Phosphorylation of tyrosine hydroxylase in protein kinase C-deficient PC12 cells.
In order to study the role of protein kinase C in the regulation of tyrosine hydroxylase phosphorylation in PC12 cells, the effects of various agonists on diacylglycerol accumulation in PC12 cells were measured and the ability of these agonists to increase the phosphorylation tyrosine hydroxylase in protein kinase C-deficient cells was evaluated. Bradykinin (10 microM) and elevated extracellular K+ (55 mM) increased the accumulation of [3H]diacylglycerol in PC12 cells that had been prelabeled with [3H]arachidonic acid, and so might be expected to activate protein kinase C in these cells; in contrast, nerve growth factor did not increase diacylglycerol accumulation in PC12 cells. Protein kinase C-deficient PC12 cells were prepared by incubating the cells for 24 h with 1 microM phorbol dibutyrate. This treatment resulted in the loss of approximately 90% of the protein kinase C activity in the cells. Control and protein kinase C-deficient cells were incubated with 32Pi for 90 min and then stimulated with various agonists. 32P-labeled tyrosine hydroxylase was isolated from the cells by polyacrylamide gel electrophoresis and subjected to tryptic hydrolysis. 32P-containing phosphopeptides were separated by two-dimensional thin-layer electrophoresis and chromatography, visualized by autoradiography, and quantitated by scintillation counting Treatment of control cells with phorbol dibutyrate increased the incorporation of 32P into one tryptic phosphopeptide (referred to as T3) in tyrosine hydroxylase. Phorbol dibutyrate did not increase the phosphorylation of this peptide in protein kinase C-deficient cells. Bradykinin or 55 mM K+ increased the incorporation of 32P into four tyrosine hydroxylase phosphopeptides, including peptide T3.(ABSTRACT TRUNCATED AT 250 WORDS)
Cahill
A L
AL
Department of Pediatrics, University of Chicago, IL 60637.
Horwitz
J
J
Perlman
R L
RL
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002460:Cell Line; D004075:Diglycerides / Q000378:metabolism; D009414:Nerve Growth Factors / Q000494:pharmacology; D010673:Pheochromocytoma; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D004075:Diglycerides; D009414:Nerve Growth Factors; D015240:Phorbol 12,13-Dibutyrate; D014446:Tyrosine 3-Monooxygenase; D011493:Protein Kinase C; D001920:Bradykinin