Diacylglycerol kinase-theta is localized in the speckle domains of the nucleus

It is well established that the nucleus is endowed with enzymes that are involved in lipid-dependent signal transduction pathways. Diacylglycerol (DAG) is a fundamental lipid second messenger that is produced in the nucleus. Previous reports have shown that the nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting DAG into phosphatidic acid (PA), terminate DAG-dependent events. Here, we show, by immunofluorescence staining and confocal analysis, that DGK-theta localizes mainly to the nucleus of various cell lines, such as MDA-MB-453, MCF-7, PC12, and HeLa. Nuclear DGK-theta co-localizes with phosphatidylinositol 4,5-bisphosphate (PIP2) in domains that correspond to nuclear speckles, as revealed by the use of an antibody to the splicing factor SC-35, a well-established marker for these structures. The spatial distribution of nuclear DGK-theta was dynamic in that it was affected by inhibition of mRNA transcription with alpha-amanitin. Immuno-electron microscopy analysis demonstrated that DGK-theta, PIP2, and phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1) associated with electron-dense particles within the nucleus that correspond to interchromatin granule clusters. Cell fractionation experiments performed in MDA-MB-453, HeLa, and PC12 cells showed a preferential association of DGK-theta with the nucleus. Western blots demonstrated that DGK-theta was enriched in the nuclear matrix fraction prepared from MDA-MB-453 cells. Immunoprecipitation experiments with an antibody to PLCbeta1 revealed in MDA-MB-453 cells an association between this enzyme and both DGK-theta and phosphatidylinositol phosphate kinase Ialpha (PIPKIalpha). Our findings strengthen the contention that speckles represent a crucial site for the nuclear-based inositol lipid cycle. We may speculate that nuclear speckle-located DGK-theta, on cell stimulation with an agonist, converts to PA the DAG derived from PLCbeta1-dependent PIP2 hydrolysis. (C) 2003 Elsevier Science (USA). All rights reserved.