Insulin selectively stimulates nuclear phosphoinositide‐specific phospholipase C (PI‐PLC) β1 activity through a mitogen‐activated protein (MAP) kinase‐dependent serine phosphorylation

Abstract

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen-activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide-specific phospholipase C (PI-PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the L L family of PI-, and L L4), insulin only activated the L L1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [32 P]orthophosphate, we ascertained that nuclear PI-PLC-L L1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-L L1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-L L1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway. ß

Increase of Nuclear Ceramide through Caspase-3-Dependent Regulation of the “Sphingomyelin Cycle” in Fas-Induced Apoptosis

“…Among the bioactive lipids, DAG is recognized as a counterpart molecule of proapoptotic ceramide because of the similarity of its chemical structure and antiapoptotic action (50). DAG is increased in the nucleus through activation of phosphatidylcholine-phospholipase C by treatment with insulin or tumor promoter 12-O-tetradecanoylphorbol-13-acetate (51,52), and phosphoinositide hydrolysis and DAG production were increased by platelet-activating factor in isolated rat liver nuclei (53). Thus, after hydrolysis of phosphatidylcholine or phosphoinositide in the nucleus, a generated DAG may mediate cell growth and survival by activating antiapoptotic molecules, such as protein kinase C, phosphoinositide 3 kinase/Akt kinase, and telomerase (52, 54 -57).…”

Possible Roles of Nuclear Lipids in Liver Regeneration

“…This could be due to a different enzyme activity such as that of phosphatidylinositol-dependent phospholipase C since various enzyme isoforms exist that may be activated by different stimuli (Martelli et al 2000, Santi et al 2001). …”

Nuclear phospholipid signaling: phosphatidylinositol-specific phospholipase C and phosphoinositide 3-kinase

“…the one operating at the cell membrane is G-protein-regulated but there are no data confirming any role of the nuclear G protein in the activation of the nuclear enzyme. However, several studies suggested the involvement and nuclear translocation of p42/p44 mitogen-activated protein kinase (MAPK) in agonist-mediated activation of the nuclear PI-PLC-β (47,48). In a detailed study, Although the Ser982 phosphorylation was prerequisite for the PI-PLC activation, as shown in mutants carrying Ser982Gly, it was not sufficient alone, and several other components of mechanism involved in the activation of the nuclear PI-PLC still remain to be determined (Fig.…”

“…The majority of PI-PLC-β 1 is probably not located in the nuclear envelope as the activity of PI-PLC-β 1 is present in both whole nuclei (21,40) and nuclei treated with different concentrations of detergents in order to remove the nuclear membrane (21,25,48,68,75,77). In rat liver nuclei, fractionation experiments indicated that both PI-PLC-β 1 and PI-PLC-γ 1 persisted in nuclear matrix and lamina, obtained after nuclease digestion and extraction with high salt and detergent (6).…”

“…Acinus is one of the recently identified targets of the activated nuclear Akt. (77,47) Increased (PLC assay) (77,47)* MAPK-mediated phosphorylation at Ser982 (77) No change (77,47) Immunoprecipitated with phospho-MAPK (77) IGF, HL-60 (56) Increased (PLC assay) (56) Increased DAG (56) No change (56) ND (56) −β 1β Insulin, NIH 3T3 (48) Increased (PLC assay) (48) MEK inhibitor-sensitive serine phosphorylation (48) No change (48) ND (48) IL-2, NK cells (75) Increased (PLC assay) (75) Increased DAG (75) MEK inhibitor-sensitive serine phosphorylation (75) No change (75) ND (75) FBS, HL-60 (42) Increased (PLC assay) (42) MEK inhibitor-sensitive serine phosphorylation (42) No change (42) ND (42) Hepatectomy, liver (16) Increased (PLC assay) (16) Serine phosphorylation (16) No change (16) ND (16) late G 1 /S FBS, HL-60 (42) Nocodazole, HL-60 (40) Increased (PLC assay) (40,42) MEK inhibitor-sensitive serine phosphorylation (40,42) No change (40,42) ND (40,…”