Regulation of human cytidine triphosphate synthetase 1 by phosphorylation and interacting proteins Public Deposited

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  • March 21, 2019
  • Higgins, Matthew James
    • Affiliation: School of Medicine, Department of Pharmacology
  • CTP is essential for the synthesis of phospholipids, DNA/RNA and the sialylation of proteins. Of the two pathways known to synthesize CTP (de novo and salvage), the de novo synthesis pathway is thought to be the key regulator of CTP pool size in mammalian cells. CTPS is the rate-limiting enzyme in the de novo synthesis of CTP. CTPS catalyzes the amination of UTP to form CTP using glutamine as the amine donor and GTP as an allosteric activator. ATP is hydrolyzed in the process and CTP is a feedback inhibitor. In S. cerevisiae CTP synthetase (CTPS) is regulated by phosphorylation by PKA and PKC. The goals of this study were to investigate phosphorylation regulation of mammalian CTPS1 and elucidate CTPS1 interacting proteins. PKA or PKC were found not to be involved in the phosphorylation of CTPS1 in intact HEK 293 cells. However, endogenous human CTPS1 was phosphorylated under low-serum conditions in a GSK3-dependent manner. S571 was identified as the major GSK3 phosphorylation site and S574 and S575 were also identified as phosphorylation sites. GSK3 phosphorylation of human CTPS1 decreased enzyme activity and in vitro dephosphorylation increased CTPS1 activity indicating that phosphorylation suppresses CTPS1 activity. CK I was also found to be involved in the phosphorylation of CTPS1. CK I inhibition did not affect CTPS1 activity indicating that CK I may regulate another function of CTPS1. Investigation of CTPS1 interacting proteins revealed that the peptidylprolyl isomerase, Pin1, can interact with CTPS1 in a S575 phosphorylation-dependent manner. However, reduction of Pin1 expression did not affect CTPS1 activity suggesting that the regulation of CTPS1 by Pin1 may be through another mechanism. Continued investigation of CTPS1 interacting proteins revealed, among other proteins, tubulin as a CTPS1 interacting proteins. Further investigation is needed to understand the significance of this interaction. CTPS1 expression was found to be highest in the brain suggesting CTPS1 may have a brain-specific function. This study is the first to characterize the phosphorylation regulation of mammalian CTPS1 expressed in mammalian cells and elucidate CTPS1 interacting proteins and CTPS1 expression in mammalian tissue.
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  • In Copyright
  • Graves, Lee
Degree granting institution
  • University of North Carolina at Chapel Hill
  • Open access

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