Induction of ornithine decarboxylase by nerve growth factor in PC12 cells: dissection by purine analogues.

Purine analogues were used in this study to dissect specific steps in the mechanism of action of nerve growth factor (NGF). Protein kinase N (PKN) is an NGF-activated serine protein kinase that is active in the presence of Mn++. The activity of PKN was inhibited in vitro by purine analogues, the most effective of which was 6-thioguanine (apparent Ki = 6 microM). Several different criteria indicated that 6-thioguanine is not a general inhibitor of protein kinases and that it is relatively specific for PKN. For instance, it did not affect protein kinases A or C and was without effect on the overall level and pattern of protein phosphorylation by either intact or broken PC12 cells. Since purine analogues rapidly and effectively enter cells, they were also assessed for their actions on both transcription-dependent and -independent responses of PC12 cells to NGF. NGF-promoted neurite regeneration was reversibly suppressed by the analogues and at concentrations very similar to those that inhibit PKN. Comparable concentrations of the analogues also blocked NGF-stimulated induction of ornithine decarboxylase activity. In contrast to its inhibition of neurite regeneration and ornithine decarboxylase induction, 6-thioguanine did not suppress NGF-dependent induction of c-fos mRNA expression. Thus, purine analogues such as 6-thioguanine appear capable of differentially suppressing some, but not other actions of NGF. These findings suggest the presence of multiple pathways in the NGF mechanism and that these can be dissected with purine analogues. Moreover, these data are compatible with a role for protein kinase N in certain of these pathways.

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The Lack of a Role for Protein Kinase C in Neurite Extension and in the Induction of Ornithine Decarboxylase by Nerve Growth Factor in PC12 Cells

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)94099-8 ◽

1989 ◽

Vol 264 (6) ◽

pp. 3538-3544 ◽

Cited By ~ 1

Author(s):

D S Reinhold ◽

K E Neet

Keyword(s):

Protein Kinase C ◽

Nerve Growth Factor ◽

Growth Factor ◽

Protein Kinase ◽

Pc12 Cells ◽

Ornithine Decarboxylase ◽

Nerve Growth ◽

Neurite Extension ◽

Kinase C

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Nerve growth factor employs multiple pathways to induce primary response genes in PC12 cells.

Molecular Biology of the Cell ◽

10.1091/mbc.3.3.363 ◽

1992 ◽

Vol 3 (3) ◽

pp. 363-371 ◽

Cited By ~ 44

Author(s):

A Batistatou ◽

C Volonté ◽

L A Greene

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Pc12 Cells ◽

Primary Response ◽

Purine Analogues ◽

Signaling Mechanism ◽

Nerve Growth ◽

Primary Response Genes ◽

Run Off ◽

Ngf Signaling

Nerve growth factor (NGF) leads to neuronal differentiation of PC12 cells and promotes their survival in serum-free medium. Past studies have shown that purine analogues block some of the effects of NGF but not others and thus that they can be used to dissect the mechanistic pathways of its action. In the present work we used 2-aminopurine (2-AP) and 6-thioguanine (6-TG) to examine whether NGF causes activation of primary response genes through a single signaling pathway or via multiple pathways. Northern blot analysis and nuclear run-off transcription assays were used to assess the activation of c-fos, c-jun, TIS1, TIS8, and TIS11 after exposure of PC12 cells to NGF in the presence or absence of 2-AP and 6-TG. Our findings indicate that NGF appears to employ at least three distinct pathways to induce early genes in PC12 cells. This suggests that the NGF signaling mechanism diverges at an early point after interaction of NGF with its receptor.

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Association of a purine-analogue-sensitive protein kinase activity with p75 nerve growth factor receptors.

Molecular Biology of the Cell ◽

10.1091/mbc.4.1.71 ◽

1993 ◽

Vol 4 (1) ◽

pp. 71-78 ◽

Cited By ~ 27

Author(s):

C Volonté ◽

A H Ross ◽

L A Greene

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Protein Kinase ◽

Pc12 Cells ◽

Kinase Activity ◽

Cellular Systems ◽

Protein Kinase Activity ◽

Purine Analogues ◽

Nerve Growth ◽

Purine Analogue

Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The analogue 6-thioguanine (6-TG) has been shown to inhibit with high specificity protein kinase N (PKN), a serine/threonine protein kinase activated by NGF in several cellular systems. In the present work, immunoprecipitates of p75 NGF receptors from PC12 cells (+/-NGF treatment) were assayed for protein kinase activity using the substrate myelin basic protein under phosphorylating conditions optimal for PKN and in the presence or absence of purine analogues. An NGF-inducible activity was detected, and approximately 80% was inhibited by purine analogues. This activity was maximally stimulated by NGF within 5-10 min, partially decreased by 60 min, and returned to basal levels after 15 h of NGF treatment. The analogue 6-TG inhibited the NGF-inducible p75-associated kinase activity with an IC50 in the range of 15-35 microM. In mutant PC12 nnr-5 cells that lack the Trk NGF receptor, the purine-analogue-sensitive p75-associated kinase activity was not inducible by NFG. In normal PC12 cells, cyclic AMP analogues and epidermal growth factor failed to induce the same activity. Application of either 2-aminopurine or 6-TG to intact cells only slightly inhibit the NGF-dependent induction of the purine-analogue-inhibited p75-associated kinase activity. This activity shares many similarities but also displays some significant differences with cytosolic PKN. Our findings therefore indicate the association of a purine-analogue-sensitive protein kinase with p75 NGF receptors.

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Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor

The Journal of Cell Biology ◽

10.1083/jcb.88.1.189 ◽

1981 ◽

Vol 88 (1) ◽

pp. 189-198 ◽

Cited By ~ 173

Author(s):

K Huff ◽

D End ◽

G Guroff

Keyword(s):

Cell Proliferation ◽

Nerve Growth Factor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Pc12 Cells ◽

Ornithine Decarboxylase ◽

Binding Sites ◽

Cellular Adhesion ◽

Nerve Growth ◽

Epidermal Growth

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.

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