Mcs6 (also called Crk1 and Mop1) is the fission yeast ortholog of CDK7, an essential 335-residue cyclin-dependent serine/threonine protein kinase of the CMGC family. With its cyclin H-like partner Mcs2 and the RING-finger MAT1-like assembly factor Pmh1 it forms the trimeric Mcs6-Mcs2-Pmh1 complex, which exists both as a free ~200 kDa trimer and as the kinase module (TFIIK) of the general transcription factor TFIIH. The complex has two experimentally separable outputs. As a CDK-activating kinase (CAK) it phosphorylates the activation-loop residue Thr167 of the major cell-cycle CDK Cdc2, a modification required for full Cdc2-cyclin activity; this CAK role is shared with the monomeric Cak1-like kinase Csk1, so Cdc2 activation and cell-cycle progression fail only when both enzymes are impaired. As the TFIIH-associated kinase it phosphorylates the heptapeptide repeats of the RNA polymerase II C-terminal domain, preferentially on Ser5 and also on Ser7, during transcription initiation; this priming phosphorylation is required to recruit the Cdk9 (P-TEFb)-Pcm1 (cap methyltransferase) complex to the transcribing polymerase and stimulates subsequent CTD phosphorylation by Cdk9, coupling initiation to early elongation and mRNA capping. Mcs6 is itself activated by phosphorylation of its T-loop residue Ser165 by Csk1, and Pmh1 can substitute for this phosphorylation. Conditional loss of Mcs6 lowers CTD Ser5 phosphorylation but reduces global transcription only modestly, instead selectively repressing a subset of genes enriched for the Sep1/Ace2-dependent cytokinesis and cell-separation program, so that mutant cells arrest as multiseptate, multinucleate cells. The protein localizes to the nucleus, with an additional cytoplasmic pool, and acts on chromatin-bound RNA polymerase II.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000785 chromatin | IDA PMID:20605454 A gene-specific requirement of RNA polymerase II CTD phospho... | ACCEPT | Summary: Coudreuse et al. (2010) studied gene-specific requirements for Pol II CTD phosphorylation in S. pombe, comparing the Ser2 kinase Lsk1 with the Ser5 kinase Mcs6. The cached record is abstract-only and the abstract foregrounds Lsk1 and ste11; the PomBase curator read the full text, which (per the paired GO:0140836 annotation from the same paper) includes analysis of Mcs6-dependent Ser5 phosphorylation and Mcs6 on chromatin. Independent full-text evidence (PMID:19328067) shows Mcs6 crosslinking to the eng1+ gene by ChIP, i.e. the kinase acts on chromatin-bound Pol II as a TFIIH subunit. Reason: Mcs6 is the kinase subunit of TFIIH and phosphorylates Pol II CTD Ser5 at promoters; ChIP in PMID:19328067 recovers Mcs6 at the eng1+ locus, and the GO-CAM model of RNA capping (gocams/66187e4700001573) already places the Mcs6 S5-kinase activity in chromatin. The abstract-only cache does not show the Mcs6 ChIP data from this paper, but the location is clearly correct for the gene, so the curator's IDA is accepted. Core site of action. Supporting Evidence: PMID:19328067 Mcs6 activity was not required for its own recruitment (Figure S3) PMID:19328067 PIC assembly occurred normally at the eng1+ promoter even when Mcs6as was inhibited PMID:20605454 We show that phosphorylation of serine 2 (S2P) in the C-terminal domain of the largest subunit of the RNA polymerase II (PolII) enzyme by the Lsk1 cyclin-dependent kinase has only a minor impact on global gene expression during vegetative growth but is critical for the induction of ste11 transcription during sexual differentiation. |
| GO:0001111 RNA polymerase II promoter clearance | ISO GO_REF:0000024 | ACCEPT | Summary: PomBase curator transfer from Drosophila Cdk7 (FB:FBgn0263237), the metazoan ortholog, to Mcs6. Promoter clearance is the transition from initiation to elongation; TFIIH-kinase phosphorylation of the CTD on Ser5 is the canonical mark of this transition. In S. pombe, chemical-genetic inhibition of Mcs6 leaves PIC assembly and promoter-proximal Pol II unchanged but depletes Pol II downstream and blocks Cdk9/Pcm1 recruitment (PMID:19328067), i.e. the defect maps precisely to the initiation-to-elongation transition. Reason: The ortholog-based transfer is consistent with the fission-yeast data: Mcs6 primes the CTD during initiation and its activity is needed for the polymerase to progress from an assembled PIC into productive elongation (PMID:19328067). This is the most mechanistically precise process term for the TFIIK/TFIIH kinase step and is compatible with the initiation (GO:0006367) and elongation-regulation (GO:0032968) annotations on either side of it. Propagation Review Root cause: NO FAILURE CORE Sources checked: FB:FBgn0263237 · Cdk7 SUPPORTS TRANSFER Drosophila Cdk7, the metazoan TFIIH kinase/CAK ortholog; the promoter-clearance role of the TFIIH kinase is conserved and the S. pombe chemical-genetic data (PMID:19328067) independently place the Mcs6 requirement at the initiation-to-elongation transition. Supporting Evidence: PMID:19328067 PIC assembly occurred normally at the eng1+ promoter even when Mcs6as was inhibited PMID:19328067 The results suggest that Mcs6 activity is specifically required to bring Cdk9/Pcm1 to the elongation complex. PMID:19328067 We propose that TFIIH primes the CTD and promotes recruitment of P-TEFb/Pcm1, serving to couple elongation and capping of select pre-mRNAs. |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the Ser/Thr protein kinase active-site signature (IPR008271) to the generic protein kinase activity term. Mcs6 is a demonstrated protein kinase (CTD and Cdk T-loop phosphorylation in immunoprecipitates; PMID:8557036, PMID:8557037). Reason: Correct but general; it is the parent of the experimentally supported cyclin-dependent Ser/Thr kinase and CTD kinase terms. Harmless electronic annotation. Supporting Evidence: PMID:8557036 In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:10226032 Cdc2 activation in fission yeast depends on Mcs6 and Csk1, t... | MODIFY | Summary: Lee et al. (1999) showed that Csk1 activates both monomeric and Mcs2-bound Mcs6, that Csk1 can also activate Cdc2-cyclin complexes, and that only the csk1-delta mcs6-ts double mutant fails to activate Cdc2 and arrests before mitosis; Cdc2-cyclin complexes from the arrested cells could be re-activated in vitro by recombinant CAK. This defines Mcs6-Mcs2 as one of two partially redundant CAKs that phosphorylate the Cdc2 T-loop (Thr167). Cached record is abstract-only. Reason: The evidence is sound (Mcs6 phosphorylates the Cdc2 activation-loop threonine) but the enzyme assayed and genetically tested here is explicitly the cyclin-bound Mcs6-Mcs2 complex, i.e. a cyclin-dependent Ser/Thr kinase, for which GO:0004693 is the exact term already used on five other rows for this gene. Recording the specific child rather than the generic parent is more informative and keeps the term set consistent (the paired GO:0004674 row from PMID:22508988 is likewise refined to the position-specific CTD kinase terms). Proposed replacements: cyclin-dependent protein serine/threonine kinase activity Supporting Evidence: PMID:10226032 Csk1 activated both the monomeric and the Mcs2-bound forms of Mcs6. PMID:10226032 When a double mutant carrying a csk1 deletion and a temperature-sensitive mcs6 allele was incubated at the restrictive temperature, Cdc2 was not activated and the cells underwent a cell division arrest prior to mitosis. PMID:10226032 Thus, fission yeast contains two partially redundant CAKs: the Mcs6-Mcs2 complex and Csk1. PMID:10226032 Cdc2-cyclin complexes isolated from the arrested cells could be activated in vitro by recombinant CAK, whereas complexes from wild-type cells or either of the single mutants were refractory to activation. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:22508988 Separate domains of fission yeast Cdk9 (P-TEFb) are required... | MODIFY | Summary: St Amour et al. (2012; full text cached) affinity-purified Mcs6 complexes from S. pombe and showed by phospho-specific immunoblotting that Mcs6 phosphorylates the Pol II CTD on Ser2, Ser5 and Ser7 in vitro; in vivo, chemical-genetic inhibition of mcs6-as reduced both Ser5-P and Ser7-P, whereas Cdk9 inhibition did not affect Ser7-P. Mcs6-primed CTD is a preferred substrate for Cdk9. Reason: The evidence is sound but the generic Ser/Thr kinase term under-describes it: this paper is the primary demonstration that Mcs6 is the fission-yeast CTD Ser7 kinase (in vitro and required in vivo), for which GO now has the dedicated term GO:0140837 (RNA polymerase II CTD heptapeptide repeat S7 kinase activity); the Ser5 activity shown in the same experiments is already captured by GO:0140836 on other rows. Recording the position-specific activities is more informative than the parent. Proposed replacements: RNA polymerase II CTD heptapeptide repeat S7 kinase activity RNA polymerase II CTD heptapeptide repeat S5 kinase activity Supporting Evidence: PMID:22508988 Mcs6 phosphorylated Ser7 in vitro and was required for Ser7-P in vivo PMID:22508988 Both Ser5-P and Ser7-P depended on Mcs6 activity in vivo PMID:22508988 Immunoblot analysis indicated that Mcs6 phosphorylates Ser2, Ser5, and Ser7, as did human Cdk7 |
| GO:0004693 cyclin-dependent protein serine/threonine kinase activity | EXP PMID:19328067 TFIIH and P-TEFb coordinate transcription with capping enzym... | ACCEPT | Summary: Viladevall et al. (2009; full text cached) engineered analog-sensitive mcs6-as and showed that Mcs6 complexes affinity-purified from yeast (via tagged Pmh1, i.e. the Mcs6-Mcs2-Pmh1 holoenzyme) phosphorylate the Pol II CTD on Ser5 in vitro and that inhibition of Mcs6 in vivo reduces Ser5-P, blocks cell division and represses specific transcripts. The discussion notes Mcs6's additional CAK role in cell-cycle control. Reason: Mcs6 is a genuine cyclin-dependent kinase: it requires its cyclin Mcs2 for activity (PMID:8557036, PMID:8557037) and the enzyme assayed here is the trimeric Mcs6-Mcs2-Pmh1 complex. The cyclin-dependent Ser/Thr kinase term is therefore exactly right for this gene (unlike the monomeric budding-yeast Cak1). Core function. Supporting Evidence: PMID:19328067 In vitro, Mcs6 generated Rpb1 CTD isoforms recognized by a monoclonal antibody specific for modification at Ser5 (H14) PMID:19328067 Inhibition of Mcs6 caused dose- and time-dependent decreases in Ser5 phosphorylation in vivo, measured with either Ser5-P or H14. PMID:19328067 The different morphological effects of inhibiting either CDK alone could be due to the additional role of Mcs6, one of two CDK-activating kinases (CAKs), in cell cycle control |
| GO:0004693 cyclin-dependent protein serine/threonine kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000624096 (the CDK7 subfamily within the CDK family PTHR24056) propagates cyclin-dependent Ser/Thr kinase activity; the node is seeded by experimentally characterised members including human/rat CDK7, Drosophila Cdk7 and Mcs6 itself (PomBase:SPBC19F8.07 in WITH/FROM, marking experimental grounding on the target). Reason: Phylogenetically sound placement: every CDK7-family kinase characterised (metazoan CDK7, fission-yeast Mcs6, and the Kin28 branch) is a cyclin H-dependent kinase. Mcs6 has direct evidence for cyclin (Mcs2)-dependent CTD kinase and CAK activity (PMID:8557036, PMID:8557037). Core function. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000624096 · PTN000624096 SUPPORTS TRANSFER CDK7-subfamily PAINT node with IBD support for cyclin-dependent Ser/Thr kinase activity, seeded by human CDK7 and by Mcs6's own IDA annotations; the target lies squarely inside the clade and shows no divergence. Supporting Evidence: PMID:8557036 In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. PMID:8557037 The Crk1-Mcs2 complex possesses CAK activity in vitro in that it phosphorylates human Cdk2 on Thr160 which results in its activation in the presence of cyclin A. |
| GO:0004693 cyclin-dependent protein serine/threonine kinase activity | IDA PMID:15829570 Impairment of the TFIIH-associated CDK-activating kinase sel... | ACCEPT | Summary: Lee et al. (2005; full text cached) reconstituted the Mcs6-Mcs2-Pmh1 trimer in insect cells, showed that HA-Mcs6 immunoprecipitates have CTD kinase activity that is stimulated by Csk1 co-expression (activating Ser165 phosphorylation) and that Pmh1 can bypass the T-loop requirement, and characterised conditional mcs6/pmh1 mutants that lose CTD Ser5 phosphorylation while still activating Cdc2 (Csk1 compensation). Reason: Direct reconstitution of the cyclin-dependent kinase holoenzyme and measurement of its activity; the paper also documents both physiological outputs (Cdc2 Thr167 CAK and TFIIH-associated CTD kinase). Core function. Supporting Evidence: PMID:15829570 The fission yeast Mcs6–Mcs2–Pmh1 complex, homologous to metazoan Cdk7–cyclin H-Mat1, has dual functions in cell division and transcription: as a partially redundant cyclin-dependent kinase (CDK)-activating kinase (CAK) that phosphorylates the major cell cycle CDK, Cdc2, on Thr-167; and as the RNA polymerase (Pol) II carboxyl-terminal domain (CTD) kinase associated with transcription factor (TF) IIH. PMID:15829570 We next reconstituted the Mcs6–Mcs2–Pmh1 complex in baculovirus-infected insect cells PMID:15829570 Activity of the wild-type and T160A proteins increased with Csk1 coexpression, consistent with activating phosphorylation at Ser-165 |
| GO:0004693 cyclin-dependent protein serine/threonine kinase activity | IDA PMID:8557036 Schizosaccharomyces pombe Mop1-Mcs2 is related to mammalian ... | ACCEPT | Summary: Damagnez et al. (1995) cloned mop1 (= mcs6/crk1), showed it is essential and rescued by Xenopus MO15 (CDK7), that Mop1 associates with the cyclin H-related Mcs2 (and each with the heterologous human partner), and that immunoprecipitated Mop1-Mcs2 acts both as a Pol II CTD kinase and as a CAK in vitro. Abstract-only in cache. Reason: Founding biochemical evidence that Mcs6 is a cyclin (Mcs2)-dependent Ser/Thr kinase with dual CTD-kinase and CAK activities, distinguishing it from budding-yeast Kin28-Ccl1. Core function. Supporting Evidence: PMID:8557036 This suggests that Mop1 would be a CDK, and indeed Mop1 associates with a previously characterized cyclin H-related cyclin Mcs2 of S.pombe. PMID:8557036 In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. PMID:8557036 The CAK activity of Mop1-Mcs2 distinguishes it from the related CDK-cyclin pair Kin28-Ccl1 from S.cerevisiae, and supports the notion that Mop1-Mcs2 may represent a homolog of MO15-cyclin H in S.pombe with apparent dual roles as a RNA polymerase CTD kinase and as a CAK. |
| GO:0004693 cyclin-dependent protein serine/threonine kinase activity | IDA PMID:8557037 Identification of a cdk-activating kinase in fission yeast. | ACCEPT | Summary: Buck et al. (1995) identified crk1 as a second fission-yeast CDK, showed it is essential, associates with the cyclin H-like Mcs2, and that the Crk1-Mcs2 complex phosphorylates human Cdk2 on Thr160 (activating it with cyclin A) and a Pol II CTD peptide; crk1 is allelic to mcs6. Abstract-only in cache. Reason: Direct in vitro demonstration of cyclin-bound Crk1/Mcs6 kinase activity toward both a CDK T-loop and the CTD. Core function. Supporting Evidence: PMID:8557037 We show that Crk1 associates with the Mcs2 mitotic catastrophe suppressor, a cyclin H-like molecule, and overexpression of crk1 rescues the cell-cycle arrest defect of a mcs2-75 cdc2-3w cdc25-22 triple mutant at high temperature. PMID:8557037 The Crk1-Mcs2 complex possesses CAK activity in vitro in that it phosphorylates human Cdk2 on Thr160 which results in its activation in the presence of cyclin A. PMID:8557037 In addition Crk1-Mcs2 effectively phosphorylates a peptide corresponding to the C-terminal repeat domain (CTD) of RNA polymerase II. PMID:8557037 We demonstrate that crk1 is allelic to the mcs6 mitotic catastrophe suppressor and that the X.laevis MO15 gene rescues the cell-cycle arrest of an mcs6-13 cdc2-3w cdc25-22 at high temperature. |
| GO:0004693 cyclin-dependent protein serine/threonine kinase activity | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learned rule assigning cyclin-dependent Ser/Thr kinase activity; concordant with multiple IDA/IBA rows. Reason: Electronic annotation that matches the experimentally established activity of the Mcs6-Mcs2 holoenzyme (PMID:8557036, PMID:8557037). Supporting Evidence: PMID:8557036 In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. |
| GO:0005515 protein binding | IPI PMID:8557037 Identification of a cdk-activating kinase in fission yeast. | MODIFY | Summary: IPI with Mcs2 (PomBase:SPBP16F5.02): Buck et al. showed that Crk1 associates with Mcs2, 'a cyclin H-like molecule', and that the Crk1-Mcs2 complex has CAK and CTD kinase activity. Damagnez et al. (PMID:8557036) independently showed Mop1 (Mcs6) binding to Mcs2 and to human cyclin H. Reason: Generic protein binding is uninformative, and the interaction it records is the defining interaction of a CDK with its cyclin: Mcs2 is the cyclin H ortholog and the obligate activating partner of Mcs6. GO:0030332 (cyclin binding) captures the same evidence as a functionally meaningful molecular function; the interaction itself is not in doubt. Proposed replacements: cyclin binding Supporting Evidence: PMID:8557037 We show that Crk1 associates with the Mcs2 mitotic catastrophe suppressor, a cyclin H-like molecule, and overexpression of crk1 rescues the cell-cycle arrest defect of a mcs2-75 cdc2-3w cdc25-22 triple mutant at high temperature. PMID:8557036 This suggests that Mop1 would be a CDK, and indeed Mop1 associates with a previously characterized cyclin H-related cyclin Mcs2 of S.pombe. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the protein kinase domain (IPR000719) and the kinase ATP-binding-site signature (IPR017441). UniProt annotates the ATP-binding glycine loop (residues 17-25) and Lys40. Reason: Mcs6 is an ATP-dependent protein kinase; ATP binding is intrinsic to the demonstrated CTD kinase and CAK activities (PMID:8557036, PMID:8557037). Correct, if generic, electronic annotation. Supporting Evidence: PMID:8557037 The Crk1-Mcs2 complex possesses CAK activity in vitro in that it phosphorylates human Cdk2 on Thr160 which results in its activation in the presence of cyclin A. |
| GO:0005634 nucleus | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: Genome-wide YFP-tagging localisation screen (Matsuyama et al. 2006) covering ~90% of the S. pombe proteome; the per-protein dataset assigns Mcs6 to the nucleus and cytoplasm, the basis of the UniProt subcellular-location entry. Reason: The nucleus is where Mcs6 functions: as the TFIIK kinase module of TFIIH it phosphorylates chromatin-bound Pol II and it is recovered at genes by ChIP (PMID:19328067). High-throughput but consistent with all functional data. Core location. Supporting Evidence: PMID:16823372 Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. PMID:19328067 Mcs6 activity was not required for its own recruitment (Figure S3) |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Deep PAINT node PTN000623091 (the CDK/CMGC clade, seeded by dozens of experimentally localised CDKs across eukaryotes) propagates nuclear localisation; Mcs6 has its own HDA nuclear localisation (PMID:16823372) and acts on nuclear Pol II. Reason: Broad but correct: the clade-level nuclear inference is confirmed for Mcs6 by direct localisation and by its function as the TFIIH kinase. Core location. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000623091 · PTN000623091 SUPPORTS TRANSFER Deep CDK-family node; nuclear localisation is a family-wide property and is directly confirmed for Mcs6 (PMID:16823372). Supporting Evidence: PMID:16823372 Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt Subcellular Location (SL-0191 Nucleus) mapped electronically; the UniProt entry cites the YFP screen (PMID:16823372). Reason: Grounded in experimental localisation and consistent with function as a TFIIH subunit. Core location. Supporting Evidence: PMID:16823372 Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. |
| GO:0005675 transcription factor TFIIH holo complex | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the CDK7 family signature (IPR037770) to the TFIIH holo complex. UniProt describes Mcs6 as one of the subunits of TFIIH, interacting with Mcs2 and Tfb3/Pmh1 (PMID:14534314, PMID:15555586). Reason: Correct: the Mcs6-Mcs2-Pmh1 kinase module associates with core TFIIH; Pmh1 fractionates in a ~700 kDa TFIIH-sized complex and binds core-TFIIH subunits (PMID:15829570). Core complex. Supporting Evidence: PMID:15829570 Pmh1 was shown to bind Mcs6, Mcs2, and components of core TFIIH when expressed in S. pombe PMID:15829570 The fission yeast Mcs6–Mcs2–Pmh1 complex, homologous to metazoan Cdk7–cyclin H-Mat1, has dual functions in cell division and transcription: as a partially redundant cyclin-dependent kinase (CDK)-activating kinase (CAK) that phosphorylates the major cell cycle CDK, Cdc2, on Thr-167; and as the RNA polymerase (Pol) II carboxyl-terminal domain (CTD) kinase associated with transcription factor (TF) IIH. |
| GO:0005675 transcription factor TFIIH holo complex | IPI PMID:8557037 Identification of a cdk-activating kinase in fission yeast. | ACCEPT | Summary: IPI with Mcs2 from Buck et al. (1995), who showed Crk1-Mcs2 association and CTD kinase activity; the 1995 abstract does not itself report TFIIH association (Pmh1/Tfb3 and the TFIIH link were established later, PMID:14534314 and PMID:15829570). PomBase records complex membership by IPI with the co-subunit. Reason: The complex assignment is correct for the gene: Mcs6 is the CDK7-like kinase of fission-yeast TFIIH (Pmh1 binds Mcs6, Mcs2 and core TFIIH; PMID:15829570). The cited paper directly supports only the Mcs6-Mcs2 (TFIIK core) interaction, so the TFIIH-holo evidence chain relies on later work, but there is no reason to overrule the curator. Supporting Evidence: PMID:8557037 We show that Crk1 associates with the Mcs2 mitotic catastrophe suppressor, a cyclin H-like molecule, and overexpression of crk1 rescues the cell-cycle arrest defect of a mcs2-75 cdc2-3w cdc25-22 triple mutant at high temperature. PMID:15829570 Pmh1 was shown to bind Mcs6, Mcs2, and components of core TFIIH when expressed in S. pombe |
| GO:0005737 cytoplasm | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | KEEP AS NON CORE | Summary: Genome-wide YFP screen (Matsuyama et al. 2006) recorded Mcs6-YFP in the cytoplasm as well as the nucleus; UniProt lists both compartments from this study. Reason: A genuine observation of a cytoplasmic pool (possibly the free Mcs6-Mcs2-Pmh1 trimer or overexpressed tagged protein), but all characterised functions (TFIIH CTD kinase, Cdc2 activation at chromatin/nucleus) are nuclear. Retained as a valid non-core localisation. Supporting Evidence: PMID:16823372 Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT node PTN000624096 (CDK7 subfamily) propagates cytoplasmic localisation from human CDK7 (P50613), Drosophila Cdk7, budding-yeast Kin28 and C9K505; metazoan CDK7-cyclin H-MAT1 exists as a free cytoplasmic/nuclear CAK pool in addition to TFIIH. Reason: The inference is consistent with the S. pombe YFP data (cytoplasm and nucleus, PMID:16823372), and a cytoplasmic CAK pool is plausible for the free trimer; but the demonstrated Mcs6 functions are executed on nuclear substrates, so this is a non-core location. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN000624096 · PTN000624096 SUPPORTS TRANSFER CDK7-subfamily node; cytoplasmic pools of CDK7-family kinases are documented for the metazoan CAK and Mcs6-YFP is seen in the cytoplasm (PMID:16823372), but the functional site is nuclear. Supporting Evidence: PMID:16823372 Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt Subcellular Location (SL-0086 Cytoplasm) mapped electronically from the YFP screen (PMID:16823372). Reason: Experimentally grounded but non-core; the functional compartment is the nucleus. Supporting Evidence: PMID:16823372 Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. |
| GO:0005829 cytosol | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | KEEP AS NON CORE | Summary: Genome-wide YFP screen (Matsuyama et al. 2006); PomBase maps the diffuse cytoplasmic signal to cytosol in addition to cytoplasm and nucleus. Reason: High-throughput localisation of a diffuse cytoplasmic pool; no Mcs6 function has been placed in the cytosol, and the protein acts in the nucleus on Pol II and on Cdc2. Kept as a non-core, screen-derived location. Supporting Evidence: PMID:16823372 Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. |
| GO:0006367 transcription initiation at RNA polymerase II promoter | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000624096 propagates transcription initiation at Pol II promoters from human CDK7, budding-yeast Kin28 and Mcs6 itself (PomBase:SPBC19F8.07 in WITH/FROM). All three are the TFIIK kinase subunit of TFIIH, a general initiation factor, and phosphorylate the CTD during initiation. Reason: Sound family-wide placement; Mcs6 is a TFIIH subunit (PMID:15829570), is recruited to promoters with the PIC (PMID:19328067) and phosphorylates CTD Ser5 at initiation. Core process. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000624096 · PTN000624096 SUPPORTS TRANSFER CDK7/Kin28/Mcs6 node; TFIIH kinase-module membership and initiation-phase CTD phosphorylation are conserved across the clade and directly shown for Mcs6. Supporting Evidence: PMID:19328067 PIC assembly occurred normally at the eng1+ promoter even when Mcs6as was inhibited PMID:15829570 The fission yeast Mcs6–Mcs2–Pmh1 complex, homologous to metazoan Cdk7–cyclin H-Mat1, has dual functions in cell division and transcription: as a partially redundant cyclin-dependent kinase (CDK)-activating kinase (CAK) that phosphorylates the major cell cycle CDK, Cdc2, on Thr-167; and as the RNA polymerase (Pol) II carboxyl-terminal domain (CTD) kinase associated with transcription factor (TF) IIH. |
| GO:0006367 transcription initiation at RNA polymerase II promoter | IDA PMID:15182371 Mediator is required for activated transcription in a Schizo... | ACCEPT | Summary: Tamayo et al. (2004) reconstituted S. pombe Pol II transcription in vitro with affinity-purified RNAPII and TFIIH plus recombinant TBP, TFIIB, TFIIE and TFIIF, supporting basal and (with Mediator) activated transcription. The abstract does not name Mcs6; the PomBase curator's IDA presumably reflects the tagged TFIIH subunit used for the affinity purification and the initiation activity of that preparation. Abstract-only in cache. Reason: Mcs6 is a bona fide subunit of the TFIIH preparation required for initiation in this system, and its role in initiation is independently established (PMID:15829570, PMID:19328067). The evidence attaches to the complex rather than to the kinase activity per se, but the process is correct for the gene; deferring to the curator on the full-text purification details. Supporting Evidence: PMID:15182371 In this report, we describe a reconstituted in vitro transcription system that consists of the following preparations of factors from the yeast Schizosaccharomyces pombe: affinity-purified RNAPII, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF. PMID:15182371 We show that this system can support basal transcription from the adenovirus major late promoter when purified RNAPII is used and activated transcription when the RNAPII holoenzyme (RNAPII plus the Mediator proteins) is included in the reaction. PMID:19328067 PIC assembly occurred normally at the eng1+ promoter even when Mcs6as was inhibited |
| GO:0008353 RNA polymerase II CTD heptapeptide repeat kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000624096 propagates Pol II CTD heptapeptide-repeat kinase activity from human CDK7, Drosophila Cdk7, budding-yeast Kin28 and Mcs6 itself (PomBase:SPBC19F8.07 in WITH/FROM). The CDK7/Kin28/Mcs6 clade is the TFIIH-associated CTD kinase in every organism examined. Reason: The defining conserved activity of the family, with direct grounding on the target (immunoprecipitated Mcs6-Mcs2 phosphorylates the CTD, PMID:8557036, PMID:8557037; Ser5-P loss on Mcs6 inhibition, PMID:19328067). Core function. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000624096 · PTN000624096 SUPPORTS TRANSFER CDK7-subfamily node seeded by CDK7, Kin28 and Mcs6 CTD-kinase evidence; the target has its own IDA support. Supporting Evidence: PMID:8557036 In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. PMID:19328067 Inhibition of Mcs6 caused dose- and time-dependent decreases in Ser5 phosphorylation in vivo, measured with either Ser5-P or H14. |
| GO:0008353 RNA polymerase II CTD heptapeptide repeat kinase activity | IDA PMID:15829570 Impairment of the TFIIH-associated CDK-activating kinase sel... | ACCEPT | Summary: Lee et al. (2005) measured CTD kinase activity of HA-Mcs6 immunoprecipitates from reconstituted insect-cell complexes and from yeast, showed it depends on Csk1 (Ser165) or Pmh1, and showed that mcs6-ts1 cells lose CTD Ser5 phosphorylation at restrictive temperature. Reason: Direct in vitro and in vivo evidence for Mcs6 CTD kinase activity. Core function. Supporting Evidence: PMID:15829570 Activity of the wild-type and T160A proteins increased with Csk1 coexpression, consistent with activating phosphorylation at Ser-165 PMID:15829570 Loss of Ser-5 phosphorylation occurred to similar degrees but with different kinetics in mcs6 ts1 and pmh1-26 cells |
| GO:0008353 RNA polymerase II CTD heptapeptide repeat kinase activity | IDA PMID:23122962 Cdk11-cyclinL controls the assembly of the RNA polymerase II... | ACCEPT | Summary: Drogat et al. (2012) characterised the Cdk11-Lcp1 complex as a Mediator kinase; the cached record is abstract-only and the abstract does not mention Mcs6. The PomBase curator read the full text, in which Mcs6 was evidently assayed as a CTD kinase (this Hermand-lab paper uses the mcs6-as strain and Mcs6 kinase preparations from their earlier work). Reason: The activity is unquestionably correct for Mcs6 (PMID:8557036, PMID:8557037, PMID:19328067); the abstract-only cache cannot show the specific experiment, so the curator's IDA is accepted rather than second-guessed. Core function. Supporting Evidence: PMID:23122962 Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. PMID:19328067 In vitro, Mcs6 generated Rpb1 CTD isoforms recognized by a monoclonal antibody specific for modification at Ser5 (H14) |
| GO:0008353 RNA polymerase II CTD heptapeptide repeat kinase activity | IDA PMID:8557036 Schizosaccharomyces pombe Mop1-Mcs2 is related to mammalian ... | ACCEPT | Summary: Damagnez et al. (1995): immunoprecipitated Mop1-Mcs2 acts as an RNA polymerase II CTD kinase (and as a CAK) in vitro. Reason: Founding direct evidence of CTD kinase activity. Core function. Supporting Evidence: PMID:8557036 In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. |
| GO:0008353 RNA polymerase II CTD heptapeptide repeat kinase activity | IDA PMID:8557037 Identification of a cdk-activating kinase in fission yeast. | ACCEPT | Summary: Buck et al. (1995): the Crk1-Mcs2 complex effectively phosphorylates a peptide corresponding to the Pol II CTD. Reason: Direct in vitro evidence of CTD kinase activity. Core function. Supporting Evidence: PMID:8557037 In addition Crk1-Mcs2 effectively phosphorylates a peptide corresponding to the C-terminal repeat domain (CTD) of RNA polymerase II. |
| GO:0008353 RNA polymerase II CTD heptapeptide repeat kinase activity | IDA PMID:9857180 Fission yeast Csk1 is a CAK-activating kinase (CAKAK). | ACCEPT | Summary: Hermand et al. (1998) identified Csk1 as the in vivo activating kinase of the Mcs6-Mcs2 CAK (phosphorylating Mcs6 Ser165); Mcs2-associated CTD kinase activity was used as the readout and fell ~3-fold in csk1-delta cells (per the deep-research summary of the full text). Abstract-only in cache. Reason: The CTD kinase activity of Mcs6-Mcs2 is the assay through which Csk1's CAKAK role was defined; consistent with all other rows. Core function. Supporting Evidence: PMID:9857180 Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK). PMID:9857180 In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). |
| GO:0008353 RNA polymerase II CTD heptapeptide repeat kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation (ARBA, InterPro IPR037770 CDK7 family, Rhea 10216 and EC 2.7.11.23) to Pol II CTD kinase activity; UniProt gives EC 2.7.11.23 for Mcs6 from PMID:8557036, PMID:8557037 and PMID:9857180. Reason: Electronic annotation fully concordant with the experimental evidence. Core function. Supporting Evidence: PMID:8557036 In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. |
| GO:0016251 RNA polymerase II general transcription initiation factor activity | IDA PMID:15182371 Mediator is required for activated transcription in a Schizo... | ACCEPT | Summary: Tamayo et al. (2004) reconstituted basal and activated Pol II transcription with affinity-purified S. pombe TFIIH; PomBase annotated Mcs6 (a TFIIH subunit) with general transcription initiation factor activity from this system. Abstract-only; Mcs6 is not named in the abstract. Reason: General transcription initiation factor activity is a property of the TFIIH holo-complex, of which Mcs6 is the kinase subunit; the annotation is correct at the complex level and PomBase's use of it for TFIIH subunits is a curation convention, not an error. In the core-function synthesis this is represented as an activity Mcs6 contributes to as part of TFIIH rather than one it has alone (the CTD kinase activity is its own). Supporting Evidence: PMID:15182371 In this report, we describe a reconstituted in vitro transcription system that consists of the following preparations of factors from the yeast Schizosaccharomyces pombe: affinity-purified RNAPII, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF. PMID:15182371 We show that this system can support basal transcription from the adenovirus major late promoter when purified RNAPII is used and activated transcription when the RNAPII holoenzyme (RNAPII plus the Mediator proteins) is included in the reaction. |
| GO:0032968 positive regulation of transcription elongation by RNA polymerase II | EXP PMID:19328067 TFIIH and P-TEFb coordinate transcription with capping enzym... | ACCEPT | Summary: Viladevall et al. (2009): Mcs6 activity is necessary and sufficient to recruit the Cdk9/Pcm1 (P-TEFb/cap methyltransferase) complex to the elongation complex at eng1+, and prior phosphorylation of the CTD by Mcs6 stimulates Cdk9 activity ~8-10-fold in vitro; inhibition of either kinase depletes Pol II downstream and increases Spt5:Pol II ratios. The authors propose that TFIIH primes the CTD and promotes P-TEFb/Pcm1 recruitment, coupling elongation and capping. Reason: Mcs6 does the work here: its Ser5/Ser7 priming phosphorylation is the molecular event that licenses Cdk9 recruitment and activity, and thus early elongation, at target genes. Consistent with the GO-CAM model gocams/6796b94c00000168 which links the Mcs6 S5-kinase activity to positive regulation of elongation. Core process. Supporting Evidence: PMID:19328067 In contrast, Mcs6 activity is sufficient-and necessary-to recruit the Cdk9/Pcm1 (mRNA cap methyltransferase) complex. PMID:19328067 In vitro, phosphorylation of the CTD by Mcs6 stimulates subsequent phosphorylation by Cdk9. PMID:19328067 Prior phosphorylation by Mcs6 stimulated Cdk9 activity ~8–10-fold PMID:19328067 The results suggest that Mcs6 activity is specifically required to bring Cdk9/Pcm1 to the elongation complex. PMID:19328067 appeared to place Mcs6 function upstream of Cdk9, and suggest sequential action by the two CDKs in promoting transcript elongation |
| GO:0032968 positive regulation of transcription elongation by RNA polymerase II | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learned rule; matches the experimental EXP annotation from PMID:19328067. Reason: Electronic annotation concordant with direct evidence (PMID:19328067). Core process. Supporting Evidence: PMID:19328067 In vitro, phosphorylation of the CTD by Mcs6 stimulates subsequent phosphorylation by Cdk9. |
| GO:0051726 regulation of cell cycle | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000624096 propagates regulation of cell cycle from human CDK7 and C9K505; CDK7-family kinases in metazoa and fission yeast are CDK-activating kinases. Mcs6-Mcs2 phosphorylates Cdc2 Thr167, and Cdc2 activation and cell division fail when both Mcs6 and the second CAK Csk1 are impaired (PMID:10226032, PMID:15829570). Reason: Correct and phylogenetically appropriate at a node that spans metazoan CDK7 and Mcs6 (the Kin28 branch, which lacks CAK activity, is not among the sources). The general term is acceptable for the IBA; the specific mechanism (positive regulation of CDK activity via T-loop phosphorylation, feeding the G2/M transition) is captured in core_functions rather than by modifying the propagated row. Core process. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000624096 · PTN000624096 SUPPORTS TRANSFER CDK7-subfamily node; the CAK role that underlies cell-cycle regulation is shared by CDK7 and Mcs6 and directly shown for the target (PMID:10226032). Supporting Evidence: PMID:10226032 When a double mutant carrying a csk1 deletion and a temperature-sensitive mcs6 allele was incubated at the restrictive temperature, Cdc2 was not activated and the cells underwent a cell division arrest prior to mitosis. PMID:10226032 Thus, fission yeast contains two partially redundant CAKs: the Mcs6-Mcs2 complex and Csk1. PMID:15829570 The fission yeast Mcs6–Mcs2–Pmh1 complex, homologous to metazoan Cdk7–cyclin H-Mat1, has dual functions in cell division and transcription: as a partially redundant cyclin-dependent kinase (CDK)-activating kinase (CAK) that phosphorylates the major cell cycle CDK, Cdc2, on Thr-167; and as the RNA polymerase (Pol) II carboxyl-terminal domain (CTD) kinase associated with transcription factor (TF) IIH. |
| GO:0060261 positive regulation of transcription initiation by RNA polymerase II | IMP PMID:19328067 TFIIH and P-TEFb coordinate transcription with capping enzym... | ACCEPT | Summary: Viladevall et al. (2009): chemical-genetic inhibition of mcs6-as reduces CTD Ser5 phosphorylation and, at the common target gene eng1+, both Mcs6 and Cdk9 must be active for normal Pol II occupancy. Notably, PIC assembly and TFIIH (Pmh1) recruitment at the eng1+ promoter were unaffected by Mcs6 inhibition; the loss of Pol II is downstream, i.e. the kinase acts after PIC formation. Reason: Mcs6 is a TFIIH subunit and its CTD phosphorylation is an initiation-phase event whose loss reduces productive Pol II output at target genes; the curator's IMP is a reasonable reading. The full text shows that the effect is on post-PIC steps (promoter clearance/early elongation, already captured by GO:0001111 and GO:0032968) rather than on PIC assembly, so this term is somewhat coarse, but it is not wrong and is retained. Supporting Evidence: PMID:19328067 At a common target gene, both CDKs must be active for normal Pol II occupancy, and Spt5-a CDK substrate and regulator of elongation-accumulates disproportionately to Pol II when either kinase is inhibited. PMID:19328067 PIC assembly occurred normally at the eng1+ promoter even when Mcs6as was inhibited PMID:19328067 Inhibition of Mcs6 caused dose- and time-dependent decreases in Ser5 phosphorylation in vivo, measured with either Ser5-P or H14. |
| GO:0070985 transcription factor TFIIK complex | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000624096 propagates TFIIK complex membership from human CDK7, budding-yeast Kin28 and Mcs6 itself. GO defines TFIIK as the CDK7/cyclin H/MAT1 (Kin28/Ccl1/Tfb3) kinase module of holo-TFIIH; the S. pombe equivalent is Mcs6-Mcs2-Pmh1. Reason: Direct evidence for the trimeric Mcs6-Mcs2-Pmh1 complex and its association with core TFIIH exists for the target (PMID:15829570). Core complex. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000624096 · PTN000624096 SUPPORTS TRANSFER CDK7-subfamily node; TFIIK membership is the conserved complex context of the clade and is directly shown for Mcs6. Supporting Evidence: PMID:15829570 We next reconstituted the Mcs6–Mcs2–Pmh1 complex in baculovirus-infected insect cells PMID:15829570 Pmh1 was shown to bind Mcs6, Mcs2, and components of core TFIIH when expressed in S. pombe |
| GO:0070985 transcription factor TFIIK complex | IDA PMID:15829570 Impairment of the TFIIH-associated CDK-activating kinase sel... | ACCEPT | Summary: Lee et al. (2005) reconstituted the Mcs6-Mcs2-Pmh1 trimer in insect cells (the ~200 kDa complex seen in yeast extracts) and showed Pmh1 binds Mcs6, Mcs2 and core TFIIH; Pmh1 is the Mat1/Tfb3 ortholog that completes TFIIK. Reason: Direct demonstration of the fission-yeast TFIIK (CAK) trimer. Core complex. Supporting Evidence: PMID:15829570 We next reconstituted the Mcs6–Mcs2–Pmh1 complex in baculovirus-infected insect cells PMID:15829570 Pmh1 was shown to bind Mcs6, Mcs2, and components of core TFIIH when expressed in S. pombe |
| GO:0070985 transcription factor TFIIK complex | IDA PMID:9857180 Fission yeast Csk1 is a CAK-activating kinase (CAKAK). | ACCEPT | Summary: Hermand et al. (1998) worked with the Mcs6-Mcs2 CAK complex (the CDK7-cyclin H equivalent) as the target of Csk1; the third subunit Pmh1 was identified later (PMID:15555586). Abstract-only in cache. Reason: Mcs6-Mcs2 is the kinase-cyclin core of TFIIK; the complex assignment is correct for the gene, and PMID:15829570 supplies the full trimer. Deferring to the curator on full-text details. Core complex. Supporting Evidence: PMID:9857180 In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). PMID:15829570 We next reconstituted the Mcs6–Mcs2–Pmh1 complex in baculovirus-infected insect cells |
| GO:0070985 transcription factor TFIIK complex | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation (ARBA and InterPro IPR037770 CDK7 family) to the TFIIK complex. Reason: Concordant with direct evidence (PMID:15829570). Core complex. Supporting Evidence: PMID:15829570 We next reconstituted the Mcs6–Mcs2–Pmh1 complex in baculovirus-infected insect cells |
| GO:0097472 cyclin-dependent protein kinase activity | IDA PMID:9857180 Fission yeast Csk1 is a CAK-activating kinase (CAKAK). | MODIFY | Summary: Hermand et al. (1998) showed that Csk1 phosphorylates Mcs6 on Ser165 and thereby activates the Mcs6-Mcs2 CAK, measured as Mcs2-associated kinase activity; Mcs6 is thus a cyclin (Mcs2)-dependent kinase whose activity is tuned by T-loop phosphorylation. Abstract-only in cache. Reason: The term is correct for Mcs6 (a genuine CDK, unlike monomeric Cak1) but is the residue-unspecified parent of GO:0004693 cyclin-dependent protein serine/threonine kinase activity, which is what Mcs6 demonstrably is (it phosphorylates Cdc2 Thr167 and CTD Ser5/Ser7) and which the same paper is already cited for on the CTD-kinase row. Recording the Ser/Thr-specific child is more informative and consistent with the other five GO:0004693 rows. Proposed replacements: cyclin-dependent protein serine/threonine kinase activity Supporting Evidence: PMID:9857180 Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK). PMID:9857180 In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). |
| GO:0140836 RNA polymerase II CTD heptapeptide repeat S5 kinase activity | EXP PMID:19328067 TFIIH and P-TEFb coordinate transcription with capping enzym... | ACCEPT | Summary: Viladevall et al. (2009): in vitro, Mcs6 generates CTD isoforms recognised by the Ser5-P-specific H14 antibody but not by the Ser2/Ser5 doubly-phosphorylated-repeat antibody H5; in vivo, inhibition of analog-sensitive Mcs6 causes dose- and time-dependent loss of Ser5 phosphorylation. Reason: Position-specific direct evidence (in vitro and in vivo) that Mcs6 is the TFIIH-associated CTD Ser5 kinase. This is the most precise MF for the transcriptional output and the activity used in both cached GO-CAM models of Mcs6. Core function. Supporting Evidence: PMID:19328067 In vitro, Mcs6 generated Rpb1 CTD isoforms recognized by a monoclonal antibody specific for modification at Ser5 (H14) PMID:19328067 Inhibition of Mcs6 caused dose- and time-dependent decreases in Ser5 phosphorylation in vivo, measured with either Ser5-P or H14. |
| GO:0140836 RNA polymerase II CTD heptapeptide repeat S5 kinase activity | IDA PMID:20605454 A gene-specific requirement of RNA polymerase II CTD phospho... | ACCEPT | Summary: Coudreuse et al. (2010) compared Lsk1-dependent Ser2 and Mcs6-dependent Ser5 phosphorylation of the CTD during vegetative growth and sexual differentiation; the cached abstract foregrounds Lsk1/S2P and ste11, and the Mcs6 Ser5 data were read from the full text by the PomBase curator. Independently, PMID:19328067 and PMID:22508988 show Mcs6-dependent Ser5-P in vitro and in vivo. Reason: The activity is firmly established for Mcs6 by full-text-cached studies (PMID:19328067, PMID:22508988); the abstract-only cache of this paper cannot display the specific experiment but there is no reason to doubt the curator. Core function. Supporting Evidence: PMID:20605454 We show that phosphorylation of serine 2 (S2P) in the C-terminal domain of the largest subunit of the RNA polymerase II (PolII) enzyme by the Lsk1 cyclin-dependent kinase has only a minor impact on global gene expression during vegetative growth but is critical for the induction of ste11 transcription during sexual differentiation. PMID:19328067 Inhibition of Mcs6 caused dose- and time-dependent decreases in Ser5 phosphorylation in vivo, measured with either Ser5-P or H14. PMID:22508988 Both Ser5-P and Ser7-P depended on Mcs6 activity in vivo |
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Download this section (compressed HTML)Q: Which Mcs6-dependent transcripts among the ~5% acutely repressed on Mcs6 inhibition require Ser5 phosphorylation per se versus Mcs6-dependent recruitment of Cdk9/Pcm1 and 5' capping, and does Ser7 phosphorylation by Mcs6 have a separable role?
Q: Do Mcs6 and Csk1 activate distinct Cdc2-cyclin subpopulations (Cdc2-Cig2 at G1/S versus Cdc2-Cdc13 at G2/M) in vivo, or do they act redundantly on all Cdc2 complexes?
Q: PomBase annotates csk1 to G2/M transition of mitotic cell cycle (GO:0000086) by EXP from the csk1-delta mcs6-ts double-mutant study (PMID:10226032), and SGD annotates Cak1 to the same term, but mcs6 carries only regulation of cell cycle (GO:0051726, IBA): was omitting GO:0000086 for mcs6 a deliberate decision (single mcs6 mutants activate Cdc2 normally because Csk1 compensates), or should the CAK contribution of Mcs6 to mitotic entry be annotated as it is for csk1?
Q: Is the Cdc2-directed CAK reaction carried out by the free Mcs6-Mcs2-Pmh1 trimer or by TFIIH-incorporated Mcs6, and where in the cell (the cytoplasmic pool seen by YFP tagging versus the nucleus) does it take place?
Experiment: Combine acute mcs6-as inhibition with nascent-RNA sequencing, Pcm1 and Cdk9 ChIP-seq and cap-methylation profiling; test whether tethering Pcm1 or Cdk9 to the eng1+/ace2+ promoters restores expression when Mcs6 is inhibited.
Hypothesis: Mcs6-dependent gene expression at the Sep1/Ace2 cell-separation cluster is mediated primarily through Cdk9/Pcm1 recruitment and cap methylation rather than through Ser5 phosphorylation alone.
Type: chemical genetics + nascent transcriptomics + ChIP-seq
Experiment: In mcs6-as and csk1-delta backgrounds, immunoprecipitate Cdc2-Cig2 and Cdc2-Cdc13 separately across a synchronised cell cycle and quantify Thr167 phosphorylation and kinase activity; complement with phosphoproteomics of T-loop sites of all fission-yeast CDKs after acute Mcs6 inhibition.
Hypothesis: Mcs6 and Csk1 preferentially phosphorylate different Cdc2-cyclin complexes in vivo.
Type: chemical genetics + phosphoproteomics
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