Insulin selectively stimulates nuclear phosphoinositide-specific phospholipase C (PI-PLC) beta1 activity through a mitogen-activated protein (MAP) kinase-dependent serine phosphorylation.
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 beta family of PI-PLC (i.e. beta1, beta2, beta3, and beta4), insulin only activated the beta1 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-beta1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-beta1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-beta1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.
Martelli
A M
AM
Cellular Signalling Laboratory, Department of Anatomical Sciences, University of Bologna, Italy.
Billi
A M
AM
Manzoli
L
L
Faenza
I
I
Aluigi
M
M
Falconi
M
M
De Pol
A
A
Gilmour
R S
RS
Cocco
L
L
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006133:Growth Substances / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000276:immunology / Q000378:metabolism; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D010738:Type C Phospholipases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004075:Diglycerides; D004791:Enzyme Inhibitors; D005419:Flavonoids; D006133:Growth Substances; D007328:Insulin; D007527:Isoenzymes; D010768:Phosphoserine; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one