Discrete subcellular localization of phosphoinositidase C beta, gamma and delta in PC12 rat pheochromocytoma cells.
Phosphoinositidase C activity was revealed in nuclei isolated from PC12 rat pheochromocytoma cells incubated with tritiated phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Phosphoinositide breakdown was found to be optimal at neutral pH and Ca++ concentrations ranging from endogenous levels to millimolar values. To characterize the enzymes involved, three monoclonal antibodies directed against the beta, gamma and delta phosphoinositidase C isoforms were employed. A combination of Western blot immunochemical analysis on cytoplasmic and nuclear fractions and of in situ immunocytochemistry on intact cells and isolated nuclei indicated that phosphoinositidase C gamma, though predominantly cytoplasmic, was present in both cell compartments. On the contrary, phosphoinositidase C beta was exclusively localized in the nucleus, whereas phosphoinositidase C delta was restricted to the cytoplasm. These data suggest that inositol lipid breakdown is controlled by different phosphoinositidase C isozymes in the various cell compartments, and support the notion that a separate phosphoinositide signalling system is located in the nucleus.
Mazzoni
M
M
Instituto di Anatomia Umana Normale, Università di Ferrara, Rome, Italy.
Bertagnolo
V
V
Neri
L M
LM
Carini
C
C
Marchisio
M
M
Milani
D
D
Manzoli
F A
FA
Capitani
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism*; D016716:PC12 Cells / Q000201:enzymology* / Q000648:ultrastructure; D010716:Phosphatidylinositols / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D010716:Phosphatidylinositols; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D002118:Calcium