The rate‐limiting enzyme in the CDP‐choline pathway is regulated by phosphorylation‐domain charge density
Bibliographic record
Abstract
Eukaryotic cell membranes are primarily composed of phospholipids, the most abundant of which is phosphatidylcholine (PC). De novo synthesis of PC by the CDP‐choline pathway is under the control of the rate‐limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT). The nuclear CCTa isoform is regulated by translocation to the inner nuclear membrane (INM) and nuclear lipid droplets (nLD) in response to lipid activators, such as oleate, or deficiency in PC content. CCTa association with the INM is mediated by a positively‐charged membrane‐binding M‐domain and a phosphorylated P‐domain containing 16 serine phospho‐sites. Mutation of all P‐domain serine residues to alanine (dephosphorylated mimic) enhanced association with nLDs in oleate‐treated U2OS cells, while mutation to glutamate (phosphorylated mimic) or mutation of M‐domain lysine residues ablated nLD association. However, it is unknown if phosphorylation regulation of CCTa association with the INM and nLDs involves specific serine residues or the net charge of the P‐domain. To address this question, we mutated 4 sets of 2‐3 serine residues in the P‐domain to alanine or aspartate. CCTa cDNAs expressing different combinations of these mutations were expressed in oleate‐treated U2OS cells and the frequency of nLD association was measured. The mutants were also expressed in CHO cells with a temperature‐sensitive CCTα isoform and INM translocation in response to oleate was determined. The greatest inhibitory effect on INM and nLD association was observed when all 4 sets of serine residues were mutated to aspartate and not by any one site, suggesting that net charge density of the P‐domain regulates affinity for the NE and nLD. Additionally, CCTa serine‐to‐alanine mutants expressed in U2OS cells were unstable compared to the corresponding aspartate mutants. We conclude that CCTa association with the INM and nLD is inhibited as overall P‐domain phosphorylation increases. In its active membrane‐associated, dephosphorylated state CCTa protein has decreased stability, a potently negative feedback mechanism to control PC synthesis. These results provide novel insight into how PC synthesis is regulated that will be essential for identifying kinases and phosphatases that act on CCTα.
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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 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".