Ppk34 (now called ckk2 on PomBase) 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 to stimulate AMPK (Ssp2) activation via T189 phosphorylation, leading to TORC1 inhibition and reduced cell size at division. 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 and Ssp2. The IBA evidence from PANTHER (PTHR43895, CaMKK-related family) is appropriate and consistent with experimental data from 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 and Ssp2.
|
|
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.
|
|
GO:0005737
cytoplasm
|
IEA
GO_REF:0000044 |
MODIFY |
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: The HDA data from PMID:16823372 supports cytosol (GO:0005829) specifically.
Proposed replacements:
cytosol
|
|
GO:0106310
protein serine kinase activity
|
IEA
GO_REF:0000116 |
KEEP AS NON CORE |
Summary: RHEA-based annotation. Ppk34/ckk2 phosphorylates serine and threonine residues, so protein serine kinase activity is a subset of its activity. Acceptable but less informative than GO:0004674 which covers both Ser and Thr. Not wrong, but redundant.
Reason: Partially redundant with GO:0004674; captures only serine-specific 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 (cmk1). This is strong experimental evidence that ppk34 has protein kinase activity, demonstrated through its functional requirement for phosphorylation of known substrates.
Supporting Evidence:
PMID:25639242
...CaMKK(Ppk34) is specifically required to stimulate AMPKalpha(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 AMPKalphaSsp2 activation following nitrogen stress...overexpression of CaMKKppk34+ can promote AMPKalphaSsp2 activation through T189 phosphorylation...
file:SCHPO/ppk34/ppk34-deep-research-falcon.md
**ppk34Δ 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. 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 nuclear–cytoplasmic shuttling of Prz1
|
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 Ssp2? 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
I begin with the InterPro architecture. The core of the protein is a canonical eukaryotic protein kinase module: IPR000719 (Protein kinase domain, residues 40–331) sits within the broader IPR011009 (Protein kinase-like domain superfamily, residues 36–346), and an additional IPR000719 signature spans residues 111–331, consistent with the C-terminal lobe and activation segment of a bilobal kinase. The catalytic center is pinpointed by IPR008271 (Serine/threonine-protein kinase, active site, residues 196–208), which marks the catalytic loop/HRD region that coordinates Mg2+-ATP and positions the substrate hydroxyl for transfer. This ordered layout—N-lobe ATP-binding, catalytic loop, and activation segment within a protein kinase-like superfamily—causes ATP-dependent transfer of phosphate to serine/threonine residues on protein substrates, establishing GO:0004674 protein serine/threonine kinase activity as the molecular function.
A serine/threonine kinase with this architecture typically controls signaling by phosphorylating effectors and upstream regulators. The stated role in calcium signaling aligns with a kinase that tunes the calcineurin pathway: phosphorylation of calcineurin targets or its regulators would dampen dephosphorylation events required for activation of calcineurin-responsive transcription factors. This mechanistic leverage explains negative regulation of calcineurin-mediated signaling (GO:0106057). Because calcium influx is the initiating signal that activates calcineurin, a kinase that restrains calcineurin will also shape the cellular response to calcium ion (GO:0071277), acting as a feedback brake that prevents excessive or prolonged calcium-driven outputs.
The same catalytic module can interface with nutrient and growth control. Kinase-mediated phosphorylation of TORC1 pathway components or their inhibitors provides a direct route to negative regulation of TORC1 signaling (GO:1904262). By attenuating TORC1, the kinase promotes entry into quiescence and supports chronological aging, consistent with a role in growth control and lifespan regulation. This causal chain—serine/threonine phosphorylation of pathway nodes leading to TORC1 downshift—connects the molecular activity to organismal aging phenotypes.
Cellular location follows from the need to access both cytosolic signaling hubs and nuclear transcriptional targets. The absence of transmembrane features and the soluble kinase fold support a non-membranous distribution. Calcineurin signaling and TORC1 regulation occur in the cytosol, while modulation of calcineurin-responsive transcription factors requires nuclear access. Thus, the protein partitions between the cytosol (GO:0005829) and nucleus (GO:0005634), enabling rapid cytosolic control of signaling and nuclear tuning of gene expression.
Mechanistically, I hypothesize that the kinase phosphorylates calcineurin’s regulatory subunit or its substrates to reduce calcineurin’s access to dephosphorylate transcription factors, thereby limiting calcium-driven transcriptional programs. In parallel, phosphorylation of TORC1 pathway inhibitors (e.g., nutrient-sensing adaptors) or activation of counteracting phosphatases would depress TORC1 activity, promoting quiescence and chronological aging. Likely interaction partners therefore include calcineurin components (the catalytic and regulatory subunits), calcineurin-responsive transcription factors, TORC1 core subunits and nutrient-sensing adaptors, 14-3-3 proteins that read phospho-motifs, and PP2A-family phosphatases that reset pathway phosphorylation states. Together, these interactions would create a feedback-controlled circuit that couples calcium influx to growth-state transitions and aging trajectories.
## Functional Summary
A soluble serine/threonine kinase in fission yeast that dampens calcium-triggered signaling and restrains growth-promoting pathways to promote entry into quiescence and chronological aging. By phosphorylating components of the calcineurin axis, it limits activation of calcium-responsive transcriptional programs, and by targeting regulators of the TORC1 pathway, it reduces anabolic signaling. Its distribution between cytosol and nucleus enables rapid control of cytosolic signaling nodes and nuclear transcription factors, integrating calcium cues with nutrient and growth-state decisions to modulate lifespan traits.
## UniProt Summary
Involved in chronological aging and growth control. Negative regulator of calcineurin signaling.
## InterPro Domains
- IPR011009: Protein kinase-like domain superfamily (homologous_superfamily) [36-346]
- IPR000719: Protein kinase domain (domain) [40-331]
- IPR000719: Protein kinase domain (domain) [111-331]
- IPR008271: Serine/threonine-protein kinase, active site (active_site) [196-208]
## GO Term Predictions
### Molecular Function
### Biological Process
### Cellular Component
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The literature retrieved consistently links Ppk34 to Schizosaccharomyces pombe ORF SPCC1919.01 and identifies it as a CaMKK-family, serine/threonine protein kinase; in a Ca2+-signaling study it was renamed Ckk2 (for CaM kinase kinase 2). (cisnerosbarroso2014negativefeedbackregulation pages 8-9, cisnerosbarroso2014negativefeedbackregulation pages 13-14, cisnerosbarroso2014negativefeedbackregulation pages 10-12)
Cisneros-Barroso et al. report that SPCC1919.01/Ppk34 (Ckk2) is most similar to mammalian CaMKK2 and also related to the other fission-yeast CaMKK-like kinase Ssp1. (cisnerosbarroso2014negativefeedbackregulation pages 10-12)
CaMKKs (Ca2+/calmodulin-dependent protein kinase kinases) are upstream kinases that activate other protein kinases—classically CaMKs and AMPK—via phosphorylation of their activation loops. In S. pombe, at least two CaMKK-like kinases are discussed in the retrieved sources: Ssp1 and Ppk34/Ckk2. (davie2015nitrogenregulatesampk pages 1-2, davie2015nitrogenregulatesampk pages 7-8, cisnerosbarroso2014negativefeedbackregulation pages 10-12)
In fission yeast, the AMPK catalytic α subunit Ssp2 is activated by phosphorylation of its T-loop site Thr189, which then can inhibit TORC1 signaling under nutrient stress, affecting growth and mitotic commitment. (davie2015nitrogenregulatesampk pages 1-2, davie2015nitrogenregulatesampk pages 5-7, davie2015nitrogenregulatesampk pages 7-8)
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), rather than only a dispensable “unknown” kinase. (bimbo2005systematicdeletionanalysis pages 2-3, cisnerosbarroso2014negativefeedbackregulation pages 13-14)
Limitations: In the retrieved primary sources, no direct in vitro kinase assay of purified Ppk34/Ckk2 (with kinetic parameters or direct substrate mapping) was captured; functional inference is therefore based on genetic requirements for downstream phosphorylation events and pathway phenotypes. (cisnerosbarroso2014negativefeedbackregulation pages 13-14, davie2015nitrogenregulatesampk pages 7-8)
A. Ca2+-stress signaling: Ckk2 → Cmk1
Cisneros-Barroso et al. identify Ckk2 (Ppk34/SPCC1919.01) as the kinase responsible for Cmk1 phosphorylation in response to Ca2+ stress. Loss of Ckk2 eliminates the Ca2+-induced Cmk1 phosphorylation mobility shift. (cisnerosbarroso2014negativefeedbackregulation pages 10-12)
B. Nitrogen-stress signaling: Ppk34 → AMPKα/Ssp2 Thr189 phosphorylation dynamics
Davie et al. show that nitrogen stress causes an increase in Ssp2 Thr189 phosphorylation (detected using a phospho-AMPK antibody cross-reactive due to activation-loop conservation) and that Ppk34 is specifically required for this stress-induced increase, while basal Thr189 phosphorylation is still present without Ppk34. (davie2015nitrogenregulatesampk pages 7-8)
Davie et al. demonstrate that nitrogen stress activates AMPK/Ssp2 and inhibits TORC1. The paper explicitly states that a second CaMKK homolog Ppk34 is specifically required to stimulate activation of Ssp2 in response to nitrogen stress, while Ssp1 is constitutively required for basal T-loop phosphorylation. (davie2015nitrogenregulatesampk pages 1-2, davie2015nitrogenregulatesampk pages 7-8)
Mechanistically, nitrogen stress led to an average 2.5-fold increase in Ssp2 Thr189 phosphorylation after 30 minutes, and this increase was absent in ppk34Δ cells. (davie2015nitrogenregulatesampk pages 7-8)
Functionally, ppk34Δ 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. (davie2015nitrogenregulatesampk pages 7-8)
Davie et al. also note that nitrogen-stress-induced AMPK activation (mitotic acceleration) occurs independently of the AMPK β and γ subunits, implying an atypical regulation of the AMPK catalytic subunit in this system and motivating models in which Ppk34 contributes to α-subunit activation without canonical AMP-sensing inputs. (davie2015nitrogenregulatesampk pages 5-7)
Cisneros-Barroso et al. place Ckk2 upstream of Cmk1 in a Ca2+-triggered signaling module that counterbalances calcineurin signaling: calcineurin promotes Prz1 activation/nuclear localization, while Ckk2 activates Cmk1, and Cmk1 phosphorylates/inactivates Prz1 (promoting nuclear export), forming a negative feedback loop in Ca2+ signaling. (cisnerosbarroso2014negativefeedbackregulation pages 13-14)
Genetically, Δckk2 phenocopies Δcmk1 in Ca2+ resistance, and the Δcmk1 Δckk2 double mutant is also Ca2+-resistant, supporting that Ckk2 and Cmk1 act in the same pathway for Ca2+ signaling outputs. (cisnerosbarroso2014negativefeedbackregulation pages 10-12)
Additionally, Ckk2 is linked to the Cdc25 protein-level response under Ca2+ stress: Δckk2 cells show Cdc25 protein behavior similar to Δcmk1 and fail to increase Cdc25 during Ca2+ response in the cited time course. (cisnerosbarroso2014negativefeedbackregulation pages 10-12)
A kinase-deletion screen for NETO (new end take-off) regulators identified ppk34 deletion as associated with increased bipolar growth, leading to its classification as a putative negative regulator of NETO in that screen. (koyano2010searchforkinases pages 3-3)
Quantitatively, the polarity fractions reported for ppk34Δ were:
- 25°C: 39.8% monopolar, 47.1% bipolar, 13.1% septated
- 36°C: 31.9% monopolar, 55.9% bipolar, 12.2% septated (koyano2010searchforkinases pages 3-3)
No direct imaging-based localization (e.g., Ppk34-GFP) was captured in the retrieved excerpts. The Ca2+ signaling model described by Cisneros-Barroso et al. treats Ckk2/Cmk1 and calcineurin as cytosolic cascades that control the nuclear–cytoplasmic shuttling of Prz1, but this is a pathway-level inference rather than direct localization of Ckk2. (cisnerosbarroso2014negativefeedbackregulation pages 13-14, cisnerosbarroso2014negativefeedbackregulation pages 10-12)
A systematic kinase deletion analysis categorized Ppk34 (SPCC1919.01) deletion as viable and listed it as “unknown” with respect to process/function in their Table 1 annotation, indicating it is non-essential under standard conditions assayed in that work. (bimbo2005systematicdeletionanalysis pages 2-3)
Across later targeted/functional screens and mechanistic studies, ppk34Δ/ckk2Δ exhibits clear condition-dependent phenotypes:
- Defective nitrogen-stress AMPK activation and mitotic acceleration (rescued by rapamycin), consistent with impaired TORC1 inhibition in that context. (davie2015nitrogenregulatesampk pages 7-8)
- Altered Ca2+ signaling outputs, including Ca2+ resistance when Ckk2/Cmk1 negative feedback on Prz1 is removed. (cisnerosbarroso2014negativefeedbackregulation pages 10-12)
- Altered growth polarity (NETO) regulation with increased bipolarity in screen conditions. (koyano2010searchforkinases pages 3-3)
A 2023 review on AMPK–TOR interplay in yeast reiterates the central role of the Ssp1–Ssp2–TORC1 axis in environmental stress responses and mitotic advancement, highlighting that stresses including nitrogen deprivation lead to Ssp2-dependent TORC1 inhibition and premature mitotic entry. (alao2023interplaysofampk pages 6-8)
While the review emphasizes Ssp1/Ssp2 regulation and downstream TOR effects, it is consistent with the primary literature establishing stress-specific upstream contributions (including Ppk34 in nitrogen-stress activation) and underscores ongoing interest in how yeast tune distinct stress responses. (alao2023interplaysofampk pages 6-8)
Within the retrieved corpus, no 2023–2024 primary studies focusing directly on Ppk34/Ckk2 molecular mechanism, structure, or localization were identified. Therefore, the most experimentally specific functional assignments remain dominated by 2014–2015 mechanistic work and earlier genetic screens. (alao2023interplaysofampk pages 6-8)
| Aspect | Key finding | Evidence type | Experimental details/conditions | Quantitative data | Source (first author year journal) | DOI/URL |
|---|---|---|---|---|---|---|
| Identity | ORF SPCC1919.01, previously named Ppk34, was identified as a second CaMKK-family kinase in S. pombe and renamed Ckk2; it is most similar to mammalian CaMKK2 and also related to Ssp1. (cisnerosbarroso2014negativefeedbackregulation pages 8-9, cisnerosbarroso2014negativefeedbackregulation pages 13-14, cisnerosbarroso2014negativefeedbackregulation pages 10-12) | Sequence comparison, genetic | BLAST/sequence comparison in Ca2+-signaling study; compared with human CaMKK1/2 and Ssp1. (cisnerosbarroso2014negativefeedbackregulation pages 10-12) | Ckk2 similarity: 34% identity to human CaMKK2, 32% to Ssp1, 28% to human CaMKK1; coverages 86%, 63%, 52%, respectively. (cisnerosbarroso2014negativefeedbackregulation pages 10-12) | Cisneros-Barroso 2014 Nucleic Acids Research | https://doi.org/10.1093/nar/gku684 |
| Molecular function | Ppk34/Ckk2 functions as an upstream serine/threonine kinase in a CaMKK-like role, activating downstream kinases in stress signaling rather than being merely an uncharacterized kinase. (cisnerosbarroso2014negativefeedbackregulation pages 13-14) | Genetic, biochemical inference | Ca2+ response assays and pathway analysis place Ckk2 upstream of Cmk1; nitrogen-stress assays place Ppk34 upstream of AMPK/Ssp2 activation. (davie2015nitrogenregulatesampk pages 7-8, cisnerosbarroso2014negativefeedbackregulation pages 13-14) | No direct catalytic constants reported for Ppk34/Ckk2 itself in retrieved sources. (davie2015nitrogenregulatesampk pages 7-8, cisnerosbarroso2014negativefeedbackregulation pages 13-14) | Cisneros-Barroso 2014 Nucleic Acids Research; Davie 2015 Current Biology | https://doi.org/10.1093/nar/gku684; https://doi.org/10.1016/j.cub.2014.12.034 |
| Substrates | In the Ca2+ pathway, Ckk2/Ppk34 is required for Cmk1 phosphorylation/activation; in the nitrogen-stress pathway, Ppk34 is required for the increase in AMPKα/Ssp2 Thr189 phosphorylation. (cisnerosbarroso2014negativefeedbackregulation pages 13-14, davie2015nitrogenregulatesampk pages 7-8) | Biochemical, genetic | For Cmk1: CaCl2 treatment and mobility-shift assays in wt vs Δckk2. For Ssp2: glutamate→proline shift, western blot with anti-phospho-AMPK antibody in wt vs ppk34Δ. (davie2015nitrogenregulatesampk pages 7-8, cisnerosbarroso2014negativefeedbackregulation pages 10-12) | AMPK/Ssp2 Thr189 phosphorylation increased ~2.5-fold after 30 min nitrogen stress in wt; this increase was absent in ppk34Δ. (davie2015nitrogenregulatesampk pages 7-8) | Davie 2015 Current Biology; Cisneros-Barroso 2014 Nucleic Acids Research | https://doi.org/10.1016/j.cub.2014.12.034; https://doi.org/10.1093/nar/gku684 |
| Pathways | Ppk34/Ckk2 participates in at least two distinct signaling modules: Ckk2→Cmk1→Prz1/Cdc25 in Ca2+ signaling and Ppk34→Ssp2/AMPK→TORC1 inhibition during nitrogen stress. (davie2015nitrogenregulatesampk pages 1-2, cisnerosbarroso2014negativefeedbackregulation pages 13-14) | Genetic, biochemical, review | Ca2+ stress: 100 mM CaCl2 time courses and mutant analysis. Nitrogen stress: shift from glutamate to proline and TORC1 readouts/Maf1 phosphorylation/rapamycin rescue. Review literature summarizes this axis in yeast physiology. (davie2015nitrogenregulatesampk pages 5-7, davie2015nitrogenregulatesampk pages 7-8, alao2023interplaysofampk pages 6-8) | Nitrogen stress activates Ssp2 and inhibits TORC1; rapamycin rescues the ppk34Δ mitotic-entry defect. (davie2015nitrogenregulatesampk pages 7-8) | Davie 2015 Current Biology; Cisneros-Barroso 2014 Nucleic Acids Research; Alao 2023 Cells | https://doi.org/10.1016/j.cub.2014.12.034; https://doi.org/10.1093/nar/gku684; https://doi.org/10.3390/cells12040519 |
| Localization | Retrieved primary papers support cytosolic signaling function for the Ckk2/Cmk1/Prz1 module but do not provide a direct subcellular localization experiment for Ppk34/Ckk2 itself. (cisnerosbarroso2014negativefeedbackregulation pages 13-14, cisnerosbarroso2014negativefeedbackregulation pages 10-12) | Inference from pathway model | Model places calcineurin, Ckk2, and Cmk1 as Ca2+-responsive cytosolic cascades acting on Prz1 nuclear shuttling; no direct GFP-localization of Ckk2 reported in retrieved excerpts. (cisnerosbarroso2014negativefeedbackregulation pages 13-14, cisnerosbarroso2014negativefeedbackregulation pages 10-12) | No direct localization percentages or compartment-enrichment values reported. (cisnerosbarroso2014negativefeedbackregulation pages 13-14, cisnerosbarroso2014negativefeedbackregulation pages 10-12) | Cisneros-Barroso 2014 Nucleic Acids Research | https://doi.org/10.1093/nar/gku684 |
| Phenotypes | ppk34Δ/ckk2Δ is viable and was initially unassigned functionally in the systematic kinase-deletion set, but later screens and mechanistic work linked it to polarity and stress signaling. (bimbo2005systematicdeletionanalysis pages 2-3, koyano2010searchforkinases pages 3-3, cisnerosbarroso2014negativefeedbackregulation pages 13-14) | Deletion screen, genetic | Genome-wide kinase deletion study; NETO/polarity screen; Ca2+ sensitivity and nitrogen-stress mitotic-entry assays. (bimbo2005systematicdeletionanalysis pages 2-3, koyano2010searchforkinases pages 3-3, davie2015nitrogenregulatesampk pages 7-8) | Among 106 kinases analyzed, 17 essential and 89 dispensable; Ppk34 was among viable deletions. (bimbo2005systematicdeletionanalysis pages 2-3) | Bimbó 2005 Eukaryotic Cell; Koyano 2010 Bioscience, Biotechnology, and Biochemistry; Cisneros-Barroso 2014 Nucleic Acids Research | https://doi.org/10.1128/ec.4.4.799-813.2005; https://doi.org/10.1271/bbb.100223; https://doi.org/10.1093/nar/gku684 |
| Phenotypes | In a growth-polarity screen, ppk34 deletion increased bipolar growth, identifying Ppk34 as a putative negative regulator of NETO. (koyano2010searchforkinases pages 3-3, koyano2010searchforkinases pages 1-3) | Genetic screen | Quantified monopolar/bipolar/septated cells at 25°C and 36°C in kinase-deletion strains. (koyano2010searchforkinases pages 3-3, koyano2010searchforkinases pages 1-3) | 25°C: 39.8% monopolar, 47.1% bipolar, 13.1% septated; 36°C: 31.9% monopolar, 55.9% bipolar, 12.2% septated. (koyano2010searchforkinases pages 3-3) | Koyano 2010 Bioscience, Biotechnology, and Biochemistry | https://doi.org/10.1271/bbb.100223 |
| Phenotypes | In Ca2+ stress, Δckk2 and Δcmk1 Δckk2 are Ca2+-resistant, similar to Δcmk1, supporting that Ckk2 and Cmk1 act in the same pathway. (cisnerosbarroso2014negativefeedbackregulation pages 13-14, cisnerosbarroso2014negativefeedbackregulation pages 10-12) | Genetic | Spot assays on YES plates containing CaCl2; comparison of wt, Δcmk1, Δckk2, and double mutant. (cisnerosbarroso2014negativefeedbackregulation pages 10-12) | Resistance phenotype observed on plates with 50–75 mM CaCl2 and with 100 mM CaCl2 in signaling assays. (cisnerosbarroso2014negativefeedbackregulation pages 9-10, cisnerosbarroso2014negativefeedbackregulation pages 10-12) | Cisneros-Barroso 2014 Nucleic Acids Research | https://doi.org/10.1093/nar/gku684 |
| Phenotypes | In nitrogen stress, ppk34Δ fails to accelerate mitosis and does not reduce cell size at division to the same extent as wild type; rapamycin rescues this defect, placing Ppk34 upstream of TORC1. (davie2015nitrogenregulatesampk pages 7-8) | Genetic, pharmacological | Early exponential cultures shifted from glutamate to proline; mutants analyzed for mitotic-entry timing and response to rapamycin. (davie2015nitrogenregulatesampk pages 7-8) | Qualitative rescue by rapamycin reported; no exact rescue percentage given in retrieved excerpt. (davie2015nitrogenregulatesampk pages 7-8) | Davie 2015 Current Biology | https://doi.org/10.1016/j.cub.2014.12.034 |
| Quantitative stats | Ppk34 specifically controls stress-induced AMPK activation, whereas Ssp1 is required for basal Ssp2 Thr189 phosphorylation; thus the two CaMKK-like kinases show nonredundant specificity. (davie2015nitrogenregulatesampk pages 7-8, alao2023interplaysofampk pages 6-8) | Biochemical, genetic, review | wt, ssp1Δ, and ppk34Δ compared under steady-state and nitrogen-stress conditions; phospho-specific westerns and Phos-tag gels used. (davie2015nitrogenregulatesampk pages 7-8) | ppk34Δ: basal Thr189 phosphorylation retained, but no stress-induced increase; ssp1Δ: Thr189 phosphorylation absent even basally. (davie2015nitrogenregulatesampk pages 7-8) | Davie 2015 Current Biology; Alao 2023 Cells | https://doi.org/10.1016/j.cub.2014.12.034; https://doi.org/10.3390/cells12040519 |
| Quantitative stats | AMPK activation by nitrogen stress in S. pombe can occur without the AMPK β and γ subunits, highlighting an unusual activation mode in which Ppk34 contributes to α-subunit activation. (davie2015nitrogenregulatesampk pages 1-2, davie2015nitrogenregulatesampk pages 5-7, alao2023interplaysofampk pages 6-8) | Biochemical, genetic, review | amk2Δ, cbs2Δ, and double mutants assayed after glutamate→proline shift; Ssp2 T189 phosphorylation and mitotic response monitored. (davie2015nitrogenregulatesampk pages 5-7, alao2023interplaysofampk pages 6-8) | β/γ subunits not essential for mitotic advancement under nitrogen stress; wt Ssp2 T189 phosphorylation rises ~2.5-fold after 30 min. (davie2015nitrogenregulatesampk pages 7-8) | Davie 2015 Current Biology; Alao 2023 Cells | https://doi.org/10.1016/j.cub.2014.12.034; https://doi.org/10.3390/cells12040519 |
| Current understanding / gap | Recent authoritative review literature continues to place S. pombe Ppk34/Ckk2 within the AMPK–TOR stress-response network, but 2023–2024 primary literature specifically focused on Ppk34/Ckk2 is sparse in the retrieved corpus. (alao2023interplaysofampk pages 6-8) | Review / literature-gap assessment | 2023 review summarizes Ssp1/Ssp2/TOR cross-talk and cites earlier work showing ppk34-related nitrogen-stress signaling. (alao2023interplaysofampk pages 6-8) | No new 2023–2024 Ppk34-specific quantitative primary dataset identified in retrieved sources. (alao2023interplaysofampk pages 6-8) | Alao 2023 Cells | https://doi.org/10.3390/cells12040519 |
Table: This table compiles the main experimentally supported findings for S. pombe Ppk34/Ckk2 (Q9UU87/SPCC1919.01), including identity, pathway placement, phenotypes, and quantitative results. It highlights where direct evidence exists and where current knowledge remains inferential or limited.
Primary supported function: Ppk34 (Ckk2) is a CaMKK-family Ser/Thr kinase that mediates stress-responsive activation of downstream kinases.
- In nitrogen stress, it is required for the stress-induced increase in AMPKα/Ssp2 activation-loop phosphorylation (Thr189), enabling efficient TORC1 inhibition and mitotic advancement under poor nitrogen. (davie2015nitrogenregulatesampk pages 7-8)
- In Ca2+ stress, it functions upstream of Cmk1 to regulate a negative-feedback loop opposing calcineurin-dependent activation of Prz1, with downstream impacts on Cdc25 dynamics and Ca2+ tolerance phenotypes. (cisnerosbarroso2014negativefeedbackregulation pages 10-12)
Localization: Not directly resolved in the retrieved literature excerpts; pathway logic suggests a cytosolic kinase acting on signaling components controlling nuclear shuttling of transcription factors, but direct tagging/localization remains a key evidence gap. (cisnerosbarroso2014negativefeedbackregulation pages 13-14)
References
(cisnerosbarroso2014negativefeedbackregulation pages 8-9): Eugenia Cisneros-Barroso, Tula Yance-Chávez, Ayako Kito, Reiko Sugiura, Alba Gómez-Hierro, David Giménez-Zaragoza, and Rosa Aligue. Negative feedback regulation of calcineurin-dependent prz1 transcription factor by the camkk-camk1 axis in fission yeast. Nucleic Acids Research, 42:9573-9587, Jul 2014. URL: https://doi.org/10.1093/nar/gku684, doi:10.1093/nar/gku684. This article has 20 citations and is from a highest quality peer-reviewed journal.
(cisnerosbarroso2014negativefeedbackregulation pages 13-14): Eugenia Cisneros-Barroso, Tula Yance-Chávez, Ayako Kito, Reiko Sugiura, Alba Gómez-Hierro, David Giménez-Zaragoza, and Rosa Aligue. Negative feedback regulation of calcineurin-dependent prz1 transcription factor by the camkk-camk1 axis in fission yeast. Nucleic Acids Research, 42:9573-9587, Jul 2014. URL: https://doi.org/10.1093/nar/gku684, doi:10.1093/nar/gku684. This article has 20 citations and is from a highest quality peer-reviewed journal.
(cisnerosbarroso2014negativefeedbackregulation pages 10-12): Eugenia Cisneros-Barroso, Tula Yance-Chávez, Ayako Kito, Reiko Sugiura, Alba Gómez-Hierro, David Giménez-Zaragoza, and Rosa Aligue. Negative feedback regulation of calcineurin-dependent prz1 transcription factor by the camkk-camk1 axis in fission yeast. Nucleic Acids Research, 42:9573-9587, Jul 2014. URL: https://doi.org/10.1093/nar/gku684, doi:10.1093/nar/gku684. This article has 20 citations and is from a highest quality peer-reviewed journal.
(davie2015nitrogenregulatesampk pages 1-2): Elizabeth Davie, Gabriella M.A. Forte, and Janni Petersen. Nitrogen regulates ampk to control torc1 signaling. Current Biology, 25:445-454, Feb 2015. URL: https://doi.org/10.1016/j.cub.2014.12.034, doi:10.1016/j.cub.2014.12.034. This article has 107 citations and is from a highest quality peer-reviewed journal.
(davie2015nitrogenregulatesampk pages 7-8): Elizabeth Davie, Gabriella M.A. Forte, and Janni Petersen. Nitrogen regulates ampk to control torc1 signaling. Current Biology, 25:445-454, Feb 2015. URL: https://doi.org/10.1016/j.cub.2014.12.034, doi:10.1016/j.cub.2014.12.034. This article has 107 citations and is from a highest quality peer-reviewed journal.
(davie2015nitrogenregulatesampk pages 5-7): Elizabeth Davie, Gabriella M.A. Forte, and Janni Petersen. Nitrogen regulates ampk to control torc1 signaling. Current Biology, 25:445-454, Feb 2015. URL: https://doi.org/10.1016/j.cub.2014.12.034, doi:10.1016/j.cub.2014.12.034. This article has 107 citations and is from a highest quality peer-reviewed journal.
(bimbo2005systematicdeletionanalysis pages 2-3): Andrea Bimbó, Yonghui Jia, Siew Lay Poh, R. Krishna Murthy Karuturi, Nicole den Elzen, Xu Peng, Liling Zheng, Matthew O'Connell, Edison T. Liu, Mohan K. Balasubramanian, and Jianhua Liu. Systematic deletion analysis of fission yeast protein kinases. Eukaryotic Cell, 4:799-813, Apr 2005. URL: https://doi.org/10.1128/ec.4.4.799-813.2005, doi:10.1128/ec.4.4.799-813.2005. This article has 124 citations and is from a peer-reviewed journal.
(koyano2010searchforkinases pages 3-3): Takayuki KOYANO, Kazunori KUME, Manabu KONISHI, Takashi TODA, and Dai HIRATA. Search for kinases related to transition of growth polarity in fission yeast. Bioscience, Biotechnology, and Biochemistry, 74:1129-1133, May 2010. URL: https://doi.org/10.1271/bbb.100223, doi:10.1271/bbb.100223. This article has 35 citations.
(alao2023interplaysofampk pages 6-8): John-Patrick Alao, Luc Legon, Aleksandra Dabrowska, Anne-Marie Tricolici, Juhi Kumar, and Charalampos Rallis. Interplays of ampk and tor in autophagy regulation in yeast. Cells, 12:519, Feb 2023. URL: https://doi.org/10.3390/cells12040519, doi:10.3390/cells12040519. This article has 33 citations.
(koyano2010searchforkinases pages 1-3): Takayuki KOYANO, Kazunori KUME, Manabu KONISHI, Takashi TODA, and Dai HIRATA. Search for kinases related to transition of growth polarity in fission yeast. Bioscience, Biotechnology, and Biochemistry, 74:1129-1133, May 2010. URL: https://doi.org/10.1271/bbb.100223, doi:10.1271/bbb.100223. This article has 35 citations.
(cisnerosbarroso2014negativefeedbackregulation pages 9-10): Eugenia Cisneros-Barroso, Tula Yance-Chávez, Ayako Kito, Reiko Sugiura, Alba Gómez-Hierro, David Giménez-Zaragoza, and Rosa Aligue. Negative feedback regulation of calcineurin-dependent prz1 transcription factor by the camkk-camk1 axis in fission yeast. Nucleic Acids Research, 42:9573-9587, Jul 2014. URL: https://doi.org/10.1093/nar/gku684, doi:10.1093/nar/gku684. This article has 20 citations and is from a highest quality peer-reviewed journal.
ppk34 was the systematic name assigned by Bimbo et al. 2005 PMID:15821139 in their "Systematic deletion analysis of fission yeast protein kinases." Cisneros-Barroso et al. 2014 PMID:25081204 renamed the gene ckk2 after identifying it as a CaMKK2 homolog: "In addition to Ssp1, we found a second sequence homologous to mammalian CaMKK: SPC1919.01, named Ppk34 for the uncharacterised putative protein kinase 34 in a systematic deletion analysis of fission yeast kinases. Due to its homology to the CaMKK proteins, we have renamed it Ckk2." PomBase now uses ckk2 as the primary gene name.
From PMID:25081204: "Ckk2 is most homologous to mammalian CaMKK2 (34%), followed by Ssp1 (32%) and CaMKK1 (28%)."
S. pombe has two CaMKK-like kinases:
1. Ssp1 - the primary CaMKK ortholog, constitutively required for AMPK (Ssp2) T-loop phosphorylation
2. Ckk2/ppk34 - a second CaMKK, with specific roles in calcium and nitrogen stress signaling
Supporting text from 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+"
Supporting text from PMID:25639242: "a second homolog CaMKK(Ppk34) is specifically required to stimulate AMPKalpha(Ssp2) activation in response to nitrogen stress"
Ca2+ stress: Ca2+/CaM -> Ckk2 -> Cmk1 -> phospho-Prz1 (inactive) = negative regulation of calcineurin signaling
N stress: N stress -> Ckk2 -> Ssp2/AMPK -> Tsc1/2 -> Rhb1 -> TORC1 inhibition
The BioReason GO Term Predictions sections (MF, BP, CC) are all empty, which is notable.
Source: ppk34-deep-research-bioreason-sft.md
The BioReason functional summary describes ppk34 as:
A soluble serine/threonine kinase in fission yeast that dampens calcium-triggered signaling and restrains growth-promoting pathways to promote entry into quiescence and chronological aging. By phosphorylating components of the calcineurin axis, it limits activation of calcium-responsive transcriptional programs, and by targeting regulators of the TORC1 pathway, it reduces anabolic signaling. Its distribution between cytosol and nucleus enables rapid control of cytosolic signaling nodes and nuclear transcription factors, integrating calcium cues with nutrient and growth-state decisions to modulate lifespan traits.
This summary captures the two main signaling axes (calcium/calcineurin and TORC1) reasonably well, but contains several inaccuracies and unsupported extrapolations.
Correctness issues:
"Chronological aging" and "lifespan traits" are unsupported. No published study demonstrates a role for ppk34/ckk2 in chronological aging or lifespan regulation. While TORC1 inhibition can affect aging in other organisms, this extrapolation to ppk34 is speculative. No aging phenotype has been reported for ckk2 deletion mutants.
"By phosphorylating components of the calcineurin axis" is mechanistically incorrect. Ckk2/ppk34 does NOT phosphorylate calcineurin or its direct components. It phosphorylates Cmk1, which in turn phosphorylates Prz1 (the calcineurin-responsive transcription factor). The BioReason model collapses a two-step cascade into a single step, losing the critical indirect nature of the regulation.
The UniProt Summary section states "Involved in chronological aging and growth control. Negative regulator of calcineurin signaling." This is presented as if it comes from UniProt, but UniProt does not contain this text. The actual UniProt entry has no FUNCTION section for ppk34 beyond the catalytic activity descriptions. This fabricated attribution is misleading.
"Promote entry into quiescence" is overstated. The PMID:25639242 paper shows that ppk34 promotes cell division at reduced size during nitrogen stress (not quiescence). Ppk34 overexpression causes growth arrest, but this is likely an artifact of overexpression rather than a normal physiological function.
What was correct:
The thinking trace demonstrates some strengths and weaknesses:
Strengths:
- The domain architecture analysis is solid, correctly identifying the canonical kinase module and catalytic loop.
- The reasoning from architecture to molecular function (GO:0004674) is appropriate.
- Recognition of the calcium and TORC1 signaling connections.
Weaknesses:
Mechanism conflation. The trace states "phosphorylation of calcineurin targets or its regulators would dampen dephosphorylation events" -- this conflates the actual mechanism. Ckk2 phosphorylates Cmk1, not calcineurin or its targets directly.
Speculative interaction partners. The trace proposes "calcineurin components (the catalytic and regulatory subunits), calcineurin-responsive transcription factors, TORC1 core subunits and nutrient-sensing adaptors, 14-3-3 proteins, and PP2A-family phosphatases" as likely interaction partners. Of these, only Cmk1 and Ssp2 are demonstrated substrates. The others are either indirect targets or entirely speculative.
Causal chain fabrication. The trace constructs a plausible-sounding narrative about "feedback-controlled circuit that couples calcium influx to growth-state transitions and aging trajectories" but this specific integration has not been demonstrated experimentally for ppk34.
Empty GO predictions. The GO Term Predictions sections (MF, BP, CC) are all blank, which is a significant incompleteness given that the narrative discusses multiple GO terms.
The existing PomBase/GOA annotations for ppk34 are well-supported by experimental evidence:
- GO:0004674 (protein Ser/Thr kinase activity) - IGI from PMID:25639242
- GO:0071277 (cellular response to calcium ion) - IMP from PMID:25081204
- GO:1904262 (negative regulation of TORC1 signaling) - IMP from PMID:25639242
- GO:0106057 (negative regulation of calcineurin-mediated signaling) - IGI from PMID:25081204
- GO:0005829 (cytosol) and GO:0005634 (nucleus) - HDA from PMID:16823372
BioReason's narrative correctly identifies all of these functions but does not add any novel predictions. The empty GO predictions section means there are no new annotations suggested beyond what is already curated.
The BioReason text does not explicitly cite PMIDs, but the claims can be traced to three real publications:
- PMID:25081204 (Cisneros-Barroso et al. 2014) - REAL, supports calcium/calcineurin function
- PMID:25639242 (Davie et al. 2015) - REAL, supports nitrogen stress/TORC1 function
- PMID:16823372 (Matsuyama et al. 2006) - REAL, supports localization data
No fabricated references were found.
The BioReason InterPro analysis lists IPR000719 twice (residues 40-331 and 111-331), which is an artifact of overlapping domain predictions. The CDD annotation (cd14008, STKc_LKB1_CaMKK) is more informative, placing ppk34 in the LKB1/CaMKK subfamily. The PANTHER classification (PTHR43895, CaMKK-related) is also correct and consistent with the experimental characterization.
The UniProt entry lists three Reactome pathways (R-SPO-111932, R-SPO-442729, R-SPO-9619229) that are ALL ortholog projections from human pathways. S. pombe does not have CREB, CaMKIV, or NMDA receptors. These Reactome annotations are misleading and should not be used to infer biological function for ppk34 in S. pombe. BioReason did not comment on these, which is appropriate since it appears to have ignored them.
The BioReason prediction correctly identifies the two main experimental functions of ppk34/ckk2 (calcium signaling via Cmk1 and TORC1 regulation via AMPK/Ssp2) but makes several unsupported extrapolations, particularly regarding chronological aging and lifespan. It also misrepresents the mechanistic details by suggesting direct phosphorylation of calcineurin components rather than the indirect Ckk2->Cmk1->Prz1 cascade. The fabricated UniProt summary and speculative interaction partners reduce overall reliability. For a moderately characterized gene like ppk34, the BioReason output is a useful starting point but requires substantial corrections to be accurate.
id: Q9UU87
gene_symbol: ppk34
product_type: PROTEIN
status: COMPLETE
taxon:
id: NCBITaxon:284812
label: Schizosaccharomyces pombe (strain 972 / ATCC 24843)
aliases:
- ckk2
description: >-
Ppk34 (now called ckk2 on PomBase) 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 to stimulate AMPK (Ssp2) activation via
T189 phosphorylation, leading to TORC1 inhibition and reduced cell size at
division. The protein localizes to both cytosol and nucleus.
existing_annotations:
- term:
id: GO:0007165
label: signal transduction
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
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.
action: KEEP_AS_NON_CORE
reason: >-
Overly broad term; more specific BP annotations exist for this gene.
supported_by:
- reference_id: file:SCHPO/ppk34/ppk34-deep-research-bioreason-sft.md
supporting_text: >-
...A soluble serine/threonine kinase in fission yeast that dampens
calcium-triggered signaling and restrains growth-promoting pathways...
- reference_id: file:SCHPO/ppk34/ppk34-deep-research-falcon.md
supporting_text: |-
identifies it as a **CaMKK-family, serine/threonine protein kinase**; in a Ca2+-signaling study it was **renamed Ckk2** (for CaM kinase kinase 2)
- term:
id: GO:0004674
label: protein serine/threonine kinase activity
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: >-
Correct molecular function. Ppk34/ckk2 is a serine/threonine kinase with
demonstrated kinase activity toward Cmk1 and Ssp2. The IBA evidence from
PANTHER (PTHR43895, CaMKK-related family) is appropriate and consistent
with experimental data from PMID:25081204 and PMID:25639242.
action: ACCEPT
supported_by:
- reference_id: file:SCHPO/ppk34/ppk34-deep-research-falcon.md
supporting_text: |-
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)
- term:
id: GO:0004672
label: protein kinase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
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.
action: KEEP_AS_NON_CORE
reason: >-
Redundant with GO:0004674 annotation; less specific.
- term:
id: GO:0004674
label: protein serine/threonine kinase activity
evidence_type: IEA
original_reference_id: GO_REF:0000003
review:
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 and Ssp2.
action: ACCEPT
- term:
id: GO:0005524
label: ATP binding
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
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.
action: ACCEPT
- term:
id: GO:0005634
label: nucleus
evidence_type: IEA
original_reference_id: GO_REF:0000044
review:
summary: >-
This IEA annotation is consistent with the experimental HDA evidence
from PMID:16823372, which showed nuclear localization by large-scale
GFP tagging.
action: ACCEPT
- term:
id: GO:0005737
label: cytoplasm
evidence_type: IEA
original_reference_id: GO_REF:0000044
review:
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.
action: MODIFY
reason: >-
The HDA data from PMID:16823372 supports cytosol (GO:0005829) specifically.
proposed_replacement_terms:
- id: GO:0005829
label: cytosol
- term:
id: GO:0106310
label: protein serine kinase activity
evidence_type: IEA
original_reference_id: GO_REF:0000116
review:
summary: >-
RHEA-based annotation. Ppk34/ckk2 phosphorylates serine and threonine
residues, so protein serine kinase activity is a subset of its activity.
Acceptable but less informative than GO:0004674 which covers both Ser
and Thr. Not wrong, but redundant.
action: KEEP_AS_NON_CORE
reason: >-
Partially redundant with GO:0004674; captures only serine-specific activity.
- term:
id: GO:0004674
label: protein serine/threonine kinase activity
evidence_type: IGI
original_reference_id: PMID:25639242
review:
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 (cmk1). This is strong experimental evidence that ppk34 has
protein kinase activity, demonstrated through its functional requirement
for phosphorylation of known substrates.
action: ACCEPT
supported_by:
- reference_id: PMID:25639242
supporting_text: >-
...CaMKK(Ppk34) is specifically required to stimulate AMPKalpha(Ssp2)
activation in response to nitrogen stress...
- reference_id: file:SCHPO/ppk34/ppk34-deep-research-falcon.md
supporting_text: |-
**Ppk34 is specifically required for this stress-induced increase**, while basal Thr189 phosphorylation is still present without Ppk34
- term:
id: GO:0071277
label: cellular response to calcium ion
evidence_type: IMP
original_reference_id: PMID:25081204
review:
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.
action: ACCEPT
supported_by:
- reference_id: PMID:25081204
supporting_text: >-
...we have identified a second CaMKK in fission yeast, the Ckk2
kinase, which is involved in the activation of Cmk1 in response
to Ca2+...
- reference_id: file:SCHPO/ppk34/ppk34-deep-research-falcon.md
supporting_text: |-
Ckk2 (Ppk34/SPCC1919.01) as the kinase responsible for **Cmk1 phosphorylation in response to Ca2+ stress**
- term:
id: GO:1904262
label: negative regulation of TORC1 signaling
evidence_type: IMP
original_reference_id: PMID:25639242
review:
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).
action: ACCEPT
supported_by:
- reference_id: PMID:25639242
supporting_text: >-
...CaMKKPpk34 is required to induce AMPKalphaSsp2 activation
following nitrogen stress...overexpression of CaMKKppk34+ can
promote AMPKalphaSsp2 activation through T189 phosphorylation...
- reference_id: file:SCHPO/ppk34/ppk34-deep-research-falcon.md
supporting_text: |-
**ppk34Δ 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
- term:
id: GO:0106057
label: negative regulation of calcineurin-mediated signaling
evidence_type: IGI
original_reference_id: PMID:25081204
review:
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. 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.
action: ACCEPT
supported_by:
- reference_id: PMID:25081204
supporting_text: >-
...Ckk2 counteracts calcineurin function by negatively regulating
Prz1 activity...
- reference_id: file:SCHPO/ppk34/ppk34-deep-research-falcon.md
supporting_text: |-
**Ckk2 activates Cmk1**, and **Cmk1 phosphorylates/inactivates Prz1** (promoting nuclear export), forming a negative feedback loop in Ca2+ signaling
- term:
id: GO:0005634
label: nucleus
evidence_type: HDA
original_reference_id: PMID:16823372
review:
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).
action: ACCEPT
supported_by:
- reference_id: PMID:16823372
supporting_text: >-
...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...
- term:
id: GO:0005829
label: cytosol
evidence_type: HDA
original_reference_id: PMID:16823372
review:
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.
action: ACCEPT
supported_by:
- reference_id: PMID:16823372
supporting_text: >-
...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...
- reference_id: file:SCHPO/ppk34/ppk34-deep-research-falcon.md
supporting_text: |-
treats Ckk2/Cmk1 and calcineurin as **cytosolic cascades** that control the nuclear–cytoplasmic shuttling of Prz1
references:
- id: file:SCHPO/ppk34/ppk34-deep-research-bioreason-sft.md
title: BioReason-Pro SFT deep research for ppk34
findings:
- statement: >-
BioReason correctly identifies ppk34 as a serine/threonine kinase involved
in calcium signaling and TORC1 regulation but makes unsupported claims
about chronological aging and incorrectly suggests direct phosphorylation
of calcineurin components.
- id: file:SCHPO/ppk34/ppk34-deep-research-falcon.md
title: Falcon (Edison) deep research report for ppk34/Ckk2
findings:
- statement: |-
Falcon confirms the gene identity: ORF SPCC1919.01/Ppk34 is a CaMKK-family
Ser/Thr protein kinase that was renamed Ckk2 (CaM kinase kinase 2) in a
calcium-signaling study, most similar to mammalian CaMKK2 and related to
the other fission-yeast CaMKK-like kinase Ssp1.
reference_section_type: OTHER
supporting_text: |-
The literature retrieved consistently links **Ppk34** to *Schizosaccharomyces pombe* ORF **SPCC1919.01** and identifies it as a **CaMKK-family, serine/threonine protein kinase**; in a Ca2+-signaling study it was **renamed Ckk2** (for CaM kinase kinase 2).
- statement: |-
Falcon corroborates the sequence relationships: Ckk2 has 34% identity to
human CaMKK2, 32% to Ssp1, and 28% to human CaMKK1, consistent with its
assignment to the CaMKK subfamily (PANTHER PTHR43895).
reference_section_type: OTHER
supporting_text: |-
Ckk2 has **34% identity** to human CaMKK2, **32%** to Ssp1, and **28%** to human CaMKK1 in the Cisneros-Barroso study.
- statement: |-
Falcon supports a CaMKK-like molecular role: Ppk34/Ckk2 acts as an upstream
Ser/Thr kinase activating downstream kinases (Cmk1, AMPK/Ssp2) in stress
signaling, rather than being a catalytically uncharacterized kinase.
reference_section_type: OTHER
supporting_text: |-
Ppk34/Ckk2 functions as an upstream **serine/threonine kinase** in a CaMKK-like role, activating downstream kinases in stress signaling rather than being merely an uncharacterized kinase.
- statement: |-
Falcon notes the two CaMKK-like kinases are non-redundant: Ppk34 is
specifically required for nitrogen-stress-induced Ssp2 activation whereas
Ssp1 is constitutively required for basal T-loop phosphorylation.
reference_section_type: OTHER
supporting_text: |-
The paper explicitly states that a **second CaMKK homolog Ppk34 is specifically required to stimulate activation of Ssp2 in response to nitrogen stress**, while **Ssp1 is constitutively required** for basal T-loop phosphorylation.
- statement: |-
Falcon quantifies the nitrogen-stress signaling phenotype: Ssp2 Thr189
phosphorylation rises ~2.5-fold at 30 min after nitrogen stress in
wild type, and this increase is absent in ppk34-delta cells.
reference_section_type: OTHER
supporting_text: |-
nitrogen stress led to an **average 2.5-fold increase** in **Ssp2 Thr189 phosphorylation after 30 minutes**, and this increase was **absent in ppk34Δ** cells.
- statement: |-
Falcon notes the Ppk34 effect on Ssp2 phosphorylation is likely indirect
(basal phosphorylation persists and Ssp1 appears to be the direct T-loop
kinase), consistent with the IGI evidence type for the kinase activity
annotation and an indirect mechanism for TORC1 inhibition.
reference_section_type: OTHER
supporting_text: |-
Davie et al. interpret the Ppk34 effect on Ssp2 phosphorylation as likely **indirect**
- statement: |-
Falcon reports that ppk34/SPCC1919.01 deletion is viable (non-essential
under standard conditions) per the systematic kinase deletion analysis.
reference_section_type: OTHER
supporting_text: |-
A systematic kinase deletion analysis categorized **Ppk34 (SPCC1919.01) deletion as viable**
- statement: |-
Falcon notes a polarity (NETO) phenotype: ppk34 deletion is associated
with increased bipolar growth, classifying Ppk34 as a putative negative
regulator of NETO (new-end take-off). This is a condition-specific
genetic-screen observation rather than a core biochemical function.
reference_section_type: OTHER
supporting_text: |-
A kinase-deletion screen for NETO (new end take-off) regulators identified **ppk34 deletion** as associated with **increased bipolar growth**, leading to its classification as a putative **negative regulator of NETO** in that screen.
- statement: |-
Falcon cautions that no direct imaging-based localization of Ppk34/Ckk2
(e.g., Ppk34-GFP) was captured; the cytosolic-cascade model is a
pathway-level inference, while the nucleus/cytosol GO annotations rest on
the large-scale GFP localization study.
reference_section_type: OTHER
supporting_text: |-
No direct imaging-based localization (e.g., Ppk34-GFP) was captured in the retrieved excerpts.
- id: GO_REF:0000002
title: >-
Gene Ontology annotation through association of InterPro records with GO
terms
findings: []
- id: GO_REF:0000003
title: Gene Ontology annotation based on Enzyme Commission mapping
findings: []
- id: GO_REF:0000033
title: Annotation inferences using phylogenetic trees
findings: []
- id: GO_REF:0000044
title: >-
Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
vocabulary mapping, accompanied by conservative changes to GO terms applied
by UniProt
findings: []
- id: GO_REF:0000116
title: Automatic Gene Ontology annotation based on Rhea mapping
findings: []
- id: PMID:11859360
title: The genome sequence of Schizosaccharomyces pombe.
findings:
- statement: >-
Genome sequencing identified ORF SPCC1919.01 encoding ppk34.
supporting_text: >-
...The genome sequence of Schizosaccharomyces pombe...
- id: PMID:15821139
title: >-
Systematic deletion analysis of fission yeast protein kinases.
full_text_unavailable: true
findings:
- statement: >-
ppk34 was identified as one of 106 protein kinases in S. pombe. Deletion
of ppk34 is viable under standard growth conditions.
supporting_text: >-
...A total of 106 eukaryotic protein kinase catalytic-domain-containing
proteins have been found in the entire fission yeast genome...Systematic
deletion analysis of all putative protein kinase-encoding genes have
revealed that 17 out of 106 were essential for viability...
reference_section_type: ABSTRACT
- id: PMID:16823372
title: >-
ORFeome cloning and global analysis of protein localization in the fission
yeast Schizosaccharomyces pombe.
findings:
- statement: >-
Large-scale GFP tagging showed ppk34/ckk2 localizes to cytosol and nucleus.
supporting_text: >-
...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...
- id: PMID:25081204
title: >-
Negative feedback regulation of calcineurin-dependent Prz1 transcription
factor by the CaMKK-CaMK1 axis in fission yeast.
findings:
- statement: >-
Identified ppk34 as Ckk2, a second CaMKK in S. pombe. Ckk2 phosphorylates
and activates Cmk1 in response to calcium. The Ckk2-Cmk1 cascade negatively
regulates Prz1 transcription factor activity, counteracting calcineurin.
supporting_text: >-
...we have identified a second CaMKK in fission yeast, the Ckk2 kinase,
which is involved in the activation of Cmk1 in response to Ca2+...
reference_section_type: ABSTRACT
- statement: >-
Ckk2 is most homologous to mammalian CaMKK2 (34%), followed by Ssp1 (32%)
and CaMKK1 (28%).
supporting_text: >-
...Ckk2 is most homologous to mammalian CaMKK2 (34%), followed by Ssp1
(32%) and CaMKK1 (28%)...
reference_section_type: RESULTS
- statement: >-
Deletion of ckk2 shows phenotypes similar to cmk1 deletion during calcium
response. Cdc25 protein levels fail to accumulate in delta-ckk2 cells
treated with Ca2+.
supporting_text: >-
...Cdc25 protein levels in Δckk2 cells were similar to those in Δcmk1
cells and, failed to increase during Ca2+ response...reinforcing the
claim that Ckk2 is in the same pathway as Cmk1 during Ca2+ signalling...
reference_section_type: RESULTS
- id: PMID:25639242
title: Nitrogen regulates AMPK to control TORC1 signaling.
findings:
- statement: >-
CaMKK(Ppk34) is specifically required for nitrogen-stress-induced AMPK
(Ssp2) activation, distinct from the constitutive role of Ssp1 in Ssp2
T-loop phosphorylation.
supporting_text: >-
...a second homolog CaMKK(Ppk34) is specifically required to stimulate
AMPKalpha(Ssp2) activation in response to nitrogen stress...
reference_section_type: ABSTRACT
- statement: >-
Overexpression of CaMKK(Ppk34) promotes Ssp2 activation through T189
phosphorylation and induces growth arrest in a nutrient-dependent manner.
supporting_text: >-
...overexpression of CaMKKppk34+ can promote AMPKalphaSsp2 activation
through T189 phosphorylation...
reference_section_type: RESULTS
- statement: >-
Nitrogen-stress-induced TORC1 inhibition requires Ssp2 (AMPK), Tsc1/2,
and Rhb1, with Ppk34 required for the nitrogen-specific activation of this
pathway.
supporting_text: >-
...CaMKKPpk34 is required to induce AMPKalphaSsp2 activation following
nitrogen stress...
reference_section_type: RESULTS
core_functions:
- description: >-
CaMKK activity in calcium signaling: phosphorylates and activates Cmk1 in
response to calcium stress, forming part of a negative feedback loop that
counteracts calcineurin-mediated activation of the Prz1 transcription factor.
molecular_function:
id: GO:0004674
label: protein serine/threonine kinase activity
directly_involved_in:
- id: GO:0071277
label: cellular response to calcium ion
- id: GO:0106057
label: negative regulation of calcineurin-mediated signaling
locations:
- id: GO:0005829
label: cytosol
- id: GO:0005634
label: nucleus
supported_by:
- reference_id: PMID:25081204
supporting_text: >-
...Ckk2 counteracts calcineurin function by negatively regulating Prz1
activity...
- description: >-
CaMKK activity in nitrogen stress signaling: specifically required for
activation of AMPK (Ssp2) via T189 phosphorylation during nitrogen stress,
leading to TORC1 inhibition and reduced cell size at division.
molecular_function:
id: GO:0004674
label: protein serine/threonine kinase activity
directly_involved_in:
- id: GO:1904262
label: negative regulation of TORC1 signaling
locations:
- id: GO:0005829
label: cytosol
supported_by:
- reference_id: PMID:25639242
supporting_text: >-
...CaMKKPpk34 is required to induce AMPKalphaSsp2 activation following
nitrogen stress...
suggested_questions:
- question: >-
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.
- question: >-
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?
- question: >-
Are there additional substrates of Ckk2 beyond Cmk1 and Ssp2? The gene
appears at the nexus of calcium and nutrient signaling, suggesting possible
additional targets.
suggested_experiments:
- hypothesis: >-
Ckk2 directly phosphorylates Ssp2 at T189 in vitro.
description: >-
Perform in vitro kinase assay with purified Ckk2 and Ssp2 to test direct
phosphorylation at T189, using Ssp2-T189A as a negative control.
experiment_type: in vitro kinase assay
- hypothesis: >-
Ckk2 activation during nitrogen stress is Ca2+/calmodulin-dependent.
description: >-
Test whether calmodulin inhibitors or Ca2+ chelators block nitrogen-stress-induced
Ssp2 phosphorylation in vivo, comparing with Ca2+-stress-induced Cmk1 activation.
experiment_type: pharmacological inhibition / epistasis analysis