Ppk34 (also called Ckk2 in the calcium-signaling literature) is a calcium/calmodulin-dependent protein kinase kinase (CaMKK) in Schizosaccharomyces pombe. It is one of two CaMKK homologs in S. pombe (the other being Ssp1) and is most similar to mammalian CaMKK2 (34% identity). Ckk2/ppk34 has two experimentally demonstrated signaling functions: (1) in calcium signaling, it phosphorylates and activates Cmk1, which in turn phosphorylates and inactivates the calcineurin-dependent transcription factor Prz1, forming a negative feedback loop; (2) in nitrogen stress, it is specifically required for the increase in AMPK (Ssp2) T189 phosphorylation and activation that leads to TORC1 inhibition and reduced cell size at division. Whether Ppk34 acts directly on Ssp2 or through an AMPK phosphatase remains unresolved. The protein localizes to both cytosol and nucleus.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0007165 signal transduction | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: This IBA annotation from phylogenetic inference is too broad. Ppk34/ckk2 is indeed involved in signal transduction, but this generic parent term does not add information beyond what is captured by the more specific annotations for calcium signaling and TORC1 regulation. The PANTHER family assignment (PTHR43895, CaMKK-related) is correct. Reason: Overly broad term; more specific BP annotations exist for this gene. Supporting Evidence: file:SCHPO/ppk34/ppk34-deep-research-bioreason-sft.md ...A soluble serine/threonine kinase in fission yeast that dampens calcium-triggered signaling and restrains growth-promoting pathways... file:SCHPO/ppk34/ppk34-deep-research-falcon.md identifies it as a **CaMKK-family, serine/threonine protein kinase**; in a Ca2+-signaling study it was **renamed Ckk2** (for CaM kinase kinase 2) |
| GO:0004674 protein serine/threonine kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Correct molecular function. Ppk34/ckk2 is a serine/threonine kinase with demonstrated kinase activity toward Cmk1. It is also required for stress-induced Ssp2 T189 phosphorylation, although a direct Ppk34-Ssp2 kinase reaction has not been established. The IBA evidence from PANTHER (PTHR43895, CaMKK-related family) is appropriate and consistent with PMID:25081204 and PMID:25639242. Supporting Evidence: file:SCHPO/ppk34/ppk34-deep-research-falcon.md Ppk34/Ckk2 is annotated as a **serine/threonine protein kinase** (EC 2.7.11.1) and is experimentally supported to function as an **upstream kinase regulator** (CaMKK-like) |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: This is a parent term of GO:0004674 (protein Ser/Thr kinase activity). Since the more specific term is already annotated with experimental evidence, this generic IEA annotation is redundant. Reason: Redundant with GO:0004674 annotation; less specific. |
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC-based annotation from EC:2.7.11.1. Correct and consistent with the IBA and IGI annotations for the same term and with demonstrated kinase activity toward Cmk1. Ppk34-dependent Ssp2 phosphorylation supports the pathway role but does not establish Ssp2 as a direct substrate. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: Appropriate. Ppk34/ckk2 has a canonical protein kinase domain (residues 40-331) with conserved ATP-binding residues (46-54, 69). ATP binding is intrinsic to its kinase activity. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: This IEA annotation is consistent with the experimental HDA evidence from PMID:16823372, which showed nuclear localization by large-scale GFP tagging. Reason: Retain this exact core-location duplicate because it independently corroborates the experimental nucleus annotation rather than introducing a broader parent term. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Consistent with the HDA evidence showing cytosol localization. Note the HDA annotation uses cytosol (GO:0005829), which is more specific than cytoplasm. This IEA annotation is less precise. Reason: Cytoplasm is a true but less-specific parent of the separately annotated HDA cytosol term, so retain it as redundant non-core localization evidence. |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | KEEP AS NON CORE | Summary: RHEA-based annotation to a serine-specific child of GO:0004674. The experimentally discussed Cmk1 T192 and Ssp2 T189 sites are threonines, so the literature reviewed here does not independently demonstrate a serine target. Retain this electronic child term as non-core rather than treating it as stronger evidence than the broader serine/threonine kinase activity. Reason: The serine-specific child is more substrate-specific than the reviewed experimental evidence establishes, so it should not be elevated to a core activity. |
| GO:0004674 protein serine/threonine kinase activity | IGI PMID:25639242 Nitrogen regulates AMPK to control TORC1 signaling. | ACCEPT | Summary: This IGI annotation from Davie et al. 2015 is based on genetic interaction data showing that ppk34 is required for Ssp2 (AMPK) T189 phosphorylation during nitrogen stress. The with/from column cites SPCC74.03c (ssp2), and the Canto extension asserts Ssp2 as the kinase input. The genetic result supports Ppk34's kinase-pathway role but does not demonstrate that Ppk34 directly phosphorylates Ssp2; the paper explicitly considers indirect control through an AMPK phosphatase. The kinase molecular function is independently supported by the conserved catalytic domain, phylogeny, and Ckk2-dependent Cmk1 phosphorylation. Supporting Evidence: PMID:25639242 ...CaMKK(Ppk34) is specifically required to stimulate AMPKΞ±(Ssp2) activation in response to nitrogen stress... file:SCHPO/ppk34/ppk34-deep-research-falcon.md **Ppk34 is specifically required for this stress-induced increase**, while basal Thr189 phosphorylation is still present without Ppk34 |
| GO:0071277 cellular response to calcium ion | IMP PMID:25081204 Negative feedback regulation of calcineurin-dependent Prz1 t... | ACCEPT | Summary: Supported by Cisneros-Barroso et al. 2014. Ckk2/ppk34 is activated by calcium and phosphorylates Cmk1 in response to Ca2+ stress. Deletion of ckk2 shows similar phenotypes to cmk1 deletion during calcium response, including failure of Cdc25 accumulation. This is a core function of the gene. Supporting Evidence: PMID:25081204 ...we have identified a second CaMKK in fission yeast, the Ckk2 kinase, which is involved in the activation of Cmk1 in response to Ca2+... file:SCHPO/ppk34/ppk34-deep-research-falcon.md Ckk2 (Ppk34/SPCC1919.01) as the kinase responsible for **Cmk1 phosphorylation in response to Ca2+ stress** |
| GO:1904262 negative regulation of TORC1 signaling | IMP PMID:25639242 Nitrogen regulates AMPK to control TORC1 signaling. | ACCEPT | Summary: Well supported by Davie et al. 2015. Ppk34/ckk2 activates AMPK (Ssp2) during nitrogen stress, which then inhibits TORC1 via the Tsc1/2-Rhb1 axis. Note the mechanism is indirect -- ppk34 does not directly phosphorylate TORC1 components but rather acts through AMPK. The IMP evidence is appropriate as it is based on mutant phenotypes (ppk34 deletion fails to reduce TORC1 activity upon nitrogen stress). Supporting Evidence: PMID:25639242 ...CaMKKPpk34 is required to induce AMPKΞ±Ssp2 activation following nitrogen stress...overexpression of CaMKKppk34+ can promote AMPKΞ±Ssp2 activation through T189 phosphorylation... file:SCHPO/ppk34/ppk34-deep-research-falcon.md **ppk34Ξ fails to accelerate mitosis and reduce cell size at division** after nitrogen stress; importantly, **rapamycin rescues** this failure, supporting a model in which Ppk34 acts **upstream of TORC1 inhibition** during nitrogen stress |
| GO:0106057 negative regulation of calcineurin-mediated signaling | IGI PMID:25081204 Negative feedback regulation of calcineurin-dependent Prz1 t... | ACCEPT | Summary: Supported by Cisneros-Barroso et al. 2014. The Ckk2/Cmk1 cascade counteracts calcineurin (Ppb1) signaling by phosphorylating Prz1, the calcineurin-responsive transcription factor. The with/from column cites SPACUNK12.02c (cmk1). Note that ckk2 does not directly phosphorylate Prz1 or calcineurin; rather, ckk2 activates Cmk1, which then phosphorylates Prz1. The IGI evidence is appropriate because the genetic interaction demonstrates the pathway function. Supporting Evidence: PMID:25081204 ...Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity... file:SCHPO/ppk34/ppk34-deep-research-falcon.md **Ckk2 activates Cmk1**, and **Cmk1 phosphorylates/inactivates Prz1** (promoting nuclear export), forming a negative feedback loop in Ca2+ signaling |
| GO:0005634 nucleus | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: From the large-scale GFP localization study by Matsuyama et al. 2006. Nuclear localization is consistent with the role of ckk2 in signaling cascades that target nuclear transcription factors (Prz1). Supporting Evidence: PMID:16823372 ...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 ... | ACCEPT | Summary: From the same large-scale GFP localization study. Cytosol localization is consistent with the role of ckk2 in cytosolic signaling cascades (AMPK activation, calcineurin pathway regulation). Falcon notes no direct Ppk34-GFP imaging was captured in its corpus, so the cytosolic role is a pathway-level inference there; the GO HDA annotation rests on the Matsuyama et al. genome-wide localization dataset. Supporting Evidence: PMID:16823372 ...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... file:SCHPO/ppk34/ppk34-deep-research-falcon.md treats Ckk2/Cmk1 and calcineurin as **cytosolic cascades** that control the nuclearβcytoplasmic shuttling of Prz1 |
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Download this section (compressed HTML)Q: Does Ckk2/ppk34 directly phosphorylate Ssp2 at T189, or does it act indirectly through an intermediate? The genetic evidence is clear but direct biochemical demonstration of kinase-substrate relationship is needed.
Q: What is the signal that activates Ckk2 specifically during nitrogen stress? Is it Ca2+/calmodulin-dependent as in the calcium stress context, or does nitrogen stress activate Ckk2 through a different mechanism?
Q: Are there additional substrates of Ckk2 beyond Cmk1, and is Ssp2 an as-yet-unconfirmed direct target? The gene appears at the nexus of calcium and nutrient signaling, suggesting possible additional targets.
Experiment: Perform in vitro kinase assay with purified Ckk2 and Ssp2 to test direct phosphorylation at T189, using Ssp2-T189A as a negative control.
Hypothesis: Ckk2 directly phosphorylates Ssp2 at T189 in vitro.
Type: in vitro kinase assay
Experiment: Test whether calmodulin inhibitors or Ca2+ chelators block nitrogen-stress-induced Ssp2 phosphorylation in vivo, comparing with Ca2+-stress-induced Cmk1 activation.
Hypothesis: Ckk2 activation during nitrogen stress is Ca2+/calmodulin-dependent.
Type: pharmacological inhibition / epistasis analysis
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