Protein Kinase C α-mediated Negative Feedback Regulation Is Responsible for the Termination of Insulin-like Growth Factor I-induced Activation of Nuclear Phospholipase C β1 in Swiss 3T3 Cells

Abstract

Previous studies from several independent laboratories have demonstrated the existence of an autonomous phosphoinositide (PI) cycle within the nucleus, where it is involved in both cell proliferation and differentiation. Stimulation of Swiss 3T3 cells with insulin-like growth factor-I (IGF-I) has been shown to induce a transient and rapid increase in the activity of nuclear-localized phospholipase C (PLC) β1, which in turn leads to the production of inositol trisphosphate and diacylglycerol in the nucleus. Nuclear diacylglycerol provides the driving force for the nuclear translocation of protein kinase C (PKC) α. Here, we report that treatment of Swiss 3T3 cells with Go6976, a selective inhibitor of PKC α, caused a sustained elevation of IGF-I-stimulated nuclear PLC activity. A time course study revealed an inverse relationship between nuclear PKC activity and the activity of nuclear PLC in IGF-1-treated cells. A time-dependent association between PKC α and PLC β1 in the nucleus was also observed following IGF-I treatment. Two-dimensional phosphopeptide mapping and site-directed mutagenesis demonstrated that PKC promoted phosphorylation of PLC β1 at serine 887 in the nucleus of IGF-I-treated cells. Overexpression of either a PLC β1 mutant in which the PKC phosphorylation site Ser887 was replaced by alanine, or a dominant-negative PKC α, resulted in a sustained activation of nuclear PLC following IGF-I stimulation. These results indicate that a negative feedback regulation of PLC β1 by PKC α plays a critical role in the termination of the IGF-I-dependent signal that activates the nuclear PI cycle.