Cln2 is one of the two closely related G1/S cyclins (Cln1 and Cln2) of the budding yeast Saccharomyces cerevisiae and, together with the upstream G1 cyclin Cln3, forms the trio of Cln proteins of which at least one is required for passage through Start, the point in late G1 at which the cell commits to a new division cycle. Cln2 has no catalytic activity of its own: it is a cyclin-box protein that binds the sole essential cyclin-dependent kinase Cdc28 (Cdk1) and, with the Cks1 subunit, forms an active Cln2-Cdc28 kinase that requires Cak1-dependent phosphorylation of Cdc28 Thr169. CLN2 is transcribed in late G1 by the SBF (Swi4-Swi6) transcription factor once Cln3-Cdc28 has begun to inactivate the SBF-bound repressor Whi5; the newly made Cln2-Cdc28 then completes Whi5 phosphorylation and displacement (and phosphorylates the SBF co-regulator Stb1), closing a positive feedback loop that makes the burst of Start transcription rapid, coherent and irreversible. Cln2-Cdc28 also primes the multisite phosphorylation of the S-phase CDK inhibitor Sic1 and phosphorylates the pheromone-induced CDK inhibitor Far1, generating phosphodegrons recognised by SCF(Cdc4), so that Clb5/6-Cdc28 is released to initiate DNA replication; it phosphorylates Cdc6, and its cytoplasmic pool, concentrated at sites of polarized growth, acts on polarity and morphogenesis regulators to trigger bud emergence and spindle pole body duplication. Cln2-Cdc28 additionally shuts off the mating MAP kinase pathway by phosphorylating the scaffold Ste5 (and Ste20), which is why cells past Start are refractory to pheromone and why Cln2 promotes recovery from pheromone arrest. Cln2 shuttles between nucleus and cytoplasm: Cdc28-dependent phosphorylation of its C terminus drives nuclear exclusion, so the unphosphorylated protein is transiently nuclear early in the cycle while the bulk is cytoplasmic. The protein is very unstable (half-life about 10 min), with a PEST-rich C terminus that is phosphorylated by Cdc28 and recognised mainly by SCF(Grr1); its mRNA and protein peak in G1 and fall as cells enter S phase, and both are lost in cells exposed to mating pheromone, whose effector Far1 also inhibits Cln-Cdc28 directly. Cln1 and Cln2 belong to a Saccharomycotina-specific cyclin family distinct from Cln3 and from the B-type Clb cyclins; cln2 single mutants are viable, cln1 cln2 double mutants grow slowly with delayed budding and DNA synthesis, and cln1 cln2 cln3 triple mutants arrest as unbudded cells at Start. The dominant CLN2-1 truncation, which removes the destabilising C terminus, advances the G1/S transition and renders cells resistant to G1-arresting signals.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000082 G1/S transition of mitotic cell cycle | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (PAINT) annotation of the G1/S transition to the G1 cyclin clade (node PTN000019791, whose experimental descendants include cyclin D, cyclin E, Cln3 and Clb5/6). Cln2 is the textbook budding-yeast G1/S cyclin: Cln2-Cdc28 closes the Whi5/SBF positive feedback loop and primes Sic1 and Far1 for SCF-dependent destruction, which is precisely the sequence GO:0000082 describes (build-up of G1 CDK, transcription of G1 cyclins, positive feedback committing the cell to S phase). Reason: The IBD node placement is sound (the G1/S-promoting function of G1 cyclin-CDKs is ancestral to the clade) and Cln2 has abundant direct experimental support for the same biology on its own (the Start/IGI/IMP rows below; Cln2-Cdc28 phosphorylation of Whi5, Stb1, Sic1 and Far1 in cached full texts). The target itself is not in this row's WITH/FROM list, but sister members Cln3, Clb5 and Clb6 are, and the transfer to Cln2 is fully consistent with its own evidence. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000019791 · PTN000019791 SUPPORTS TRANSFER G1/S-promoting activity is ancestral to the G1 cyclin node and is directly demonstrated for Cln2 by genetics and biochemistry. Supporting Evidence: PMID:8387915 the G1 cyclins Cln1, Cln2 and Cln3 regulate entry into the cell cycle (Start) by activating the Cdc28 protein kinase PMID:2142620 The S. cerevisiae CLN genes encode cyclin homologs essential for progression from G1 to S phase PMID:21993622 We propose that in late G1, Clb5-Cdk1 is inhibited by Sic1, and the cascade of phosphorylation events begins with T5 phosphorylation by Cln2-Cdk1 |
| GO:0000307 cyclin-dependent protein kinase holoenzyme complex | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic annotation that Cln2 is a subunit of a cyclin-CDK holoenzyme. Cln2 forms an active Cln2-Cdc28(-Cks1) kinase complex, shown by co-immunoprecipitation from yeast, by reconstitution in insect cells and in cyclin-depleted extracts; the complex is catalogued as CPX-342 (CLN2-CDC28 kinase complex) in the Complex Portal. Reason: Being the cyclin subunit of a CDK holoenzyme is the defining, ancestral property of the cyclin family, and it is demonstrated directly for Cln2. The target (SGD:S000006177, CLN2) appears in its own WITH/FROM list, as expected when its own experimental annotation contributed to the IBD. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000019791 · PTN000019791 SUPPORTS TRANSFER Cyclin-CDK holoenzyme membership is ancestral to the whole cyclin family and directly shown for Cln2. Supporting Evidence: PMID:2142620 we demonstrate that the Cln2 polypeptide interacts with p34CDC28 to form an active protein kinase complex PMID:7862657 A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase |
| GO:0000307 cyclin-dependent protein kinase holoenzyme complex | IDA PMID:2142620 G1-specific cyclins of S. cerevisiae: cell cycle periodicity... | ACCEPT | Summary: Wittenberg, Sugimoto & Reed 1990: the Cln2 polypeptide co-immunoprecipitates with p34CDC28 in an active histone H1 kinase complex whose abundance peaks in G1 and collapses on pheromone exposure. Reason: The abstract states the result explicitly; this is the founding demonstration of the Cln2-Cdc28 holoenzyme (abstract-only cache, but the claim needs no further verification and is corroborated by the in vitro reconstitution work). Supporting Evidence: PMID:2142620 we demonstrate that the Cln2 polypeptide interacts with p34CDC28 to form an active protein kinase complex PMID:2142620 suggests that Cln proteins are an essential component of the active protein kinase complex required for the G1 to S transition |
| GO:0000307 cyclin-dependent protein kinase holoenzyme complex | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning annotation of CDK holoenzyme complex membership derived from the cyclin family signature. Reason: Correct family-level inference, fully corroborated by the IDA, IPI and IBA rows for the same term. |
| GO:0000307 cyclin-dependent protein kinase holoenzyme complex | IPI PMID:10913169 Cks1 is required for G(1) cyclin-cyclin-dependent kinase act... | ACCEPT | Summary: Reynard et al. 2000: Cln2 and Cdc28 co-expressed in baculovirus-infected insect cells form complexes whose protein kinase activity requires the third subunit Cks1, which both stabilises Cln2-Cdc28 complexes and activates pre-formed ones. Reason: Direct physical evidence for the Cln2-Cdc28-Cks1 holoenzyme; the study also defines Cks1 as an obligate component of the active G1 cyclin-CDK, which distinguishes Cln-Cdc28 from Clb-Cdc28 complexes. Supporting Evidence: PMID:10913169 Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro PMID:10913169 Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1 |
| GO:0000321 re-entry into mitotic cell cycle after pheromone arrest | IGI PMID:9927449 POG1, a novel yeast gene, promotes recovery from pheromone a... | KEEP AS NON CORE | Summary: Leza & Elion 1999 (genetic interaction with POG1): pheromone-arrested cells that fail to mate recover by re-entering the cycle; POG1 overexpression blocks alpha-factor repression of CLN1/CLN2 transcription, and epistasis shows that POG1 promotes recovery through CLN2, with the resulting Cln2 protein acting primarily on Ste20, an activator of the mating MAPK cascade. Reason: Cln2-Cdc28 does the work of recovery - it phosphorylates Far1 to trigger its degradation and phosphorylates the MAPK scaffold Ste5 (and Ste20) to shut off pheromone signalling - so the participation test is met and the annotation is correct. It is, however, a context-specific application of the same G1 CDK activity that executes Start, and the pheromone-recovery process is downstream of the core G1/S function; it is therefore retained as non-core, in line with the treatment of substrate-derived downstream processes for the other Cln cyclins. The cached record is abstract-only, so the specific epistasis experiments are deferred to the SGD curator. Supporting Evidence: PMID:9927449 Genetic tests strongly argue that POG1 promotes recovery through upregulation of the CLN2 gene and that the resulting Cln2 protein promotes recovery primarily through an effect on Ste20, an activator of the mating MAPK cascade PMID:17289571 In this study, we report that G1 CDK activity inhibits pheromone signaling by inhibiting Ste5 membrane recruitment PMID:11080155 ubiquitylation of Far1-nls1 or Far1-nls1/2 was dependent on phosphorylation of serine 87 by Cdc28–Cln2 |
| GO:0000321 re-entry into mitotic cell cycle after pheromone arrest | IGI PMID:9927449 POG1, a novel yeast gene, promotes recovery from pheromone a... | KEEP AS NON CORE | Summary: Second IGI row from the same study, recording the genetic interaction with the MAPK phosphatase gene MSG5, whose promotion of recovery parallels that of POG1 and which acts on the same Cln2-dependent recovery route. Reason: Same evidence base and same biology as the POG1 row - Cln2-Cdc28 phosphorylation of Ste5/Ste20 and Far1 genuinely executes recovery, but this is a downstream, pheromone-specific output of the core G1 CDK activity rather than a core function; retained as non-core. Supporting Evidence: PMID:9927449 We have isolated a novel gene, POG1, whose promotion of recovery parallels that of the MAPK phosphatase Msg5 PMID:9927449 Genetic tests strongly argue that POG1 promotes recovery through upregulation of the CLN2 gene and that the resulting Cln2 protein promotes recovery primarily through an effect on Ste20, an activator of the mating MAPK cascade |
| GO:0005515 protein binding | IPI PMID:10913169 Cks1 is required for G(1) cyclin-cyclin-dependent kinase act... | MODIFY | Summary: Protein-binding annotation with Cdc28 from Reynard et al. 2000, in which recombinant Cln2-Cdc28 complexes are assembled and assayed for kinase activity: the evidence is an active Cln2-Cdc28 kinase, i.e. Cln2 acting as the CDK activator. Reason: The paper measures Cln2-Cdc28 protein kinase activity (in insect-cell complexes and in yeast extracts), which directly supports cyclin-dependent protein serine/threonine kinase activator activity (GO:0061575) rather than uninformative generic protein binding. Removing the generic term does not question the interaction. Proposed replacements: cyclin-dependent protein serine/threonine kinase activator activity Supporting Evidence: PMID:10913169 The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts PMID:7862657 A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase |
| GO:0005515 protein binding | IPI PMID:11805826 Functional organization of the yeast proteome by systematic ... | MODIFY | Summary: Protein-binding annotation with Cdc28 from the Gavin et al. 2002 proteome-wide tandem-affinity purification/mass spectrometry survey of yeast complexes; Cln2 co-purifies with its cognate CDK. Reason: High-throughput co-purification with a protein kinase supports protein kinase binding (GO:0019901) as the minimal informative molecular function for this evidence; the generic term adds nothing beyond the holoenzyme and activator rows. The interaction is real and independently established biochemically. Proposed replacements: protein kinase binding Supporting Evidence: PMID:11805826 We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae PMID:2142620 we demonstrate that the Cln2 polypeptide interacts with p34CDC28 to form an active protein kinase complex |
| GO:0005515 protein binding | IPI PMID:11805837 Systematic identification of protein complexes in Saccharomy... | MODIFY | Summary: Protein-binding annotation with Cdc28 from the Ho et al. 2002 high-throughput mass-spectrometric protein complex identification (HMS-PCI) screen. Reason: Same reasoning as the other high-throughput rows - the partner is the CDK catalytic subunit, so protein kinase binding (GO:0019901) is the informative replacement; the activator function itself is captured by the GO:0016538/GO:0061575 rows. Proposed replacements: protein kinase binding Supporting Evidence: PMID:11805837 Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome PMID:2142620 we demonstrate that the Cln2 polypeptide interacts with p34CDC28 to form an active protein kinase complex |
| GO:0005515 protein binding | IPI PMID:16429126 Proteome survey reveals modularity of the yeast cell machine... | MODIFY | Summary: Protein-binding annotation with Cdc28 from the Gavin et al. 2006 genome-wide affinity-purification/MS survey of the yeast complexome. Reason: High-throughput interaction evidence with a protein kinase partner; protein kinase binding (GO:0019901) is the informative MF supported by this kind of evidence. Removal of the generic term does not question the Cln2-Cdc28 interaction. Proposed replacements: protein kinase binding Supporting Evidence: PMID:16429126 Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry PMID:2142620 we demonstrate that the Cln2 polypeptide interacts with p34CDC28 to form an active protein kinase complex |
| GO:0005515 protein binding | IPI PMID:17289571 A mechanism for cell-cycle regulation of MAP kinase signalin... | MODIFY | Summary: Protein-binding annotation with Cdc28 from Strickfaden et al. 2007, which shows that G1 CDK (Cln2/Cdc28) activity phosphorylates a cluster of sites flanking the membrane-binding motif of the MAPK scaffold Ste5, blocking its membrane recruitment and thereby shutting off pheromone signalling once cells pass Start. The evidence is Cln2-Cdc28 kinase activity toward a physiological substrate. Reason: The study assays Cln2/CDK-dependent phosphorylation of Ste5 in vivo and in vitro, i.e. Cln2 functioning as the activating and substrate-directing subunit of Cdc28; cyclin-dependent protein serine/threonine kinase activator activity (GO:0061575) is the informative replacement for generic protein binding. Proposed replacements: cyclin-dependent protein serine/threonine kinase activator activity Supporting Evidence: PMID:17289571 In this study, we report that G1 CDK activity inhibits pheromone signaling by inhibiting Ste5 membrane recruitment PMID:17289571 Cln2/CDK can phosphorylate the PAK-family kinase Ste20 |
| GO:0005515 protein binding | IPI PMID:20489023 A global protein kinase and phosphatase interaction network ... | MODIFY | Summary: Protein-binding annotation with Cdc28 from the Breitkreutz et al. 2010 kinase and phosphatase interaction network (AP-MS); Cln2 co-purifies with its cognate CDK. Reason: Mass-spectrometric co-purification with a protein kinase supports protein kinase binding (GO:0019901); the generic term adds nothing beyond the holoenzyme and activator annotations. Proposed replacements: protein kinase binding Supporting Evidence: PMID:20489023 We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes PMID:2142620 we demonstrate that the Cln2 polypeptide interacts with p34CDC28 to form an active protein kinase complex |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic annotation that Cln2 is active in the nucleus. Although most Cln2 is cytoplasmic at steady state, the protein shuttles: hypophosphorylated Cln2-Cdc28 is imported into the nucleus, is nuclear in small newborn cells, and an appended NES weakens Cln2 function in complementation and cell-size assays. Its key Start substrates (Whi5/SBF at promoters, Sic1, Far1) are nuclear and Far1 is degraded specifically in the nucleus after Cln2-Cdc28 phosphorylation. Reason: Nuclear activity of G1 cyclin-CDKs is ancestral for the node and target-specific evidence (NES-Cln2 loses functions; forced nuclear localization supports specific Cln2 functions; nuclear substrates) confirms that a nuclear pool of Cln2-Cdc28 acts. The target (SGD:S000006177) appears in its own WITH/FROM list, as expected. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000019791 · PTN000019791 SUPPORTS TRANSFER Nuclear site of action is ancestral for G1 cyclin-CDKs and directly supported for Cln2 by NES/NLS forced-localization experiments. Supporting Evidence: PMID:10611233 Overall, it appears that Cln2p is required in both the cytoplasm and the nucleus of the cell for maximal function PMID:11509671 These data are consistent with regulated shuttling of Cln2p in and out of the nucleus, where nonphosphorylated Cln2p is enriched in the nucleus and phosphorylated Cln2p is enriched in the cytoplasm PMID:11792824 Forced localization showed that some functions of Cln2 required a cytoplasmic location, while other functions required a nuclear location |
| GO:0005634 nucleus | IDA PMID:10611233 Distinct subcellular localization patterns contribute to fun... | ACCEPT | Summary: Miller & Cross 2000: indirect immunofluorescence and fractionation show Cln2 to be primarily cytoplasmic, but adding a nuclear export signal reduces Cln2 activity in cln complementation and cell-size assays (reversed by a point mutation in the NES), indicating that a functionally important fraction of Cln2 resides in the nucleus. Reason: Full text cached. The nuclear component is inferred here from the NES effect rather than seen directly, but the same group''s 2001 follow-up and Edgington & Futcher 2001 detect nuclear Cln2 directly, so the located_in nucleus annotation is sound. Supporting Evidence: PMID:10611233 While the majority of Cln2 myc p is clearly cytoplasmic, it is likely that some proportion of Cln2p also localizes to the nucleus, since limiting the ability of Cln2p to reside in the nucleus with an NES inhibits some Cln2p functions PMID:10611233 We found NES-dependent decreases in Cln2p and Cln3-1p activity in both cln complementation and cell size assays, indicating that the presence of Cln proteins in the nucleus is critical for some CLN functions |
| GO:0005634 nucleus | IDA PMID:11080155 Nuclear-specific degradation of Far1 is controlled by the lo... | ACCEPT | Summary: Blondel et al. 2000 (Far1 degradation): as part of showing that Far1 is degraded in the nucleus by SCF(Cdc4) after phosphorylation by Cdc28-Cln2, Cln1-GFP and Cln2-GFP were imaged and found in both nucleus and cytoplasm; purified Cdc28-Cln2 phosphorylates Far1 Ser87 to license its ubiquitylation. Reason: Full text cached; direct GFP localization of Cln2 in the nucleus and cytoplasm, and a mechanistic reason (nuclear Far1 phosphorylation) for a nuclear pool. Supporting Evidence: PMID:11080155 Cln1–GFP as well as Cln2–GFP were localized in both the nucleus and the cytoplasm PMID:11080155 ubiquitylation of Far1-nls1 or Far1-nls1/2 was dependent on phosphorylation of serine 87 by Cdc28–Cln2 |
| GO:0005634 nucleus | IDA PMID:11792824 Relationship between the function and the location of G1 cyc... | ACCEPT | Summary: Edgington & Futcher 2001: by fractionation and microscopy Cln2 was found in both nucleus and cytoplasm, as was its substrate Sic1; forced-localization cassettes showed some Cln2 functions require a nuclear location and one requires shuttling between compartments. Reason: Abstract-only cache but the abstract states the localization result explicitly and it is concordant with the Miller & Cross studies. Supporting Evidence: PMID:11792824 Cln2 was found in both nucleus and cytoplasm PMID:11792824 A substrate of Cln2, Sic1, was also in both compartments |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic annotation that Cln2 is active in the cytoplasm. Cln2 is the cytoplasmic G1 cyclin of budding yeast: the bulk of the protein is cytoplasmic, concentrated at sites of polarized growth, cytoplasmic Cln2 is required for its budding/polarity functions, and Miller & Cross describe this as the first indication of a cytoplasmic function for a CDK. Reason: Unlike its paralog Cln3 (for which the same IBA row is over-stated), cytoplasmic activity is a documented, Cln2-specific property confirmed by forced-localization genetics. The target (SGD:S000006177) appears in its own WITH/FROM list, as expected. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000019791 · PTN000019791 SUPPORTS TRANSFER Cytoplasmic activity of G1 cyclin-CDKs (cyclin D, Cln1/Cln2) is a node-level property that Cln2 itself exemplifies. Supporting Evidence: PMID:10611233 In contrast, Cln2p localizes to the cytoplasm. PMID:10611233 This is the first indication of a cytoplasmic function for a cyclin-dependent kinase PMID:11792824 Cytoplasmic Cln2 was concentrated at sites of polarized growth file:yeast/CLN2/CLN2-deep-research-falcon.md Cln2 is predominantly **cytoplasmic** and can concentrate at sites of polarized growth, consistent with its role in budding. |
| GO:0005737 cytoplasm | IDA PMID:10611233 Distinct subcellular localization patterns contribute to fun... | ACCEPT | Summary: Miller & Cross 2000: by indirect immunofluorescence and biochemical fractionation Cln2 is predominantly cytoplasmic, in contrast to the nuclear Cln3, and forcing Cln3 into the cytoplasm partially converts it to a Cln2-like functional profile. Reason: Full text cached; the cytoplasmic localization of Cln2 is shown by two independent methods and is functionally meaningful. Supporting Evidence: PMID:10611233 In contrast, Cln2p localizes to the cytoplasm. PMID:10611233 Constitutively expressed Cln2p remains cytoplasmic in large budded cells (data not shown), while Cln3p localizes to the nuclei |
| GO:0005737 cytoplasm | IDA PMID:11080155 Nuclear-specific degradation of Far1 is controlled by the lo... | ACCEPT | Summary: Blondel et al. 2000: Cln2-GFP is observed in both the cytoplasm and the nucleus. Reason: Full text cached; direct GFP localization, concordant with the immunofluorescence studies. Supporting Evidence: PMID:11080155 Cln1–GFP as well as Cln2–GFP were localized in both the nucleus and the cytoplasm |
| GO:0005737 cytoplasm | IDA PMID:11509671 Mechanisms controlling subcellular localization of the G(1) ... | ACCEPT | Summary: Miller & Cross 2001: wild-type Cln2 is primarily cytoplasmic with clear nuclear depletion; cytoplasmic localization requires both binding to Cdc28 and Cdc28-dependent phosphorylation of the Cln2 C terminus, and hypophosphorylated Cln2 (phospho-site mutant, cdc28-4 cells, or small newborn cells) accumulates in the nucleus. Reason: Full text cached; immunofluorescence and fractionation establish the cytoplasmic steady-state location and define the mechanism (phosphorylation-dependent nuclear exclusion of the Cln2-Cdc28 complex). Supporting Evidence: PMID:11509671 The Cln3p G 1 cyclin is localized primarily to the nucleus, while Cln2p is localized primarily to the cytoplasm PMID:11509671 Both binding to Cdc28p and Cdc28p-dependent phosphorylation in the C-terminal region of Cln2p are independently required for efficient nuclear depletion of Cln2p, suggesting that this process may be physiologically regulated |
| GO:0005737 cytoplasm | IDA PMID:11792824 Relationship between the function and the location of G1 cyc... | ACCEPT | Summary: Edgington & Futcher 2001: Cln2 is found in both nucleus and cytoplasm, with cytoplasmic Cln2 concentrated at sites of polarized growth; some Cln2 functions require a cytoplasmic location. Reason: Abstract states the result explicitly; independent confirmation of the cytoplasmic pool and its enrichment at polarized growth sites. Supporting Evidence: PMID:11792824 Cln2 was found in both nucleus and cytoplasm PMID:11792824 Cytoplasmic Cln2 was concentrated at sites of polarized growth |
| GO:0005737 cytoplasm | IMP PMID:11509671 Mechanisms controlling subcellular localization of the G(1) ... | ACCEPT | Summary: Mutant-based localization evidence from Miller & Cross 2001: mutating all seven Cdc28 consensus sites in Cln2 (Cln2-4t3s), inactivating Cdc28 (cdc28-4) or crippling the cyclin box (Cln2-KAEA) abolishes nuclear depletion and shifts Cln2 into the nucleus, showing that the cytoplasmic residence of wild-type Cln2 is an actively maintained, Cdc28-dependent state. Reason: Loss-of-function alleles that relocalize the protein define the cytoplasm as the regulated default compartment of the Cln2-Cdc28 complex. Supporting Evidence: PMID:11509671 Using a mutant Cln2p in which all potential Cdc28p target sites have been mutated, we find a partial accumulation of Cln2p in the nucleus PMID:11509671 Consistent with these data, Cln2p also accumulates in the nucleus when CDC28 has been inactivated by using the temperature-sensitive allele cdc28 - 4 |
| GO:0007089 traversing start control point of mitotic cell cycle | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning annotation to the Start-transit term derived from the Cln cyclin family signature. Reason: Correct and specific; the term's definition (commitment to S phase via positive feedback between transcription and G1 CDK activity) describes exactly what Cln2-Cdc28 does, and the experimental rows below confirm it. |
| GO:0007089 traversing start control point of mitotic cell cycle | IGI PMID:2147225 Cell cycle arrest caused by CLN gene deficiency in Saccharom... | ACCEPT | Summary: Cross 1990 (genetic interaction with CLN1 and CLN3): cln1 cln2 cln3 triple mutants kept alive by GAL-CLN3 arrest as unbudded G1 cells when CLN3 is shut off; the arrest resembles START-I arrest, is independent of the pheromone signalling pathway, and completes cycles already past S phase, showing a specific CLN requirement for START transit. Reason: Classical genetic evidence that the redundant Cln proteins, Cln2 among them, are required for passage through Start; abstract-only cache but the abstract states the conclusion explicitly. Supporting Evidence: PMID:2147225 Null mutations in three genes encoding cyclin-like proteins (CLN1, CLN2, and CLN3) in Saccharomyces cerevisiae cause cell cycle arrest in G1 (cln arrest) PMID:2147225 These results are consistent with a specific CLN requirement for START transit |
| GO:0007089 traversing start control point of mitotic cell cycle | IGI PMID:2574633 An essential G1 function for cyclin-like proteins in yeast. | ACCEPT | Summary: Richardson et al. 1989 (genetic interaction with CLN1 and CLN3): eliminating CLN1, CLN2 and DAF1/WHI1 (renamed CLN3) causes G1 arrest that any single CLN gene rescues; conditional CLN1 expression showed the essential Cln function is confined to G1 and decays rapidly when Cln synthesis stops. Reason: Founding demonstration of the redundant, essential G1 function of the Cln proteins, interpreted as activation of Cdc28 for the G1-to-S transition. Supporting Evidence: PMID:2574633 Mutational elimination of the CLN1, CLN2, and DAF1/WHI1 products leads to cell cycle arrest independent of cell type, while expression of any one of the genes allows cell proliferation PMID:2574633 The data are consistent with the hypothesis that Cln proteins activate the Cdc28 protein kinase, shown to be essential for the G1 to S phase transition in S. cerevisiae |
| GO:0007089 traversing start control point of mitotic cell cycle | IMP PMID:2569741 A family of cyclin homologs that control the G1 phase in yea... | ACCEPT | Summary: Hadwiger et al. 1989: CLN1 and CLN2 were cloned as dosage suppressors of cdc28-ts, and the dominant CLN2-1 allele (a C-terminal truncation) advances the G1-to-S transition and prevents G1 arrest in response to external signals. Reason: Gain-of-function genetics placing Cln2 at the G1 control point; corroborated by the loss-of-function IGI rows. Supporting Evidence: PMID:2569741 A dominant mutation in the CLN2 gene, CLN2-1, advances the G1- to S-phase transition in cycling cells and impairs the ability of cells to arrest in G1 phase in response to external signals PMID:2569741 Two Saccharomyces cerevisiae genes were isolated based upon their dosage-dependent rescue of a temperature-sensitive mutation of the gene CDC28 |
| GO:0016538 cyclin-dependent protein serine/threonine kinase regulator activity | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic annotation of CDK regulator activity to the G1 cyclin node. The regulator term is correct for Cln2 but is the generic parent; every experimental study of Cln2 shows that it activates Cdc28 (GST-Cln2 activates Cdc28 as an H1 kinase in cyclin-depleted extracts; Cln2-Cdc28 immunoprecipitates are active kinases), which the child term GO:0061575 captures. Reason: Direction of regulation is unambiguous: cyclins of this node are CDK activators, and for Cln2 the activation is demonstrated directly in vitro and in vivo. Following the treatment of the sister G1 cyclin Cln3, the more specific activator child term is proposed while the parent remains biologically true. The target (SGD:S000006177) appears in its own WITH/FROM list, as expected. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000019791 · PTN000019791 SUPPORTS TRANSFER The node-level regulator assertion transfers correctly; the only issue is that the activator child term is the accurate level of specificity for this clade. Proposed replacements: cyclin-dependent protein serine/threonine kinase activator activity Supporting Evidence: PMID:7862657 A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase PMID:8387915 the G1 cyclins Cln1, Cln2 and Cln3 regulate entry into the cell cycle (Start) by activating the Cdc28 protein kinase |
| GO:0016538 cyclin-dependent protein serine/threonine kinase regulator activity | IDA PMID:10409718 Regulation of transcription at the Saccharomyces cerevisiae ... | MODIFY | Summary: Ho et al. 1999 (Stb1): Cln2-Cdc28 kinase immunoprecipitated from HA-CLN2 cells phosphorylates the Swi6-binding protein Stb1 in vitro (a better substrate than histone H1), and Stb1 phosphoforms in vivo depend on CLN1/CLN2/CLN3, with Cln1/Cln2-Cdc28 identified as the physiological Stb1 kinases; full text cached. Reason: The assay is of an active Cln2-Cdc28 kinase acting on a Start transcription regulator, which supports the activator child term GO:0061575 rather than the generic regulator parent; the regulator term is not wrong, but the activator term is the accurate description and is used consistently across the Cln2 and Cln3 reviews. Proposed replacements: cyclin-dependent protein serine/threonine kinase activator activity Supporting Evidence: PMID:10409718 Stb1 was an excellent substrate for both the Cln1-Cdc28 and Cln2-Cdc28 kinases in vitro and was a better in vitro substrate than histone H1 PMID:10409718 several observations suggest that Cln1-Cdc28 and Cln2-Cdc28 are the physiological kinases for Stb1 phosphorylation |
| GO:0016538 cyclin-dependent protein serine/threonine kinase regulator activity | IDA PMID:7862657 G1 cyclin-dependent activation of p34CDC28 (Cdc28p) in vitro... | MODIFY | Summary: Deshaies & Kirschner 1995: in a cyclin-depleted G1 extract, a GST-Cln2 chimera binds Cdc28 and activates it as a histone H1 kinase; activation requires ATP, cytosol and the CAK-phosphorylated Thr169 of Cdc28. This is the cleanest biochemical demonstration that Cln2 is a Cdc28 activator. Reason: The experiment directly measures Cln2-dependent activation of Cdc28, which is the definition of GO:0061575 (binds to and increases the activity of a CDK); the generic regulator parent understates what was shown. Proposed replacements: cyclin-dependent protein serine/threonine kinase activator activity Supporting Evidence: PMID:7862657 A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase PMID:7862657 Activation of Cdc28p by GST-Cln2p requires ATP, crude yeast cytosol, and the conserved Thr-169 residue that serves in other organisms as a substrate for phosphorylation by cyclin-dependent protein kinase-activating kinase |
| GO:0016538 cyclin-dependent protein serine/threonine kinase regulator activity | IDA PMID:8387915 Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 ... | MODIFY | Summary: Tyers, Tokiwa & Futcher 1993: Cln1- and Cln2-associated histone H1 kinase activity is much stronger than that of the rarer Cln3, and the Cln1/Cln2 kinases are proposed to be the cyclins that directly catalyse Start downstream of Cln3. Reason: The comparison of cyclin-associated Cdc28 kinase activities is direct evidence that Cln2 activates Cdc28; the activator child term GO:0061575 is the accurate level. Proposed replacements: cyclin-dependent protein serine/threonine kinase activator activity Supporting Evidence: PMID:8387915 the G1 cyclins Cln1, Cln2 and Cln3 regulate entry into the cell cycle (Start) by activating the Cdc28 protein kinase PMID:8387915 Cln3 is a much rarer protein than Cln1 or Cln2 and has a much weaker associated histone H1 kinase activity |
| GO:0016538 cyclin-dependent protein serine/threonine kinase regulator activity | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning annotation of CDK regulator activity derived from the cyclin family signature. Correct at family level and redundant with the IBA and experimental rows. Reason: The automated inference is correct but stops at the generic parent; the experimental evidence shows Cln2 is an activator of Cdc28, so the activator child term (GO:0061575) proposed on the IBA and IDA rows is the consistent replacement. Proposed replacements: cyclin-dependent protein serine/threonine kinase activator activity Supporting Evidence: PMID:7862657 A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IDA PMID:19823668 Dual regulation by pairs of cyclin-dependent protein kinases... | NEW | Summary: Proposed annotation for the positive-feedback arm of Start transcription. Recombinant Cln2-Cdc28 phosphorylates the SBF-bound repressor Whi5 and releases it from a preassembled Whi5-Swi4-Swi6 complex in vitro (an activity that purified Cln3-Cdc28 and Pcl9-Pho85 lack), and in vivo the slow-migrating Whi5 phosphoforms are completely absent in cln1 cln2 cells (Huang et al. 2009, full text cached; the same in vitro result was first reported by de Bruin et al. 2004). Cln2-Cdc28 also phosphorylates the SBF co-regulator Stb1 (Ho et al. 1999). The G1/S term definitions (GO:0000082, GO:0007089) presuppose this positive feedback of G1 cyclins on SBF-driven transcription, but no transcription-branch term is currently annotated to CLN2. Reason: Participation test met: Cln2-Cdc28 is the kinase that phosphorylates Whi5 and Stb1, dissociating the repressor from SBF and enabling the burst of SBF/MBF-dependent RNA polymerase II transcription at Start, so the gene product does the work of the positive regulation rather than merely being required for it. Comparator check: the sister cyclin Cln3 carries SGD experimental annotations to the regulation-of-transcription branch (GO:0006357, IGI/IMP from the same Huang et al. 2009 study), so the term is one curators use for the cyclin that phosphorylates Whi5; the historical caveat that CLN1/CLN2 are dispensable for the initial activation of SBF (Cln3 suffices, as noted in Ho et al. 1999) does not contradict a positive-regulatory role, since Cln1/Cln2 feedback is what makes the transcriptional burst complete and coherent. The positive term is used to match the treatment of Cln3 (GO:0006357 modified to GO:0045944) and of the core_functions block; it sits on a different branch from GO:0000082 and GO:0007089, so it is not redundant with them. Retained at IDA strength because the in vitro dissociation assay and the in vivo phosphoform dependence are in the cached full text. Supporting Evidence: PMID:19823668 As expected, Cln2-Cdc28 phosphorylation caused most of the SBF-bound Whi5 to be released into the soluble fraction PMID:19823668 Whi5 is then further phosphorylated by Cln1- and Cln2-Cdc28 complexes leading to complete disassembly of the Whi5-SBF complex, Whi5 nuclear export and a burst in gene expression necessary for the G1/S phase transition PMID:19823668 slow migrating Whi5 isoforms present in asynchronous wt extracts (Figure 2B, lane 1) were modestly reduced in cells lacking CLN3 (Figure 2B, lane 7) and completely absent in a cln1Δ cln2Δ double mutant (Figure 2B, lane 6), confirming that Whi5 phosphorylation depends on Cln-Cdc28 kinase complexes PMID:15210110 Cln/CDK phosphorylation of Whi5 in vitro promotes its dissociation from SBF complexes PMID:10409718 several observations suggest that Cln1-Cdc28 and Cln2-Cdc28 are the physiological kinases for Stb1 phosphorylation file:yeast/CLN2/CLN2-deep-research-falcon.md SBF induces **CLN1** and **CLN2**, after which Cln1/2–Cdc28 reinforces the transition by phosphorylating the SBF repressor Whi5. This creates positive feedback that sharpens late-G1 transcription and promotes commitment. |
| GO:1902806 regulation of cell cycle G1/S phase transition | NAS PMID:2569741 A family of cyclin homologs that control the G1 phase in yea... | MODIFY | Summary: Complex Portal author-statement annotation (for the CLN2-CDC28 kinase complex, CPX-342) citing the Hadwiger et al. 1989 cloning paper. The complex does regulate the G1/S transition, but the term is the generic, direction-less parent and is not specific to the mitotic cycle. Reason: The direction is unambiguous (Cln2-Cdc28 activates Start and the CLN2-1 gain-of-function allele advances G1/S), so the more precise positive regulation of G1/S transition of mitotic cell cycle (GO:1900087) is the appropriate term, as used for Cln3. The process itself is already captured by the GO:0000082 and GO:0007089 rows. Proposed replacements: positive regulation of G1/S transition of mitotic cell cycle Supporting Evidence: PMID:2569741 A dominant mutation in the CLN2 gene, CLN2-1, advances the G1- to S-phase transition in cycling cells and impairs the ability of cells to arrest in G1 phase in response to external signals PMID:7862657 transient accumulation of G1 cyclin/p34CDC28 (Cdc28p) complexes induces cells to traverse the cell cycle Start checkpoint and commit to a round of cell division |
| GO:2000045 regulation of G1/S transition of mitotic cell cycle | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO electronic mapping from the Cyclin_CLN family entry (IPR014399, the Saccharomycotina Cln1/Cln2-type cyclins) to regulation of the mitotic G1/S transition. Reason: The family-level mapping is correct but direction-less; Cln2-Cdc28 is an activator of the transition, so positive regulation of G1/S transition of mitotic cell cycle (GO:1900087) is the accurate term. Cln2 does the work of the transition itself (Whi5/Sic1/Far1 phosphorylation), which is why the direct GO:0000082 rows are also accepted. Proposed replacements: positive regulation of G1/S transition of mitotic cell cycle Supporting Evidence: PMID:2569741 A dominant mutation in the CLN2 gene, CLN2-1, advances the G1- to S-phase transition in cycling cells and impairs the ability of cells to arrest in G1 phase in response to external signals |
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Download this section (compressed HTML)Q: Which Cln2-Cdc28 substrates in the cytoplasm account for its Cln2-specific, non-interchangeable role in polarization and bud emergence, and does docking through the LP motif or localization at polarized-growth sites explain why a mitotic cyclin cannot substitute?
Q: Is the transient nuclear residence of hypophosphorylated Cln2-Cdc28 in newborn cells the pool that phosphorylates Whi5 and Sic1, or do cytoplasmic Cln2-Cdc28 complexes act on these substrates during their own nucleocytoplasmic shuttling?
Q: To what extent is Cln2 turnover physiologically shared between SCF(Grr1) and SCF(Cdc4), and is the F-box choice determined by the compartment in which phosphorylated Cln2 resides?
Experiment: Combine analog-sensitive cdc28-as1 with rapidly degradable (AID) Cln2 and NLS- versus NES-tethered Cln2 variants, then perform quantitative phosphoproteomics within minutes of Cln2 release in synchronized small daughter cells to map nuclear (Whi5, Stb1, Sic1, Far1) versus cytoplasmic (polarity, Ste5) targets.
Hypothesis: Cln2-Cdc28 has a compartment-specific substrate repertoire that underlies the Cln2/Cln3 functional split.
Experiment: Using a cyclin-replacement strain in which Cln1/2/3 are replaced by an inducible mitotic cyclin, test whether Cln2 docking-pocket mutants, Cln2-Clb chimeras or LP-motif mutants of candidate polarity substrates restore or abolish polarized growth, scoring bud emergence and Cdc42 polarization by live imaging.
Hypothesis: Cln2-specific docking (VLLPP-type LP motifs) rather than bulk CDK activity is required for bud emergence.
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