Wis1 is the sole MAP kinase kinase (MAP2K/MAPKK; EC 2.7.12.2) of the fission-yeast stress-activated protein kinase (SAPK) pathway. It is a dual-specificity protein kinase that phosphorylates and activates the p38/Hog1-related MAP kinase Spc1 (also called Sty1/Phh1) on the activation-loop residues threonine-171 and tyrosine-173 in response to osmotic, oxidative, heat and nutritional stress. Wis1 is itself activated by the two partially redundant MAPKKKs Wis4 (Wik1/Wak1) and Win1, which phosphorylate the conserved Wis1 activation-loop residues serine-469 and threonine-473; Win1 is the major transducer of osmotic-stress signals. The upstream module is organized around the Mcs4 response regulator, which forms a stable heteromer with Wis4 and Win1 and receives peroxide-stress signals through a Mak2/Mak3-Mpr1 multistep phosphorelay. Wis1 is predominantly cytoplasmic, retained there by an N-terminal nuclear export signal acting via the exportin Crm1, and its cytoplasmic localization is required for stress-induced nuclear accumulation of Spc1. Downstream, activated Spc1 phosphorylates the bZIP transcription factor Atf1 to drive stress-response gene expression. The pathway also couples environmental conditions to G2/M cell-cycle progression, and Wis1 was originally identified as a dosage-dependent inducer of mitosis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000165 MAPK cascade | IBA GO_REF:0000033 | ACCEPT | Summary: Wis1 is the MAPKK tier of the fission-yeast stress-activated MAPK cascade, directly transmitting signals from the Wis4/Win1 MAPKKKs to the Spc1 MAPK. Participation in a MAPK cascade is a core, well-supported function; the more specific term p38MAPK cascade (GO:0038066) is also annotated and preferred. Reason: Phylogenetic inference is consistent with extensive experimental evidence that Wis1 is the MAPKK of the Spc1 SAPK cascade. Supporting Evidence: PMID:12181336 Wis1 is the only MAPKK responsible for the phosphorylation and activation of Spc1 MAPK under various stress conditions |
| GO:0071474 cellular hyperosmotic response | IBA GO_REF:0000033 | ACCEPT | Summary: Wis1 is essential for survival and the adaptive response to high osmolarity; delta wis1 cells cannot grow on high-osmolarity media. The hyperosmotic response is a central biological role of this MAPKK and the annotation is well supported by both phylogeny and S. pombe genetics. Reason: Loss-of-function and phylogenetic evidence both support a core role in the hyperosmotic stress response. Supporting Evidence: PMID:7657164 we find that sty2 is allelic to the wis1 MAP kinase kinase and that delta sty1 and delta wis1 cells are unable to grow in high osmolarity medium |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Wis1 is a bona fide protein kinase, but this is a generic parent term. The precise molecular function is MAP kinase kinase activity (GO:0004708), which is separately annotated with strong experimental evidence and should be used. Reason: Correct but uninformatively general; the specific MAP kinase kinase activity term is supported and preferred. Proposed replacements: MAP kinase kinase activity Supporting Evidence: PMID:12181336 Wis1 is the only MAPKK responsible for the phosphorylation and activation of Spc1 MAPK under various stress conditions |
| GO:0004708 MAP kinase kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Core molecular function. Wis1 is a dual-specificity MAP kinase kinase (EC 2.7.12.2) that phosphorylates Spc1 on Thr-171 and Tyr-173. This term is strongly supported experimentally and by the EC mapping behind this IEA. Reason: Electronic EC-based assignment agrees with multiple experimental annotations of the same term. Supporting Evidence: PMID:10749922 Spc1 is activated through phosphorylation of Thr-171 and Tyr-173, which is carried out by a MEK homologue, Wis1. |
| GO:0004713 protein tyrosine kinase activity | IEA GO_REF:0000116 | MARK AS OVER ANNOTATED | Summary: As a dual-specificity MAP2K, Wis1 does phosphorylate the tyrosine (Tyr-173) of its substrate Spc1, so tyrosine-kinase activity is biochemically real. However, in the GO function hierarchy this is captured more precisely and appropriately by MAP kinase kinase activity (GO:0004708); the bare protein-tyrosine-kinase term mischaracterizes Wis1 as a classical PTK. Reason: Reflects the dual-specificity chemistry but is misleading as a standalone function; MAP kinase kinase activity is the correct precise term. Proposed replacements: MAP kinase kinase activity Supporting Evidence: PMID:10749922 Spc1 is activated through phosphorylation of Thr-171 and Tyr-173, which is carried out by a MEK homologue, Wis1. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: Wis1 has a canonical protein-kinase domain (residues 320-579) with a conserved ATP-binding P-loop (residues 326-334) and is an active kinase, so ATP binding is a supported molecular function consistent with the InterPro domain signature. Reason: ATP binding is an expected, sequence- and structure-supported feature of the active kinase domain. Supporting Evidence: PMID:12181336 Wis1 is a 605-amino acid protein with a C-terminal kinase catalytic domain |
| GO:0005737 cytoplasm | IEA GO_REF:0000117 | ACCEPT | Summary: Wis1 is predominantly cytoplasmic at steady state, retained there by an N-terminal NES acting through Crm1; only a fraction translocates to the nucleus upon stress. Cytoplasmic localization is well supported and is the principal site of function. Reason: Localization is corroborated by direct experimental immunofluorescence and GFP studies. Supporting Evidence: PMID:9585506 We have established that Wis1 is detected completely in the cytoplasm, whereas Atf1 is found exclusively in the nucleus. |
| GO:0032991 protein-containing complex | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: Wis1 associates with the Mcs4-Wis4-Win1 MAPKKK module and docks onto its Spc1 substrate, so membership in a protein-containing complex is true but this root-level term is uninformative. The specific Mcs4 RR-MAPKKK complex (GO:1990315) annotation is preferred. Reason: Generic complex term superseded by the specific Mcs4 RR-MAPKKK complex annotation. Proposed replacements: Mcs4 RR-MAPKKK complex Supporting Evidence: PMID:24255738 The Wis1 MAPKK and the Tdh1 GAPDH are also found on the complex |
| GO:0038066 p38MAPK cascade | IEA GO_REF:0000117 | ACCEPT | Summary: The Spc1 SAPK pathway is the fission-yeast p38/Hog1-class MAPK cascade, and Wis1 is its dedicated MAPKK. This is a core biological process, supported by numerous experimental annotations of the same term. Reason: Electronic assignment agrees with multiple experimental annotations of the p38MAPK cascade. Supporting Evidence: PMID:9321395 integrity of the Wis1-Spc1 pathway is required for survival in extreme conditions of heat, osmolarity, oxidation or limited nutrition |
| GO:0071470 cellular response to osmotic stress | IEA GO_REF:0000117 | ACCEPT | Summary: Wis1 is required for the cellular response to osmotic stress; this is a core process supported by direct S. pombe genetics (delta wis1 cells fail to grow at high osmolarity) and corroborated by the redundant IMP annotation. Reason: Electronic assignment matches experimentally supported osmotic-stress phenotypes of wis1 mutants. Supporting Evidence: PMID:7859738 One locus is identical to wis1+, encoding a MAP kinase kinase (MEK) homolog. |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | MARK AS OVER ANNOTATED | Summary: Wis1 phosphorylates the serine/threonine (Thr-171) of Spc1 in addition to tyrosine, so serine-kinase activity is biochemically consistent with its dual-specificity chemistry. As with the tyrosine-kinase term, the precise function is MAP kinase kinase activity (GO:0004708). Reason: Reflects the dual-specificity chemistry but is less precise than MAP kinase kinase activity. Proposed replacements: MAP kinase kinase activity Supporting Evidence: PMID:10749922 Spc1 is activated through phosphorylation of Thr-171 and Tyr-173, which is carried out by a MEK homologue, Wis1. |
| GO:0090055 positive regulation of silent mating-type cassette heterochromatin formation | EXP PMID:15292231 Regulation of Swi6/HP1-dependent heterochromatin assembly by... | KEEP AS NON CORE | Summary: The Sty1/Atf1-Pcr1 MAPK pathway contributes to Swi6/HP1-dependent heterochromatin assembly at the silent mating-type region, and a phosphorylation event by the conserved MAPK pathway is implicated. This is a downstream, indirect consequence of Spc1/Atf1 signaling rather than a direct Wis1 biochemical activity, so it is best retained as a non-core, pleiotropic role. Reason: Real but indirect/downstream effect of the Sty1-Atf1 pathway on heterochromatin; not a core Wis1 function. Supporting Evidence: PMID:15292231 a phosphorylation event catalyzed by the conserved mitogen-activated protein kinase pathway is important for regulation of heterochromatin silencing by Atf1 and Pcr1 |
| GO:0004708 MAP kinase kinase activity | EXP PMID:9585506 Phosphorylation and association with the transcription facto... | ACCEPT | Summary: Core molecular function, supported experimentally. Wis1 is the activator kinase of Spc1 and dissociates from Spc1 upon stress-induced nuclear translocation, consistent with MAPKK activity toward Spc1. Reason: Directly supported experimental annotation of the core MAPKK function. Supporting Evidence: PMID:9585506 Nuclear translocation of Spc1 is coupled with disassociation from its activator kinase Wis1. |
| GO:0038066 p38MAPK cascade | EXP PMID:10749922 Multistep phosphorelay proteins transmit oxidative stress si... | ACCEPT | Summary: Core process. Wis1 is the MAPKK relaying osmotic and oxidative stress signals (the latter via the Mcs4 phosphorelay) to the Spc1 p38-class MAPK. Reason: Experimentally supported placement of Wis1 in the Spc1 SAPK (p38-class) cascade. Supporting Evidence: PMID:10749922 oxidative stress stimuli are transmitted by a multistep phosphorelay system to the Spc1/Sty1 stress-activated protein kinase |
| GO:0004708 MAP kinase kinase activity | EXP PMID:12181336 Cytoplasmic localization of Wis1 MAPKK by nuclear export sig... | ACCEPT | Summary: Core molecular function. Wis1 is explicitly the only MAPKK that phosphorylates and activates Spc1 under stress; its activity depends on a MAPK-docking region that binds Spc1. Reason: Strong direct experimental support for the core MAPKK activity. Supporting Evidence: PMID:12181336 Wis1 is the only MAPKK responsible for the phosphorylation and activation of Spc1 MAPK under various stress conditions |
| GO:0005829 cytosol | IDA PMID:12181336 Cytoplasmic localization of Wis1 MAPKK by nuclear export sig... | ACCEPT | Summary: Wis1 is maintained in the cytoplasm/cytosol by its N-terminal NES via Crm1 and is active there, where it is activated by upstream MAPKKKs and phosphorylates Spc1. Cytosolic activity is well supported. Reason: Direct localization evidence shows cytoplasmic Wis1 is the functional pool. Supporting Evidence: PMID:12181336 cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus |
| GO:0004708 MAP kinase kinase activity | IDA PMID:9321395 Multiple modes of activation of the stress-responsive MAP ki... | ACCEPT | Summary: Core molecular function, supported by direct evidence that Wis1 is required to activate Spc1 and is itself phosphorylated/activated by the Wis4 MAPKKK. Reason: Direct experimental support for the core MAPKK activity. Supporting Evidence: PMID:9321395 The MAPKK homologue Wis1 is required for activation of the MAPK homologue Spc1 |
| GO:0005515 protein binding | IPI PMID:9585506 Phosphorylation and association with the transcription facto... | REMOVE | Summary: This generic protein-binding annotation reflects the Wis1-Spc1 interaction (Wis1 docks and dissociates from Spc1). The bare protein-binding term is uninformative; the functionally meaningful relationship is Wis1 acting as the MAP kinase kinase for Spc1, captured by GO:0004708. Reason: Uninformative protein binding term; the relevant Wis1-Spc1 interaction is already represented by the MAP kinase kinase activity annotations. Supporting Evidence: PMID:9585506 Nuclear translocation of Spc1 is coupled with disassociation from its activator kinase Wis1. |
| GO:0004708 MAP kinase kinase activity | IMP PMID:7657164 Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kin... | ACCEPT | Summary: Core molecular function inferred from mutant phenotype; sty2/wis1 mutants act upstream of Sty1 and are unable to grow at high osmolarity, consistent with loss of MAPKK activity toward Sty1/Spc1. Reason: Mutant-phenotype evidence supports Wis1 functioning as the MAPKK upstream of Sty1. Supporting Evidence: PMID:7657164 Sty1, which acts downstream of the Wis1 MAP kinase kinase to control cell size at division in fission yeast |
| GO:0038066 p38MAPK cascade | IMP PMID:23389634 Response regulator-mediated MAPKKK heteromer promotes stress... | ACCEPT | Summary: Core process. Wis1 is the MAPKK that receives input from the Mcs4-Wis4-Win1 heteromer and signals to Spc1; integrity of that complex promotes Wis1-Spc1 signaling. Reason: Mutant analysis places Wis1 centrally in the Spc1 (p38-class) cascade. Supporting Evidence: PMID:23389634 whose integrity promotes physical interaction with the Wis1 MAPKK and, thus, signaling to the Spc1 MAPK |
| GO:0038066 p38MAPK cascade | IMP PMID:7657164 Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kin... | ACCEPT | Summary: Core process. Genetic evidence places Wis1 in the stress MAPK cascade controlling Sty1/Spc1, with osmotic-stress phenotypes for wis1 mutants. Reason: Mutant-phenotype evidence supports Wis1 participation in the Sty1/Spc1 SAPK cascade. Supporting Evidence: PMID:7657164 Sty1, which acts downstream of the Wis1 MAP kinase kinase to control cell size at division in fission yeast |
| GO:1990315 Mcs4 RR-MAPKKK complex | IDA PMID:23389634 Response regulator-mediated MAPKKK heteromer promotes stress... | ACCEPT | Summary: Wis1 physically associates with the Mcs4 response-regulator/Wis4-Win1 MAPKKK complex, which serves as the activating platform for the MAPKK. Wis1 docks on this complex and is released upon stress, so the part_of annotation is reasonable, though Wis1 behaves as a transient, regulated partner rather than a constitutive core subunit. Reason: Direct co-purification evidence supports Wis1 association with the Mcs4 RR-MAPKKK complex. Supporting Evidence: PMID:23389634 whose integrity promotes physical interaction with the Wis1 MAPKK and, thus, signaling to the Spc1 MAPK |
| GO:1990315 Mcs4 RR-MAPKKK complex | IPI PMID:24255738 Phosphorelay-dependent and -independent regulation of MAPKKK... | ACCEPT | Summary: Co-precipitation evidence shows the Wis1 MAPKK is found on the Mcs4-Wis4-Win1 complex and dissociates in response to both oxidative and osmotic stress signals, consistent with the part_of annotation. Reason: Physical-interaction evidence directly supports Wis1 membership in the Mcs4 RR-MAPKKK complex. Supporting Evidence: PMID:24255738 The Wis1 MAPKK is released from the Mcs4-MAPKKK complex in response to phosphorelay-dependent (H2O2) and -independent (osmostress) signals |
| GO:0071470 cellular response to osmotic stress | IMP PMID:7859738 Counteractive roles of protein phosphatase 2C (PP2C) and a M... | ACCEPT | Summary: Core process. wis1 mutations render cells hypersensitive to high osmolarity, directly demonstrating a requirement for Wis1 in osmotic stress signaling; Wis1 is the S. pombe ortholog of the osmoregulatory MEK Pbs2. Reason: Mutant-phenotype evidence demonstrates a core requirement in the osmotic stress response. Supporting Evidence: PMID:7859738 The Wis1 sequence is most closely related to the Saccharomyces cerevisiae MEK encoded by PBS2, which is required for osmoregulation. |
| GO:0038066 p38MAPK cascade | IMP PMID:9693384 The fission yeast mitotic regulator win1+ encodes an MAP kin... | ACCEPT | Summary: Core process. Win1 (MAPKKK) phosphorylates and activates Wis1, which signals to Spc1; loss of upstream input reduces Wis1 activity and Spc1 activation, confirming Wis1 in the SAPK cascade. Reason: Mutant analysis confirms Wis1 in the Spc1 (p38-class) stress MAPK cascade. Supporting Evidence: PMID:9693384 Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1 |
| GO:0004672 protein kinase activity | IDA PMID:9693384 The fission yeast mitotic regulator win1+ encodes an MAP kin... | MARK AS OVER ANNOTATED | Summary: Direct evidence that Wis1 has protein-kinase activity, but the parent term is generic; the specific MAP kinase kinase activity (GO:0004708) is the precise function and is independently well supported. Reason: Correct but too general; MAP kinase kinase activity is the precise supported term. Proposed replacements: MAP kinase kinase activity Supporting Evidence: PMID:9693384 Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1 |
| GO:0004708 MAP kinase kinase activity | IDA PMID:9693384 The fission yeast mitotic regulator win1+ encodes an MAP kin... | ACCEPT | Summary: Core molecular function, directly supported. Wis1 is the MAPKK activated by the Win1 MAPKKK that in turn phosphorylates/activates the Spc1 MAPK. Reason: Direct experimental support for the core MAPKK activity. Supporting Evidence: PMID:9693384 Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1 |
| GO:0038066 p38MAPK cascade | IMP PMID:8824588 The Atf1 transcription factor is a target for the Sty1 stres... | ACCEPT | Summary: Core process. The Sty1/Wis1 pathway is activated by osmotic, oxidative and heat stress and signals to the Atf1 transcription factor; Pyp2 induction provides negative feedback on Sty1/Wis1 signaling. Reason: Establishes Wis1 within the Sty1 stress-MAPK cascade that controls Atf1-dependent transcription. Supporting Evidence: PMID:8824588 results in a negative feedback loop that serves to control signaling through the Sty1/Wis1 pathway |
| GO:0005737 cytoplasm | IDA PMID:9585506 Phosphorylation and association with the transcription facto... | ACCEPT | Summary: Wis1 is detected completely in the cytoplasm at steady state and is active there as the activator kinase of Spc1. Cytoplasmic localization/activity is directly supported. Reason: Direct immunolocalization shows cytoplasmic Wis1 is the functional pool. Supporting Evidence: PMID:9585506 We have established that Wis1 is detected completely in the cytoplasm, whereas Atf1 is found exclusively in the nucleus. |
| GO:0004708 MAP kinase kinase activity | IDA PMID:10398679 Heat-shock-induced activation of stress MAP kinase is regula... | ACCEPT | Summary: Core molecular function. Wis1 (MEK) activates Spc1 through Thr-171/Tyr-173 phosphorylation; activation is strong under osmotic/oxidative stress and weak and transient under heat shock. Reason: Direct biochemical demonstration of Wis1 MAPKK activity toward Spc1. Supporting Evidence: PMID:10398679 osmostress and oxidative stress induce strong activation of the Wis1 MAPK kinase (MEK), which activates Spc1 through Thr-171/Tyr-173 phosphorylation |
| GO:0038066 p38MAPK cascade | IDA PMID:10398679 Heat-shock-induced activation of stress MAP kinase is regula... | ACCEPT | Summary: Core process. Wis1 is the MEK of the Spc1 p38/Hog1-class SAPK cascade, with stress-dependent activation kinetics. Reason: Direct evidence places Wis1 in the Spc1 (p38-class) SAPK cascade. Supporting Evidence: PMID:10398679 osmostress and oxidative stress induce strong activation of the Wis1 MAPK kinase (MEK), which activates Spc1 through Thr-171/Tyr-173 phosphorylation |
| GO:1990315 Mcs4 RR-MAPKKK complex | IDA PMID:24255738 Phosphorelay-dependent and -independent regulation of MAPKKK... | ACCEPT | Summary: Direct evidence that the Wis1 MAPKK is found on the Mcs4-Wis4-Win1 complex and is released upon stress; supports the part_of annotation. Reason: Direct co-purification evidence supports Wis1 association with the Mcs4 RR-MAPKKK complex. Supporting Evidence: PMID:24255738 The Wis1 MAPKK and the Tdh1 GAPDH are also found on the complex |
| GO:0004708 MAP kinase kinase activity | IDA PMID:9718372 Evidence for a novel MAPKKK-independent pathway controlling ... | ACCEPT | Summary: Core molecular function. Sty1/Spc1 is activated by the single MAPKK Wis1; Wis1 activation normally requires its conserved Ser-469/Thr-473 activation-loop phosphosites, with a minor MAPKKK-independent activation mode. Reason: Direct support for Wis1 as the sole MAPKK activating Sty1. Supporting Evidence: PMID:9718372 Sty1 is activated by a single MAPKK, Wis1. |
| GO:0038066 p38MAPK cascade | IMP PMID:9718372 Evidence for a novel MAPKKK-independent pathway controlling ... | ACCEPT | Summary: Core process. Wis1 is the sole MAPKK of the Sty1/Spc1 SAPK cascade responsive to osmotic, oxidative, heat, UV and anisomycin stress. Reason: Mutant analysis of the conserved activation-loop sites confirms Wis1 in the Sty1 SAPK cascade. Supporting Evidence: PMID:9718372 the conserved MAPKKK phosphorylation sites Ser 469 and Thr 473 in the catalytic domain of Wis1 are normally essential for Sty1 activation |
| GO:0004708 MAP kinase kinase activity | IDA PMID:7501024 Cell-cycle control linked to extracellular environment by MA... | ACCEPT | Summary: Core molecular function. Spc1 is a MAP kinase activated by Wis1 kinase in response to osmotic stress and nutrient limitation, supporting Wis1 MAPKK activity toward Spc1. Reason: Direct evidence that Wis1 is the activating kinase of Spc1. Supporting Evidence: PMID:7501024 Spc1 is a MAP kinase homologue that is activated by Wis1 kinase in response to osmotic stress and nutrient limitation. |
| GO:0038066 p38MAPK cascade | IMP PMID:7501024 Cell-cycle control linked to extracellular environment by MA... | ACCEPT | Summary: Core process. The Wis1-Spc1 MAPK pathway links cell-cycle G2/M control to the extracellular environment; Wis1 acts upstream of Spc1 in this SAPK cascade. Reason: Genetic/biochemical evidence places Wis1 in the Spc1 SAPK cascade. Supporting Evidence: PMID:7501024 a fission yeast MAP kinase pathway links the cell-cycle G2/M control with changes in the extracellular environment |
| GO:0038066 p38MAPK cascade | IMP PMID:8649397 Activation and regulation of the Spc1 stress-activated prote... | ACCEPT | Summary: Core process. Spc1 is activated by Wis1 MAPK kinase and inhibited by Pyp1; Spc1 is the p38/Hog1-related SAPK responsive to multiple stresses. Wis1 is the upstream MAPKK in this cascade. Reason: Establishes Wis1 as the activating MAPKK in the Spc1 p38-class SAPK cascade. Supporting Evidence: PMID:8649397 Spc1, also known as Sty1, is activated by Wis1 MAPK kinase and inhibited by Pyp1 tyrosine phosphatase. |
| GO:0010971 positive regulation of G2/M transition of mitotic cell cycle | IMP PMID:1756736 The wis1 protein kinase is a dosage-dependent regulator of m... | KEEP AS NON CORE | Summary: Wis1 was originally identified as a dosage-dependent inducer of mitosis: overexpression advances mitosis (small cell size) and loss delays it (elongated cells). This G2/M role is genuine but operates through the downstream Sty1/Spc1 cascade and reflects the pathway's coupling of stress and nutritional sensing to mitotic timing; it is a non-core, pleiotropic aspect relative to the core MAPKK signaling function. Reason: Genuine mitotic-timing role but a downstream, pleiotropic output of the SAPK pathway rather than a core biochemical function. Supporting Evidence: PMID:1756736 wis1+ plays an important role in the regulation of entry into mitosis, as it shares with cdc25+ and nim1+/cdr1+ the property of inducing mitosis in a dosage-dependent manner |
| GO:0005829 cytosol | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: High-throughput localization assigns Wis1 to the cytosol, consistent with the targeted studies showing Wis1 is predominantly cytoplasmic and active there. Reason: High-throughput cytosolic localization is concordant with focused experimental localization data. Supporting Evidence: PMID:9585506 We have established that Wis1 is detected completely in the cytoplasm, whereas Atf1 is found exclusively in the nucleus. |
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Download this section (compressed HTML)Q: What distinguishes the substrate selectivity of Wis1 for both Thr-171 and Tyr-173 of Spc1, and is the order of dual phosphorylation regulated?
Q: Does the stress-induced nuclear translocation of a fraction of Wis1 have a function beyond delivering/retaining Spc1, such as nuclear substrate phosphorylation?
Q: How is release of Wis1 from the Mcs4-Wis4-Win1 complex coupled to its activation, and is complex disassembly required for productive Spc1 phosphorylation?
Experiment: Quantitative in vitro kinase assays with recombinant active Wis1 and Spc1 activation-loop variants (T171A, Y173F, double mutant) to measure the kinetics and order of dual Thr/Tyr phosphorylation.
Experiment: Phosphoproteomic comparison of wild-type versus analog-sensitive wis1 (or constitutively active wis1DD) strains to define the direct and pathway-dependent Wis1 substrate/target landscape under osmotic versus oxidative stress.
Experiment: Live-cell imaging of endogenously tagged Wis1, Spc1 and Mcs4-Wis4-Win1 to track complex assembly/disassembly and Wis1 nucleocytoplasmic shuttling kinetics during a defined stress time course.
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