Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 275, Issue 13, Pages 9699-9708Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.13.9699
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- NHLBI NIH HHS [HL55584] Funding Source: Medline
- NIGMS NIH HHS [GM45737] Funding Source: Medline
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We investigated the effects of tumor necrosis factor cu (TNF alpha), a key cytokine involved in inflammatory lung disease, on phosphatidylcholine (PtdCho) biosynthesis in a murine alveolar type II epithelial cell line (MLE-12). TNF alpha significantly inhibited [H-3]choline incorporation into PtdCho after 24 h of exposure. TNF alpha reduced the activity of CTP:phosphocholine cytidylyltransferase (CCT), the rate-regulatory enzyme within the CDP-choline pathway, by 40% compared with control, but it did not alter activities of choline kinase or cholinephosphotransferase. Immunoblotting revealed that TNF alpha inhibition of CCT activity was associated with a uniform decrease in the mass of CCT alpha in total cell lysates, cytosolic, microsomal, and nuclear subfractions of RILE cells, Northern blotting revealed no effects of the cytokine on steady-state levels of CCT alpha mRNA, and CCT beta mRNA was not detected. Incorporation of [S-35]methionine into immunoprecipitable CCT alpha protein in pulse and pulse-chase studies revealed that TNF alpha did not alter de novo synthesis of enzyme, but it substantially accelerated turnover of CCTa. Addition of N-acetyl-Leu-Leu-Nle-CHO (ALLN), the calpain I inhibitor, or lactacystin, the 20 S proteasome inhibitor, blocked the inhibition of PtdCho biosynthesis mediated by TNF alpha. TNF alpha-induced degradation of CCTa protein was partially blocked by ALLN or lactacystin, CCT was ubiquitinated, and ubiquitination increased after TNF alpha exposure. m-Calpain degraded both purified CCT and CCT in cellular extracts. Thus, TNF alpha inhibits PtdCho synthesis by modulating CCT protein stability via the ubiquitin-proteasome and calpain-mediated proteolytic pathways.
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