Autophosphorylation-based Calcium (Ca2+) Sensitivity Priming and Ca2+/Calmodulin Inhibition of Arabidopsis thaliana Ca2+-dependent Protein Kinase 28 (CPK28)
Bibliographic record
Abstract
Plant calcium (Ca 2+ )-dependent protein kinases (CPKs) represent the primary Ca 2+ -dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca 2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca 2+ /CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca 2+ /CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca 2+ -responsive and was inhibited by Ca 2+ /CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca 2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca 2+ activation and might also allow CPK28 to remain active when Ca 2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca 2+ /CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca 2+ signaling specificity through Ca 2+ sensor priming.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".