Protein kinase C alpha-mediated negative feedback regulation is responsible for the termination of insulin-like growth factor I-induced activation ofnuclear phospholipase C beta 1 in Swiss 3T3 cells

Previous studies from several independent laboratories have demonstrated th e existence of an autonomous phosphoinositide (PI) cycle within the nucleus , where it is involved in both cell proliferation and differentiation. Stim ulation of Swiss 3T3 cells with insulin-like growth factor-I (IGF-I) has be en shown to induce a transient and rapid increase in the activity of nuclea r-localized phospholipase C (PLC) beta1, which in turn leads to the product ion of inositol trisphosphate and diacylglycerol in the nucleus. Nuclear di acylglycerol provides the driving force for the nuclear translocation of pr otein kinase C (PKC) alpha. Here, we report that treatment of Swiss 3T3 cel ls with Go6976, a selective inhibitor of PKC alpha, caused a sustained elev ation of IGF-I-stimulated nuclear PLC activity. A time course study reveale d an inverse relationship between nuclear PKC activity and the activity of nuclear PLC in IGF-I-treated cells. A time-dependent association between PK C alpha and PLC beta1 in the nucleus was also observed following IGF-I trea tment. Two-dimensional phosphopeptide mapping and site-directed mutagenesis demonstrated that PKC promoted phosphorylation of PLC beta1 at serine 887 in the nucleus of IGF-I-treated cells. Overexpression of either a PLC beta1 mutant in which the PKC phosphorylation site Ser(887) was replaced by alan ine, or a dominant-negative PKC alpha, resulted in a sustained activation o f nuclear PLC following IGF-I stimulation. These results indicate that a ne gative feedback regulation of PLC beta1 by PKC a plays a critical role in t he termination of the IGF-I-dependent signal that activates the nuclear PI cycle.