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DISTINCTIVE ROLES AND MOLECULAR REGULATION OF CTP:PHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE ALPHA AND GAMMA SPLICE VARIANTS

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Title: DISTINCTIVE ROLES AND MOLECULAR REGULATION OF CTP:PHOSPHOETHANOLAMINE CYTIDYLYLTRANSFERASE ALPHA AND GAMMA SPLICE VARIANTS
Author: Pavlovic, Zvezdan
Department: Department of Human Health and Nutritional Sciences
Program: Human Health and Nutritional Sciences
Advisor: Bakovic, Marica
Abstract: Phosphatidylethanolamine (PE) is the most abundant lipid in the inner leaflet of cellular membranes. CTP: phosphoethanolamine cytidylyltransferase (Pcyt2) controls the rate-limiting step of de novo biosynthesis of PE via the CDP-ethanolamine pathway (Kennedy pathway). Pcyt2 gene is ubiquitously expressed and essential for embryonic development and metabolic health. Alternative splicing was previously identified as a molecular mechanism through which two active Pcyt2 isoforms (Pcyt2α and –β) are produced. This thesis includes a study of cloning and characterization of the third murine Pcyt2 isoform (Pcyt2γ) made by a distinct alternative splicing mechanism. Although inactive per se, the novel Pcyt2 variant was determined to be a post-translational repressor and an indicator that both cytidylyltransferase domains are required for activity. Pcyt2γ was shown to restrain Pcyt2α activity through the formation of unproductive enzyme complexes that are degraded via the proteasome. The N-domain (H35Y, H35A) and C-domain (H244Y, H244A) catalytic site mutants were inactive and similarly to Pcyt2γ they reduced dimerization and activity of Pcyt2α. In addition, the protein kinase C (PKC)-mediated stimulation of Pcyt2 activity by phosphorylation was investigated. Single (S215A and S223A) and double mutations (S215.223A) of PKC consensus sites reduced Pcyt2α phosphorylation, activity, and PE synthesis. Since the identified phosphorylations cluster within the linker segment, we propose that phosphorylation could affect the ordering of  helices, which in turn could impact cooperativity between active sites and alter the enzyme’s activity. In sight of the existence of multiple Pcyt2 isoforms, cyto-nuclear partitioning of Pcyt2α and Pcyt2γ was examined. Despite the presence of a putative nuclear localization signal (NLS, 343PKRRGIF349) only ~10% of Pcyt2α localized to the nucleus of COS-7 cells. Interestingly, deletion of the NLS did not significantly alter sub-cellular distribution of Pcyt2α, while ~35% of Pcyt2γ localized to the nucleus despite the absence of the predicted NLS. This work shows that multiple molecular mechanisms regulate Pcyt2 activity and PE synthesis. Pcyt2 activity was shown to be influenced by interactions among different isoforms produced by alternative splicing and by phosphorylation of the linker region, a unique regulatory segment of Pcyt2, not present in other member of cytidylyltransferase family.
URI: http://hdl.handle.net/10214/8314
Date: 2014-08


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