Nerve Growth Factor‐Induced Transient Increase in the Phosphorylation of Ribosomal Protein S6 Mediated Through a Mechanism Independent of Cyclic AMP‐Dependent Protein Kinase and Protein Kinase C

Abstract

Treatment of PC12h cells with nerve growth factor (NGF) induced a transient increase in the phosphorylation of a 35,000-dalton protein. This transient increase was observed also when extracts of NGF-treated cells were incubated with [gamma-32P]ATP. In the intact-cell phosphorylation system, treatment with N,2'-dibutyryladenosine 3',5'-cyclic monophosphate (dBcAMP) or 12-O-tetradecanoylphorbol 13-acetate (TPA) also induced a transient increase in the phosphorylation of the 35,000-dalton protein, but the effect was less than that of NGF. An effect comparable to that of NGF was obtained by the combination of dBcAMP and TPA. Pretreatment of PC12h cells with dBcAMP plus TPA for 3 days, which deprived the cells of their ability to respond to a rechallenge with dBcAMP, TPA, or dBcAMP plus TPA by increasing the rate of 35,000-dalton protein phosphorylation, caused only a slight attenuation of the NGF effect, directly indicating a minimal role of cyclic AMP (cAMP)-dependent protein kinase and protein kinase C in the mechanism of the NGF action. Pretreatment of the cells with K-252a, a protein kinase inhibitor, at a concentration of 300 nM almost completely blocked the action of NGF, but scarcely affected the action of dBcAMP, TPA, or dBcAMP plus TPA in intact-cell phosphorylation experiments. This NGF-sensitive 35,000-dalton protein was a ribosomal protein and identified as ribosomal protein S6. The results lead us to conclude that NGF activates some NGF-sensitive component(s), probably some specific protein kinase(s) other than cAMP-dependent protein kinase or protein kinase C, which is suppressed by K-252a and directly or indirectly activates a 35,000-dalton protein kinase(s) [S6 kinase(s)] to increase the rate of phosphorylation of the 35,000-dalton ribosomal protein (S6).

Role of Protein Kinase C α in Nerve Growth Factor‐Induced Arachidonic Acid Release from PC12 Cells

“…These data lend biochemical support to the previous finding that the tumor promoter PMA enhances the action of low concentrations of NGF on the formation of neurites in these cells (Burstein et a!., 1982). In contrast, PMA down-regulation has no effect on the NGF-associated stimulations of S6 kinase activity or ornithine decarboxylase activity (Reinhold and Neet, 1989;Hashimoto and Hagino, 1990), and may even increase FIG. 6.…”

Growth factors protect PC12 cells against ischemia by a mechanism that is independent of PKA, PKC, and protein synthesis

“…In addition to stimulating differentiation, NGF or bFGF promotes survival of PC 12 cells in serum-free medium, and the activation of intracellular protein kinases plays a key role in this process (Greene, 1978;Rukenstein et al, 199 1). NGF has been shown to activate several protein kinases, including its cell surface receptor (Klein et al,199 1;Meakin and Shooter,199 l), N-kinase (Volonte et al, 1989), the S6 kinases (Blenis and Erikson, 1986;Hashimoto and Hagino, 1990) a proline-directed kinase (Vulliet et al, 1989) MAP2/ERK kinases (Miyasaka et al, 1990;Boulton et al, 1991), and PKC (Hama et al, 1986;Heasley and Johnson, 1989). Activators of protein kinase A (PKA, i.e., the CAMP-dependent protein kinase) have been shown to mimic some of NGF's effects, including protection against serum deprivation.…”

Involvement of protein kinase C in nerve growth factor- and K-252a-stimulated calcium uptake into PC12 cells

“…C, control; N, NGF; S, staurosporine; CC, calphostin C. Lloyd and Wooten, 1992). Clearly, however, kinase C does not mediate all the actions of NGF; PMA downregulation of kinase C does not interfere with the ability of NGF to induce ornithine decarboxylase (Reinhold and Neet, 1989) or increase S6 kinase activity (Hashimoto and Hagino, 1990), and NGF-induced neurite outgrowth, itself, is not inhibited (Burstein et al, 1982;Reinhold and Neet, 1989;O'Driscoll et al, 1995). Nevertheless, the evidence that NGF activates protein kinase C, which, in turn, mediates some of the actions of NGF, is quite persuasive.…”