Enhanced nuclear diacylglycerol kinase activity in response to a mitogenicstimulation of quiescent Swiss 3T3 cells with insulin-like growth factor I

Results from several laboratories have established the existence in the nuc leus of an autonomous polyphosphoinositide cycle, which is involved in both cell proliferation and differentiation. A key step of intranuclear polypho sphoinositide metabolism is the phospholipase C-mediated generation of diac ylglycerol (DAG), In insulin-like growth factor (IGF)-I-stimulated Swiss 3T 3 cells, a transient elevation of intranuclear DAG levels is essential for attracting the cw isoform of protein kinase C (PKC) to the nucleus. Previou s evidence has shown that the nucleus also contains DAG kinase, ie., the en zyme that yields phosphatidic acid from DAG, thus terminating PKC-mediated signaling events. Here we show that IGF-I treatment of quiescent Swiss 3T3 cells results in the stimulation of nuclear DAG kinase activity. Time cours e analysis showed an inverse relationship between nuclear DAG mass and DAG kinase activity Levels. After IGF-I treatment, maximal enhancement of DAG k inase activity was measured in the internal matrix domain of the nucleus. P KC-alpha remained within the nuclear compartment, even when nuclear DAG mas s returned to basal levels. This was conceivably due to interactions with s pecific nuclear PKC-binding proteins, some of which were identified as lami ns A, B, and C and protein C23/nucleolin. Treatment of cells with two DAG k inase inhibitors, R59022 and R59949, blocked the IGF-I-dependent rise in nu clear DAG kinase activity and maintained elevated intranuclear levels of DA G, The two inhibitors also markedly potentiated the mitogenic effect of IGF -I, These results suggest that nuclear DAG kinase plays a key role in regul ating the levels of DAG present in the nucleus and that DAG is a key molecu le for the mitogenic effect that IGF-I exerts on Swiss 3T3 cells.