Overexpressed protein kinase C-delta and -epsilon subtypes in NIH 3T3 cells exhibit differential subcellular localization and differential regulation of sodium-dependent phosphate uptake.
To examine the biological properties of protein kinase C (PKC)-delta and -epsilon NIH 3T3 cells were stably transfected with metallothionein-based expression vectors that overexpressed these isoforms. In addition to their inducibility by Zn2+, the protein levels of these two PKC subtypes, but not that of endogenous PKC-alpha, increased with increasing cell density. An unexpected role for Mg2+ in the subcellular localization of PKC-delta was found. This isoenzyme was predominantly membrane-associated when cell fractionation was carried out in the absence of Mg2+ but cytosolic when the fractionation was performed in the presence of 10 mM Mg2+. In contrast, the predominant localization of cytosolic PKC-alpha and of membrane-associated PKC-epsilon was not influenced by Mg2+. In vivo and in vitro studies of [3H]phorbol 12,13-dibutyrate binding in the overexpressing cell lines confirmed the cytosolic localization of PKC-alpha, the membrane-associated state of PKC-epsilon, and the presence of PKC-delta at both locations. Readdition of serum for 5 min to serum-starved, quiescent cell lines initiated the redistribution of PKC-alpha to the particulate fraction, while the location of PKC-delta and PKC-epsilon was not affected. Zn(2+)-induced overexpression of PKC-delta- and PKC-epsilon-stimulated sodium-dependent phosphate uptake. Overexpression of PKC-delta caused an increase in the Vmax of Na+/P(i) uptake, while overexpression of PKC-epsilon resulted in a decrease in Km for orthophosphate. A further stimulation of Na+/P(i) uptake in the overexpressing cells could be achieved by phorbol ester activation of endogenous PKC-alpha. These results suggest that each of the three PKC isotypes contribute to the regulation of sodium-dependent phosphate uptake, but through distinct mechanisms.
Lehel
C
C
Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Olah
Z
Z
Mischak
H
H
Mushinski
J F
JF
Anderson
W B
WB
D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D015870:Gene Expression / Q000187:drug effects; D007527:Isoenzymes / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology*; D013347:Subcellular Fractions / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D015032:Zinc / Q000494:pharmacology
D007527:Isoenzymes; D010710:Phosphates; D015240:Phorbol 12,13-Dibutyrate; D012964:Sodium; D011493:Protein Kinase C; D015032:Zinc; D013755:Tetradecanoylphorbol Acetate