Characterization of Cellular DGK-θ

In this report we have summarized the data regarding the regulation of DGK-θ by two phospholipids: PtdSer and PtdOH. Our previous data has shown that stimulation of quiescent fibroblasts with a potent mitogen (α-thrombin) leads to an increase in nuclear localized DGK-θ (Bregoli, L., Baldassare, J. J., and Raben, D. M., 2001; Bregoli, L., Tu-Sekine, B., and Raben, D. M., 2002), as has been seen in neuronal cells (Tabellini, G., Bortul, R., Santi, S., Riccio, M., Baldini, G., Cappellini, A.et al., 2003; Tabellini, G., Billi, A. M., Fala, F., Cappellini, A., Evagelisti, C., Manzoli, L.et al., 2004). Furthermore, these previous studies demonstrated that DGK-θ is actually localized to the nuclear matrix (Bregoli, L., Tu-Sekine, B., and Raben, D. M., 2002; Tabellini, G., Bortul, R., Santi, S., Riccio, M., Baldini, G., Cappellini, A.et al., 2003). As we have also previously shown that PtdSer and PtdOH modulate DGK-θ activity (Tu-Sekine, B., Ostroski, M., and Raben, D. M., 2006; Tu-Sekine, B., Ostroski, M., and Raben, D. M., 2007), we examined the phospholipid composition of the nuclear matrix as well as the phospholipid composition of the intact nuclei and non-nuclear membranes. This analysis has revealed that there are phospholipids in the “matrix” of nuclei and the composition of these lipids largely resembles those of the other membranes. The physical form and localization of the matrix-associated lipids has not been established, though the nearly identical percent composition of the non-nuclear membranes and the nuclear matrix lend credence to the hypothesis that at least some of the internal nuclear lipid is derived from invaginations of the cellular membranes through the nuclear interior known as nuclear tubules (Fricker, M., Hollinshead, M., White, N., and Vaux, D., 1997; Lee, R. K., Lui, P. P., Ngan, E. K., Lui, J. C., Suen, Y. K., Chan, F.et al., 2006). An important finding resulting from this analysis is that the nuclear envelope is enriched in PtdSer relative to the nuclear matrix and to cellular membranes, and that over-expression of DGK-θ reduces both the PtdSer and PtdEth levels of the nuclear envelope. While the mechanism behind these fluctuations is unknown, the data are consistent with a DGK-regulated phosphatidylcholine-dependent phosphatidylserine synthase (PSS-1) (EC 2.7.8.8) activity at the nuclear envelope. PtdSer synthesis in mammalian cells proceeds by headgroup exchange with PtdCho or PtdEth, catalyzed by PSS-1 or PSS-2, respectively. Interestingly, the decrease in both PtdSer and PtdEth at the nuclear membrane mirrors effects observed in the CHO-K1 mutant cell lines M.6.1.1 and PSA-3, which have been shown to be deficient in PSS-1 activity (Vance, J. E. and Steenbergen, R., 2005). While there is very little information on the regulation of mammalian PSS enzymes, phosphorylation has been shown to regulate serine exchange activities in rat brain (Kanfer, J. N., McCartney, D., and Hattori, H., 1988), and we have shown that nuclear DGK-θ regulates the exit of PKC-α from the nucleus (see Regulation of PKC-α). To our knowledge, there is currently no reported study of nuclear phosphatidylyserine synthases. One other notable fluctuation in cellular lipids from DGK-θ expression is an increase in plasmalogen PtdEth in both the NNM and nuclear matrix. While the majority of studies conducted on plasmalogen synthesis agree in that there is an almost complete dependence on the classical CDP-ethanolamine pathway to provide the headgroup for precursor plasmalogen synthesized in the peroxisome, there are several studies utilizing radiolabeled serine that report approximately 20-30% of cellular plasmalogen-PtdEth can be derived from PtdSer in at least some cell models (Yorek, M. A., Rosario, R. T., Dudley, D. T., and Spector, A. A., 1985; Xu, Z. L., Byers, D. M., Palmer, F. B., Spence, M. W., and Cook, H. W., 1991). Whether nuclear PtdSer-derived PtdEth a precursor for the membrane plasmalogen-PtdEth present in DGK-θ expressing cells is not evident from the data presented here, though one potential mechanism for an increase in flux through this pathway could be an increase in PtdSer-derived PtdEth, At this juncture we can only speculate on the cause of the lipid perturbations, and further work is required to support our hypothesis that DGK-θ impacts PtdSer and plasmalogen metabolism. The physiological role for nuclear DGK-θ has remained a mystery. It has long been recognized that an obvious role for DGKs is to modulate cellular levels of its DAG substrate thereby modulating DAG-sensitive proteins like such as many PKCs (Merida, I., Avila-Flores, A., and Merino, E., 2008). Our data support this notion as suppression of DGK-θ, either by expression of RhoA or a dominant-negative construct of DGK-θ, leads to a sustained increase in nuclear PKC-α (Figure 2). In addition to the regulation by PtdSer and PtdOH, we report here that the regulation of nuclear DGK-θ by RhoA is modulated by a Class I PI 3-kinase. Furthermore, we have examined the effect of various inhibitors on the activity of DGK-θ in lysates over-expressing this isoform. This is important as many studies often use pharmacologic inhibitors to determine the role of selected enzymes in signaling pathways. While this approach is useful for comparative analyses, there are no data pertaining to the effect of these inhibitors on DGK-θ. We have found that one well-established PI-PLC inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, but not its inactive analog {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343, competitively inhibits this isoform with respect to its diacylglycerol (DAG) substrate. These data indicate that studies designed to examine the role of PI-PLC in modulating DGK-θ activity using this inhibitor should be interpreted with caution. Major Topics Diacylglycerol kinase DGKtheta Lipids Enzymology Diacylglycerol Phosphatidylserin Phophatidic acid Signal Transduction Kinases Enzymes Mixed Micelles Competitive Inhibition PI 3-Kinase DGK-θ RhoA ​ TABLE 1 Phospholipid composition of non-nuclear membranes, nuclear envelope and nuclear matrix of embryonic fibroblasts Acknowledgements This work was supported by Grant GM059251 from the National Institutes of Health (D.M.R.).