Delta-protein kinase C phosphorylation parallels inhibition of nerve growth factor-induced differentiation independent of changes in Trk A and MAP kinase signalling in PC12 cells.
We investigated the ability of bryostatin 1 to block nerve growth factor (NGF)-induced differentiation of pheochromocytoma PC12 cells and to effect expression of protein kinase C (PKC) isoforms. Compared with phorbol myristate acetate (PMA), a likewise potent activator of PKC, high doses of bryostatin (> 200 nM) failed to down-regulate delta-PKC, as with zeta-PKC, whereas, alpha-PKC was completely down-regulated. Two forms of delta-PKC were expressed in PC12 cells, a phosphorylated 78.000 M(r) species and a de-phosphorylated 76.000 M(r) form. High-dose bryostatin treatment resulted in a 4.5-fold increase in phosphorylated delta-PKC and a 2.5-fold increase in phosphotyrosine. Inhibition of tyrosine kinase activity, with either herbimycin or genistein, prior to addition of bryostatin abrogated protection from down-regulation and led to simultaneous increases in ubiquitinated 110.000 M(r)-delta-PKC. Similarly, pre-treatment of cells with N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal, an inhibitor of the proteasome pathway, prior to low-dose treatment with bryostatin resulted in a dose-dependent accumulation of delta-PKC and inhibition of down-regulation. Protection of delta-PKC from down-regulation by high-dose bryostatin requires a counter-balance between protein tyrosine kinase and phosphatase systems. High doses of bryostatin blocked NGF-induced neurite outgrowth without altering Y-490 TrK A phosphorylation or an alteration in pp44/42 mitogen-activated protein kinase. Our findings suggest that the phosphorylation state of delta-PKC may regulate its ability to participate in signal coupling and modulation of cell growth and differentiation pathways. Moreover, these data reveal the existence of a signalling pathway independent of MAP kinase that affects NGF differentiation in a negative fashion.
Wooten
M W
MW
Department of Zoology, Auburn University, AL 36849. mwwooten@acesag.auburn.edu
Seibenhener
M L
ML
Heikkila
J E
JE
Mischak
H
H
D000818:Animals; D054713:Bryostatins; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D009414:Nerve Growth Factors / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051745:Protein Kinase C-delta; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000502:physiology*; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D014443:Tyrosine / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology
D054713:Bryostatins; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D009414:Nerve Growth Factors; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D014475:Uncoupling Agents; C046785:bryostatin 1; D014443:Tyrosine; C497260:Prkcd protein, rat; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D017871:Calcium-Calmodulin-Dependent Protein Kinases