δ-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

Abstract

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 δ-PKC, as with ζ-PKC, whereas, α-PKC was completely down-regulated. Two forms of δ-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 δ-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 -δ-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 δ-PKC and inhibition of down-regulation. Protection of δ-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 δ-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.