Phosphatidylcholine-specific phospholipase C in mitogen-stimulated fibroblasts.
To investigate expression, subcellular localization and mechanisms of translocation of phosphatidylcholine-specific phospholipase C (PC-PLC) during the cell proliferative response, biochemical, immunoblotting, and immunofluorescence analyses were performed on quiescent and mitogen-stimulated NIH-3T3 fibroblasts. Platelet-derived growth factor (PDGF), insulin and 12-O-tetradecanoylphorbol-13-acetate induced, in 10-60 min, PC-PLC translocation from a perinuclear cytoplasmic area to the plasma membrane. Following cell exposure to PDGF (60 min), the overall PC-PLC expression increased up to 2-3x, while the enzyme activity increased 5x in total cell lysates, 2x in the plasma membrane, and 4x in the nucleus; moreover, confocal laser scanning microscopy showed a progressive externalization of PC-PLC on the outer plasma membrane surface and its accumulation in the nuclear matrix. Pre-incubation of cells with the PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609), before PDGF-stimulation, not only reduced the enzyme activity in total cell lysates as well as in plasma membrane and nuclear fractions, but also blocked the mechanisms of PC-PLC subcellular redistribution. These effects were associated with a D609-induced long-lasting cell cycle block in Go.
Ramoni
Carlo
C
Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.
Spadaro
Francesca
F
Barletta
Bianca
B
Dupuis
Maria Luisa
ML
Podo
Franca
F
D016475:3T3 Cells; D000818:Animals; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology*; D007328:Insulin / Q000378:metabolism; D007834:Lasers; D051379:Mice; D018613:Microscopy, Confocal; D008934:Mitogens / Q000494:pharmacology*; D009636:Norbornanes; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D016192:Resting Phase, Cell Cycle / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D013859:Thiocarbamates; D013871:Thiones / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
D001952:Bridged-Ring Compounds; D007328:Insulin; D008934:Mitogens; D009636:Norbornanes; D010982:Platelet-Derived Growth Factor; D013859:Thiocarbamates; D013871:Thiones; C046498:tricyclodecane-9-yl-xanthogenate; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C; D013755:Tetradecanoylphorbol Acetate