Ceramide-1-phosphate (C1P) has proven to be a bioactive sphingolipid with diverse functions within the cell. At this time, ceramide kinase (CERK) is the only known enzyme known to generate C1P in mammalian cells, and this bioactive lipid is responsible for the activation and translocation of cytosolic phospholipase A2, the initial rate limiting step in eicosanoid synthesis. These studies investigate the regulation of ceramide kinase by calcium. While CERK activity has been shown to be calcium sensitive, little is known about how CERK is activated within the cell; one possibility is the interaction with calcium "sensor" proteins such as calmodulin. In this study, we develop two protocols to efficiently examine the interaction of CERK with calcium dependent proteins: V5 co-immunoprecipitation and Ni-NTA affinity purification. The methods utilize either adenoviral infection or Effectene© transfection of cells to ectopically express CERK with both a 6x His and V5 tag on its C terminus. Unlike the report of Igarashi and co-workers, our findings reveal that CERK does not specifically interact with the calcium sensor, calmodulin, in three different cell types. We also show that the calcium dependent membrane organizer, annexin A2, also does not bind to CERK. In light of these findings, we illustrate that while CERK may be sensitive to calcium, it does not, as previously reported by another laboratory, specifically bind to calmodulin. These studies eliminate possible calcium mediator proteins and are suggestive of another method for the calcium sensitive regulation of CERK lending to new avenues of investigation (i.e. CaMKII). This report also firmly established two successful protocols for investigating protein partners of CERK. Ultimately, through providing a clearer picture behind the calcium regulation of CERK, we can elucidate possible novel therapeutic targets within the inflammatory pathway.

名古屋大学博士学位論文 学位の種類:博士（医療技術学） (課程) 学位授与年月日　平成22年3月25日

Ceramide-1-phosphate (C1P) is a bioactive lipid that has been implicated in many biological processes. Our laboratory has conclusively demonstrated its role in inflammation via activation of cPLA2α. The only known enzyme to date responsible for direct synthesis of C1P is ceramide kinase. Very little was known about this enzyme in terms of its enzyme kinetics and substrate specificity. As CERK is an enzyme that acts on membrane lipids, its kinetics cannot be studied using standard bulk dilutions methods. Thus we developed a surface dilution approach using Triton X 100 mixed micelles for studying the kinetics of CERK. We discovered that ceramide kinase has an affinity for naturally occurring long chain ceramides while ceramides containing shorter than 8 carbons are very poor substrates for the enzyme. Also of note is the discovery that there is no discrimination between the naturally occurring long chain ceramides leading to the conclusion that the preponderance of D-e-C16 C1P in cells are due to an availability effect. We also investigated the chain length specificity of interaction between C1P and cPLA2α. Our data indicate that cPLA2α is activated by C1P’s containing acyl chains longer than two carbons. The study showed C2 C1P as being unable to activate cPLA2α thus establishing a tool for the investigation of cPLA2α dependent and independent effects of C1P. In the course of the study we investigated the ethanol/dodecane delivery system as a means of safely delivering lipids to cells. Our data conclusively demonstrate that this delivery system successfully delivers lipids to the internal membranes where their biological action takes place and that at low lipid concentration (<1µM), is non toxic to cells. A significant technical hurdle in the study of C1P was the lack of accurate and reproducible method of quantitatively and qualitatively analyzing the lipid. Using a mass spectrometric approach we developed an accurate technique that now allows us to quantify the lipids in cells. Using this and radiolabeling studies we discovered evidence for production of C1P from S1P via an acyl transferase pathway. Further studies are currently being carried out to identify the enzyme/s responsible for this pathway.

Using the known sequences for serine palmitoyltransferase (SPT) and ceramide kinase (CERK) from Arabidopsis, candidates for the corresponding genes in Loblolly pine were cloned and examined during embryogenesis. The cloned two cDNA sequences from Loblolly pine, which has similarity of 81% and 88% respectively to two subunits of SPT1 and SPT2 in Arabidopsis, were presumed as the Loblolly pine SPT1 and SPT2 (Pt-SPT1 and Pt-SPT2). A few different versions of Pt-SPT1 mRNAs (2223 nts, 756 nts, 822 nts, and 754 nts respectively), most likely the alternative splicing results, were found. Three of these mRNAs are capable of encoding proteins. The long version (2223 nts) encodes a protein with 484 amino acids (Pt-SPT1); two short versions (822 nts, 756 nts) encode a 90 a.a. protein. Another cDNA sequence of 2396 nts encodes a protein of 493 a.a. (Pt-SPT2). Both predicted Pt-SPT1 and Pt-SPT2 proteins possess highly conserved serine palmitoyltransferase functional domains (E value 5.7e-61). Their expression patterns are different between somatic and zygotic embryogenesis. Two different versions of mRNAs, with 2786 nts (long), and 2320 nts (short) respectively, of ceramide kinases in Loblolly pine (Pt-CERKs) have been cloned. The long version encodes a protein with 721 a.a.; the short version with 560 a.a. The expression patterns for these two CERK mRNAs are different during embryo development. The long version is constitutively expressed, while the short one is only expressed in some stages with much lower expression level. Overexpression Pt-CERKL, Pt-CERKS, and Pt-CERKF in E.coli and function analysis in vitro show that all Pt-CERKs appear to have the same catalytic functions as their homologs in human and Arabidopsis, but with different efficiency. The catalytic efficiency was dramatically lower in the short Pt-CERK protein compared with the long Pt-CERK protein and Pt-CERKF. The membrane system is not necessary for the catalytic reactions of these three Pt-CERKs in vitro and Pt-CERKs were less dependent on the Ca2+ ions. Thus, these studies have provided the first information about SPT- and CERK- like proteins in loblolly pine, and open new avenues of investigation for the roles of sphingolipids in embryonic development.