human ENSA. Reason: The node placement at the endosulfine family root is well supported - every characterised member from yeast Igo1/2 to human ARPP19 is a Greatwall/Rim15-phosphorylated PP2A-B55 inhibitor - and the activity is demonstrated for human ENSA itself (Ser67-phosphorylated ENSA inhibits PP2A in HeLa extracts and platelet lysates; structural work places the Ser67 region at the B55/C-subunit interface). This is the core molecular function of the gene. Supporting Evidence: PMID:21164013 We report that inhibition of PP2A-B55Ξ΄ results from a small protein, known as Ξ±-endosulfine (Ensa), that is phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of PP2A-B55Ξ΄. PMID:24618897 As the name suggests, during M phase PP2A-B55's attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. PMID:32085646 Our data also established that both MASTL- and PKA/PKG-phosphorylated ENSA/ARPP19 are PP2A substrates, whereas only MASTL-phosphorylated ENSA/ARPP19 are PP2A inhibitors. PMID:34346186 ENSA inhibits PP2A activity when it is phosphorylated at Ser67 by Greatwall (Gwl) kinase. file:human/ENSA/ENSA-deep-research-falcon.md Phosphorylated ENSA then binds PP2A-B55 as a high-affinity inhibitory substrate. Its phosphoserine occupies the phosphatase catalytic machinery, but dephosphorylation is unusually slow relative to ordinary substrates. |
| GO:0005102 signaling receptor binding | IDA PMID:9653196 Human alpha-endosulfine, a possible regulator of sulfonylure... | MODIFY | Summary: Recombinant human alpha-endosulfine displaced [3H]glibenclamide from beta-cell membranes, which was read as binding to the sulfonylurea receptor SUR1 (ABCC8); direct binding was not shown and the authors could not exclude that the peptide acts on Kir6.2 instead. Reason: The evidence is a competitive ligand-displacement assay, not a demonstration of binding to a signalling receptor. SUR1 is an ABC-transporter regulatory subunit of the K-ATP channel that acts as a drug receptor, not a signalling receptor in the GO sense, and the paper itself states that binding to Kir6.2 cannot be excluded. What the study does establish directly is that ENSA reduces cloned Kir6.2/SUR1 K-ATP channel currents, which is captured by the potassium channel inhibitor activity annotation the gene already carries; that is the informative molecular-function reading of these data. The channel-regulatory role remains historical and non-core (see the GO:0019870 review). Proposed replacements: potassium channel inhibitor activity Supporting Evidence: PMID:9653196 The recombinant protein displaces binding of the sulfonylurea [3H]glibenclamide to beta cell membranes, inhibits cloned KATP channel currents, and stimulates insulin secretion. PMID:9653196 We therefore cannot exclude the alternative possibility that Ξ±-endosulfine binds to Kir6.2 and thereby displaces glibenclamide binding to SUR1. |
| GO:0005102 signaling receptor binding | TAS PMID:9653196 Human alpha-endosulfine, a possible regulator of sulfonylure... | MODIFY | Summary: Legacy ProtInc statement of the same sulfonylurea-receptor interaction inferred from glibenclamide displacement in the cloning paper. Reason: Same evidence and same problem as the IDA row - an indirect displacement assay on a drug receptor subunit rather than binding to a signalling receptor. The direct functional readout in the paper is inhibition of K-ATP channel current, so the potassium channel inhibitor activity term is the appropriate molecular-function statement. Proposed replacements: potassium channel inhibitor activity Supporting Evidence: PMID:9653196 The recombinant protein displaces binding of the sulfonylurea [3H]glibenclamide to beta cell membranes, inhibits cloned KATP channel currents, and stimulates insulin secretion. PMID:9653196 We therefore cannot exclude the alternative possibility that Ξ±-endosulfine binds to Kir6.2 and thereby displaces glibenclamide binding to SUR1. |
| GO:0005515 protein binding | IPI PMID:21044950 Genome-wide YFP fluorescence complementation screen identifi... | REMOVE | Summary: ENSA scored as a bimolecular fluorescence complementation hit with the shelterin subunit ACD (TPP1) in a genome-wide arrayed screen of ~12,000 human proteins against six telomeric baits; ENSA is not discussed in the text and there is no follow-up. Reason: Generic protein binding conveys no function, and this particular interaction is a single untargeted high-throughput screen hit for which the authors themselves note that secondary validation is needed and that high expression can give false positives. There is no independent evidence linking ENSA to telomere biology or to ACD. Removal reflects that the annotation is uninformative, not an assertion that the fluorescence complementation signal was false. Supporting Evidence: PMID:21044950 Although PCA/BiFC does appear to be more tolerant of differences in expression, high expression levels of bait and prey may still result in false positives. Therefore, secondary screens are needed to validate the identified interactions. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-2430535 | ACCEPT | Summary: Reactome places the MASTL-ENSA Ser67 phosphorylation reaction in the nucleoplasm; consistent with the nuclear pool of ENSA seen in human cells, which increases in S phase alongside nuclear active Greatwall. Reason: Endogenous ENSA is detected in nuclei of HeLa cells and its nuclear localisation rises significantly in S-phase cells, where it is phosphorylated by nuclear Greatwall (Charrasse et al. 2017). MASTL is nuclear in interphase, so the Reactome compartment assignment for the phosphorylation reaction is reasonable; ENSA is additionally cytoplasmic (see the cytoplasm rows), which Reactome does not model. Supporting Evidence: Reactome:R-HSA-2430535 MASTL (GWL) activates ENSA by phosphorylating it on serine residue S67 (Mochida et al. 2010, Gharbi-Ayachi et al. 2010). PMID:28785014 In contrast, nuclear localisation of Ensa was significantly increased in S-phase cells suggesting that Ensa is relocalised into the nucleus during DNA replication. |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT propagation of cytoplasmic localisation across the endosulfine family, seeded by rat Ensa/Arpp19 and yeast Igo; human ENSA is a soluble protein with no signal peptide, is abundant in anucleate platelets, and acts on cytoplasmic PP2A-B55 after nuclear envelope breakdown. Reason: The family-level placement is sound and the human protein fits it - ENSA lacks a signal peptide and was predicted intracellular from the cDNA, is readily detected (about 7,800 copies per cell) in anucleate human platelets where it is phosphorylated at Ser67 and Ser109, and its mitotic target PP2A-B55 is cytoplasmic once the nuclear envelope has broken down. UniProt records the same location. Supporting Evidence: PMID:9653196 There is no obvious signal peptide sequence at the N terminus, suggesting that Ξ±-endosulfine is an intracellular protein. PMID:32085646 We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. file:human/ENSA/ENSA-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location keyword mapping (Cytoplasm). Reason: Mirrors the phylogenetic and experimental evidence for a cytoplasmic pool of ENSA. Supporting Evidence: PMID:9653196 There is no obvious signal peptide sequence at the N terminus, suggesting that Ξ±-endosulfine is an intracellular protein. PMID:32085646 We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. file:human/ENSA/ENSA-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0007584 response to nutrient | TAS PMID:9653196 Human alpha-endosulfine, a possible regulator of sulfonylure... | REMOVE | Summary: Legacy ProtInc process annotation from the cloning paper; the full text contains no experiment in which ENSA expression, modification or activity changes in response to a nutrient - the only nutrient manipulation is the glucose concentration used as the baseline of the insulin-release assay. Reason: The full text is available and shows that this annotation rests on the authors' speculative discussion that an endogenous K-ATP ligand might couple cell metabolism to electrical activity, not on any assay of a nutrient response by ENSA. Recombinant ENSA was applied at a fixed non-stimulatory 1 mM glucose to elicit insulin release; nothing was measured about ENSA responding to glucose or any other nutrient, and no later work has established such a response. The annotation is unsupported rather than merely too broad. Supporting Evidence: PMID:9653196 When tested at a nonstimulatory glucose concentration (1 mM), Ξ±-endosulfine induced a dose-dependent stimulation of insulin release. PMID:9653196 In particular it will be important to address the possibility that Ξ±-endosulfine is involved in the metabolic regulation of K ATP channel activity. |
| GO:0008200 ion channel inhibitor activity | TAS PMID:9653196 Human alpha-endosulfine, a possible regulator of sulfonylure... | MODIFY | Summary: Legacy ProtInc statement of the K-ATP channel inhibition reported in the cloning paper, at the generic ion-channel level. Reason: The activity described is specifically inhibition of the Kir6.2/SUR1 potassium channel (2 ΞΌM recombinant ENSA reduced K-ATP conductance by about 75% in inside-out patches and was without effect on Kir1.1), so the more specific potassium channel inhibitor activity term, already carried by IDA, should be used in place of this parent. As for that row, the activity is historical and non-core. Proposed replacements: potassium channel inhibitor activity Supporting Evidence: PMID:9653196 The recombinant protein displaces binding of the sulfonylurea [3H]glibenclamide to beta cell membranes, inhibits cloned KATP channel currents, and stimulates insulin secretion. PMID:9653196 Ξ±-Endosuline (2 ΞΌM) was without effect on Kir1.1a, a related inwardly rectifying K channel (data not shown), suggesting that the peptide may interact specifically with the Kir6.2/SUR1 channel. |
| GO:0019212 phosphatase inhibitor activity | ISS GO_REF:0000024 | MODIFY | Summary: Transferred from Xenopus Ensa; the inhibited enzyme is the PP2A-B55 protein serine/threonine phosphatase, so the generic phosphatase-inhibitor term is less precise than the protein phosphatase inhibitor term the gene already carries by IBA. Reason: The underlying claim is correct and central, but the term is one level too shallow - phospho-ENSA inhibits a protein phosphatase (PP2A-B55), not phosphatases in general. Use GO:0004864 protein phosphatase inhibitor activity. Proposed replacements: protein phosphatase inhibitor activity Supporting Evidence: PMID:21164013 We report that inhibition of PP2A-B55Ξ΄ results from a small protein, known as Ξ±-endosulfine (Ensa), that is phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of PP2A-B55Ξ΄. PMID:24618897 As the name suggests, during M phase PP2A-B55's attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. PMID:32085646 Our data also established that both MASTL- and PKA/PKG-phosphorylated ENSA/ARPP19 are PP2A substrates, whereas only MASTL-phosphorylated ENSA/ARPP19 are PP2A inhibitors. PMID:34346186 ENSA inhibits PP2A activity when it is phosphorylated at Ser67 by Greatwall (Gwl) kinase. file:human/ENSA/ENSA-deep-research-falcon.md Phosphorylated ENSA then binds PP2A-B55 as a high-affinity inhibitory substrate. Its phosphoserine occupies the phosphatase catalytic machinery, but dephosphorylation is unusually slow relative to ordinary substrates. |
| GO:0019870 potassium channel inhibitor activity | IDA PMID:9653196 Human alpha-endosulfine, a possible regulator of sulfonylure... | KEEP AS NON CORE | Summary: Recombinant human alpha-endosulfine applied to inside-out patches from oocytes expressing Kir6.2/SUR1 reduced K-ATP conductance by about 75% at 2 ΞΌM (EC50 about 1 ΞΌM) and did not affect the related Kir1.1 channel. Reason: This is a direct assay with the human protein on the cloned channel and the effect was channel-selective, so the annotation is retained. It is graded non-core because the affinity is micromolar (three orders of magnitude weaker than sulfonylureas), the protein blocked currents whether applied inside or outside the membrane, the interacting subunit was never identified, no direct ENSA-channel binding has since been shown, and later work found no evidence that ENSA is extracellular. Subsequent genetic work in mouse brain does link Ensa to a SUR1/Kir6.2 K-ATP-dependent regulation of neprilysin, so a channel-modulatory activity remains plausible, but the well-established core function of the protein is PP2A-B55 inhibition. Supporting Evidence: PMID:9653196 The recombinant protein displaces binding of the sulfonylurea [3H]glibenclamide to beta cell membranes, inhibits cloned KATP channel currents, and stimulates insulin secretion. PMID:9653196 Ξ±-Endosuline (2 ΞΌM) was without effect on Kir1.1a, a related inwardly rectifying K channel (data not shown), suggesting that the peptide may interact specifically with the Kir6.2/SUR1 channel. PMID:9653196 On the other hand, we observed that Ξ±-endosulfine blocked K ATP currents when applied both intracellularly and extracellularly, and it stimulated insulin secretion when added to the extracellular solution. PMID:28167675 However, there is no evidence that ENSA is found extracellularly. PMID:34737456 Here, we identified Ξ±-endosulfine (ENSA), an endogenous ligand of the ATP-sensitive potassium (KATP) channel, as a negative regulator of NEP downstream of SST signaling. |
| GO:0019888 protein phosphatase regulator activity | ISS GO_REF:0000024 | MODIFY | Summary: Transferred from Xenopus Ensa; the regulation is specifically inhibition of PP2A-B55, so the more specific inhibitor term is preferable to the generic regulator term. Reason: Correct in essence but too general - the mode of regulation is known (Ser67-phosphorylated ENSA inhibits PP2A-B55 by occupying the active site) and is captured by the child term GO:0004864 protein phosphatase inhibitor activity that the gene already carries by IBA. Proposed replacements: protein phosphatase inhibitor activity Supporting Evidence: PMID:21164013 We report that inhibition of PP2A-B55Ξ΄ results from a small protein, known as Ξ±-endosulfine (Ensa), that is phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of PP2A-B55Ξ΄. PMID:24618897 As the name suggests, during M phase PP2A-B55's attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. PMID:32085646 Our data also established that both MASTL- and PKA/PKG-phosphorylated ENSA/ARPP19 are PP2A substrates, whereas only MASTL-phosphorylated ENSA/ARPP19 are PP2A inhibitors. PMID:34346186 ENSA inhibits PP2A activity when it is phosphorylated at Ser67 by Greatwall (Gwl) kinase. file:human/ENSA/ENSA-deep-research-falcon.md Phosphorylated ENSA then binds PP2A-B55 as a high-affinity inhibitory substrate. Its phosphoserine occupies the phosphatase catalytic machinery, but dephosphorylation is unusually slow relative to ordinary substrates. |
| GO:0050796 regulation of insulin secretion | IDA PMID:9653196 Human alpha-endosulfine, a possible regulator of sulfonylure... | MARK AS OVER ANNOTATED | Summary: Recombinant alpha-endosulfine added to the extracellular medium of MIN6 insulinoma cells at non-stimulatory glucose caused a dose-dependent increase in insulin release, interpreted as a consequence of K-ATP channel closure. Reason: The observation is real, but a physiological role for ENSA in regulating insulin secretion has not been established. The effect was obtained by adding micromolar recombinant protein to the outside of a cell line, whereas ENSA is an intracellular protein with no signal peptide and no evidence of secretion; the authors themselves noted that either membrane crossing or an unidentified second site would be needed to explain it. No loss-of-function evidence (Ensa-deficient mice exist) has connected endogenous ENSA to insulin secretion in the intervening decades, and the field now treats the sulfonylurea-receptor ligand role as historical. The annotation therefore over-states a pharmacological observation as a process role of the gene. Supporting Evidence: PMID:9653196 When tested at a nonstimulatory glucose concentration (1 mM), Ξ±-endosulfine induced a dose-dependent stimulation of insulin release. PMID:9653196 On the other hand, we observed that Ξ±-endosulfine blocked K ATP currents when applied both intracellularly and extracellularly, and it stimulated insulin secretion when added to the extracellular solution. PMID:28167675 However, there is no evidence that ENSA is found extracellularly. |
| GO:0051721 protein phosphatase 2A binding | ISS GO_REF:0000024 | ACCEPT | Summary: Transferred from Xenopus Ensa, which binds PP2A-B55 when phosphorylated at Ser67; direct binding of human ENSA to PP2A subunits is confirmed by NMR/SAXS and pull-down work and by the finding that PP2A-B55 is the ENSA phosphatase in HeLa extracts. Reason: Unlike generic protein binding, this term names the specific physiological partner and is mechanistically informative - the PP2A-B55 interaction is the basis of the inhibitor activity and of the unfair-competition substrate mechanism. Human ENSA binds the PP2A A subunit in vitro, and Ser67-phosphorylated ENSA is both bound and slowly dephosphorylated by PP2A-B55 in human cell extracts, so the transfer is well founded. Supporting Evidence: PMID:24120663 Furthermore, the removal of the PP2A regulatory subunits PPP2R2AβPPP2R2D stabilized both PRC1 pT481 and ENSA phosphorylation (Figure 4B), indicating that PP2A-B55 is the ENSA phosphatase in this system. PMID:34346186 ENSA was observed to interact PP2A mainly via A-subunit, as the affinity with the A-subunit is significantly stronger than with any of the B56 subunits. PMID:24354984 Structural analyses revealed that the most-conserved middle region of ENSA containing S67 physically interacts with PP2A-B55 at the interface of the B55 and C subunits, where the catalytic centre of PP2A is located. |
| GO:0010923 negative regulation of phosphatase activity | IDA PMID:24120663 The BEG (PP2A-B55/ENSA/Greatwall) pathway ensures cytokinesi... | NEW | Summary: Proposed process annotation. Ser67-phosphorylated ENSA inhibits PP2A-B55 in human cells; removing ENSA (or MASTL) from HeLa extracts abolishes the delay in PP2A-B55 substrate dephosphorylation, and recombinant MASTL-phosphorylated human ENSA inhibits PP2A in platelet lysates. This is the biological-process counterpart of the inhibitor activity and is the process term used for the paralog ARPP19 in GOA and in the GO-CAM for the MAST3-ARPP19-PP2A module. Reason: ENSA itself performs the inhibition (it is the entity that occupies the PP2A-B55 active site), so the participation test is met, and the comparator check passes - human ARPP19 carries GO:0010923 by IDA and the ARPP19 GO-CAM (gocams/69729a3800000481) uses this term as the process of its protein phosphatase inhibitor activity. The evidence is human (HeLa extracts and platelet lysates with recombinant human protein) rather than transferred from Xenopus. Note the deliberate species divergence, in which the S. pombe igo1 review declines the same term because PomBase captures endosulfine inhibition through the molecular-function annotation alone (its comparator Sds23 carries only the MF term), whereas in human GOA the process term is an established convention for ARPP19. Supporting Evidence: PMID:24120663 Removal of the ENSA/Greatwall (EG) timer module eliminates this second threshold, as well as associated delay in PRC1 dephosphorylation and initiation of cytokinesis, by uncoupling PP2A-B55 from Cdk1-cyclin B activity. PMID:24120663 Given that ARPP19 was not detected (Figure S3E) and that ARPP19 siRNA had little effect (Figure 4B), ENSA is the major target of Greatwall/MASTL in these cells. PMID:32085646 Our data also established that both MASTL- and PKA/PKG-phosphorylated ENSA/ARPP19 are PP2A substrates, whereas only MASTL-phosphorylated ENSA/ARPP19 are PP2A inhibitors. file:human/ENSA/ENSA-deep-research-falcon.md Consequently, a comparatively small inhibitory pool can block access of many conventional PP2A-B55 substrates. |
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Download this section (compressed HTML)Q: In human somatic cells, which of ENSA and ARPP19 is the physiologically limiting PP2A-B55 inhibitor at mitotic entry, and does this vary by cell type? HeLa data point to ENSA, Xenopus extract and mouse fibroblast data point to ARPP19, and most studies deplete both together.
Suggested experts: Francis A. Barr, Anna Castro, Thierry Lorca
Q: Is the S-phase function of ENSA (stabilisation of Treslin against PP2A-mediated dephosphorylation) a candidate for a regulation-of-DNA-replication annotation, and is it exerted through PP2A-B55 or a different PP2A holoenzyme? The mechanism is a single human study and no replication-related term is currently annotated.
Suggested experts: Anna Castro, Thierry Lorca
Q: Does ENSA bind directly to any K-ATP channel subunit (SUR1 or Kir6.2), and by what route would an intracellular protein without a signal peptide reach an extracellular or channel site in vivo? The 1998 channel and insulin data and the 2022 mouse neprilysin axis both presuppose a channel interaction that has never been shown biochemically.
Suggested experts: Frances M. Ashcroft, Takaomi C. Saido
Experiment: Generate single and double CRISPR knockouts (or acute degron alleles) of ENSA and ARPP19 in several non-transformed and transformed human lines; measure G2/M entry timing, S-phase length, Treslin levels and PP2A-B55 substrate (PRC1 pT481) dephosphorylation kinetics, with rescue by Ser67 phospho-mimetic ENSA.
Hypothesis: ENSA and ARPP19 are partially redundant PP2A-B55 inhibitors whose relative importance for mitotic entry and for S-phase length differs between human cell types.
Type: genome editing / live-cell imaging
Experiment: Beta-cell-specific Ensa knockout mice and ENSA-null human stem-cell-derived beta cells assayed for K-ATP current density, glibenclamide sensitivity and glucose-stimulated insulin secretion; complement with proximity labelling or cross-linking to test for a physical ENSA-SUR1/Kir6.2 interaction in intact cells.
Hypothesis: Endogenous ENSA does not regulate insulin secretion through K-ATP channels in beta cells.
Type: mouse genetics / electrophysiology
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