Protein kinase C regulation of IEC-6 cell ornithine decarboxylase.
Experiments were performed to immunologically identify protein kinase C (PKC) in cultured IEC-6 cells. Polyclonal antibodies specific to PKC revealed an immunoreactive band of approximately 84 kDa in both cytosolic and solubilized particulate fractions. Treatment with phorbol 12-myristate 13-acetate (PMA; 10 nM x 60 min) increased the intensity of the 84-kDa band by 25% in the solubilized particulate fraction while decreasing it by 36% in the cytosolic fraction. Prolonged 24-h treatment with 300 nM PMA completely abolished the 84-kDa band in both fractions. Isoform-specific antisera demonstrated that alpha- and epsilon-isoforms of PKC were expressed in IEC-6 cells. Treatment of quiescent cultures with PMA induced a maximal 400% increase in ornithine decarboxylase (ODC) activity. Similarly, addition of exogenous phospholipase C (PLC) to quiescent cells stimulated ODC activity. Downregulation of PKC with 300 nM PMA x 24 h inhibited basal, serum, and PLC-stimulated ODC activity by 70%. Northern analysis revealed that PKC downregulation was correlated with a marked reduction in ODC mRNA levels, suggesting regulation of ODC enzyme at this level. Despite their ability to modulate ODC activity in quiescent cultures, neither PMA nor PLC induced [3H]thymidine incorporation at 24 h. Furthermore, downregulation of PKC did not attenuate thymidine incorporation. However, chronic PMA treatment caused the cells to contact-inhibit at a 30% lower cell density, 3.16 x 10(6) vs. 2.1 x 10(6) cells/35-mm plate, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Groblewski
G E
GE
Department of Physiology, School of Medicine, East Carolina University, Greenville, North Carolina 27858.
Ways
D K
DK
Seidel
E R
ER
D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004386:Duodenum / Q000166:cytology / Q000201:enzymology*; D004790:Enzyme Induction / Q000187:drug effects; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D012333:RNA, Messenger; D011493:Protein Kinase C; D009955:Ornithine Decarboxylase; D013755:Tetradecanoylphorbol Acetate