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) ␤1, 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) ␣. Here, we report that treatment of Swiss 3T3 cells with Go6976, a selective inhibitor of PKC ␣, 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-I-treated cells. A time-dependent association between PKC ␣ and PLC ␤1 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 ␤1 at serine 887 in the nucleus of IGF-I-treated cells. Overexpression of either a PLC ␤1 mutant in which the PKC phosphorylation site Ser 887 was replaced by alanine, or a dominant-negative PKC ␣, resulted in a sustained activation of nuclear PLC following IGF-I stimulation. These results indicate that a negative feedback regulation of PLC ␤1 by PKC ␣ plays a critical role in the termination of the IGF-I-dependent signal that activates the nuclear PI cycle.Phospholipase C ␤ isoforms (␤1, ␤2, ␤3 and ␤4) at the plasma membrane are regulated by G protein-coupled seven-transmembrane receptors which activate heterotrimeric G␣␤␥ protein complexes upon ligand stimulation (1-3). The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 by PLC ␤s generates two well recognized second messengers, inositol 1,4,5-trisphosphate and DG. Inositol 1,4,5-trisphosphate evokes release of Ca 2ϩ from intracellular stores, while DG alone, or in concert with Ca 2ϩ , activates some isoforms of PKC (4). Mounting evidence suggests that an analogous phosphoinositide (PI) signaling, which is distinct from classic PI signaling at plasma membrane, exists in the nucleus (5-7). The presence of such a nuclear PI cycle has been demonstrated in several different cell lines (8 -11, 12), and has been shown to be important for both cell proliferation (13) and differentiation (14).The key enzyme for the initiation of nuclear PI signaling is phospholipase C (PLC) ␤1, which is the major isoform present in the nucleus of 3T3 cells (15) as well as other cell lines (16 -19). It exists as two alternatively spliced subtypes, PLC ␤1a (150 kDa) and PLC ␤1b (140 kDa) which differ only in a short region of their carboxyl termini (20). Recent studies suggest that the ␤1b form predominantly localizes in the nucleus while the ␤1a form distributes equally between the nucleus and plasma membrane (21). A nuclear locali...

K562 cell proliferation is modulated by PLCβ1 through a PKCα-mediated pathway

“…12,13 Indeed, we demonstrated that upregulation of both the isoforms of PLCβ1 correlated with the activation of G 1 -specific cyclin D3/cdk4 complex, leading cells to an increased progression through G 1 phase. 11 Further evidence about PLCβ1 involvement in cell cycle control was reported again in FELC; in fact, it has been shown that PLCβ1 was involved in lamin B1 phosphorylation and G 2 /M progression via PKCα activation in the nucleus. 12 The direct target of PLC signaling is represented by protein kinases C (PKC), which are activated by the second messengers DAG and IP3.…”

“…strengthened by the finding that its overexpression was sufficient to drive Swiss 3T3 in S phase of the cell cycle. 11 Subsequent studies on Friend murine erythroleukemia cells (FELC) showed a role for PLCβ1 in cell cycle control in both G 0 -G 1 /S transition and G 2 /M progression. 12,13 Indeed, we demonstrated that upregulation of both the isoforms of PLCβ1 correlated with the activation of G 1 -specific cyclin D3/cdk4 complex, leading cells to an increased progression through G 1 phase.…”

Testosterone Selectively Reduces the High Molecular Weight Form of Adiponectin by Inhibiting Its Secretion from Adipocytes

“…To this end, rat adipocytes were pulse-labeled with [ 35 S]methionine and [ 35 S]cysteine for 2 h and then chased in a "cold" medium for different time periods. Cell lysates and culture medium were collected and subjected to immunoprecipitation using the anti-mouse adiponectin antibody as we previously described (28). A non-heating and non-reducing SDS-PAGE analysis revealed that all the three oligomeric complexes of adiponectin were immunoprecipitated by the rabbit anti adiponectin IgG (Fig.…”

Novel Mechanisms for Feedback Regulation of Phospholipase C‐β Activity

“…PKC confers negative feedback regulation of IGF-1 signaling, terminating activation of nuclear PLC-β 1 by IGF-1 (43). Overexpression of either a PLC-β 1 mutant, in which the Ser 887 was replaced by Ala or a dominant negative PKCα, resulted in a sustained activation of nuclear PLC-β 1 following IGF-1 stimulation.…”

“…GPCR stimulation of PLC-β generates diacylglycerol, which activates PKC. PKC phosphorylates PLC-β 1 in vivo and in vitro at Ser 887 (18,42,43). Both the convention and novel PKC isoforms phosphorylate PLC-β 1 .…”