Nuclear diacylglycerol kinase-theta is activated in response to alpha-thrombin

Currently, there is substantial evidence that nuclear lipid metabolism play s a critical role in a number of signal transduction cascades. Previous wor k from our laboratory showed that stimulation of quiescent fibroblasts with Lu-thrombin leads to the production of two lipid second messengers in the nucleus: an increase in nuclear diacylglycerol mass and an activation of ph ospholipase D, which catalyzes the hydrolysis of phosphatidylcholine to gen erate phosphatidic acid. Diacylglycerol kinase (DGK) catalyzes the conversi on of diacylglycerol to phosphatidic acid, making it an attractive candidat e for a signal transduction component. There is substantial evidence that t his activity is indeed regulated in a number of signaling cascades (reviewe d by van Blitterswijk, W.J., and Houssa, B. (1999) Chem. Phys. Lipids 98, 9 5-108). In this report, we show that the addition of cr-thrombin to quiesce nt IIC9 fibroblasts results in an increase in nuclear DGK activity. The exa mination of nuclei isolated from quiescent IIC9 cells indicates that DGK-th eta and DGK-delta are both present. We took advantage of the previous obser vations that phosphatidylserine inhibits DGK-delta (reviewed by Sakane, F., Imai, S., Kai, M., Wada, I., and Kanoh, H. (1996) J. Biol. Chem. 271, 8394 -8401), and constitutively active RhoA inhibits DGK-theta (reviewed by Hous sa, B., de Widt, J., Kranenburg, O., Moolenaar, W. H., and van Blitterswijk , W. J. (1999) J. Biol Chem. 274, 6820-6822) to identify the activity induc ed by Lv-thrombin. Constitutively active RhoA inhibited the nuclear stimula ted activity, whereas phosphatidylserine did not have an inhibitory effect. In addition, a monoclonal anti-DGK-theta antibody inhibited the alpha -thr ombin-stimulated nuclear activity in vitro. These results demonstrate that DGK-theta is the isoform responsive to alpha -thrombin stimulation. Western blot and immunofluorescence microscopy analyses showed that cr-thrombin in duced the translocation of DGK-theta to the nucleus, implicating that this translocation is at least partly responsible for the increased nuclear acti vity. Taken together, these data are the first to demonstrate an agonist-in duced activity of nuclear DGK-theta activity and a nuclear localization of DGK-delta.