Chromatographic Resolution and Characterization of a Nerve Growth Factor-Dependent Kinase That Phosphorylates Microtubule-Associated Proteins 1 and 2 in PC12 Cells

When the supernatant fractions from extracts of control and nerve growth factor (NGF)- or dibutyryl cyclic AMP-treated PC12D cells were applied to DEAE-Sepharose columns and proteins were eluted with a gradient of NaCl, three separate peaks of kinase activity that phosphorylated microtubule-associated proteins (MAPs) were recovered. Enhancement of the kinase activity in peak 1 was noted in the case of dibutyryl cyclic AMP-treated cells. In contrast, the kinase activity in the third peak was markedly elevated, in terms of the ability to phosphorylate MAP1 and MAP2, in the case of the extract from NGF-treated cells. This activity was designated previously as NGF-dependent MAP kinase. The apparent molecular mass of the active kinase was 45-50 kDa. The apparent Km value was 35 microM for ATP with either MAP1 or MAP2 as substrate. When the kinase activity in the fractions from the DEAE-Sepharose column was assayed in the presence of Mn2+ instead of Mg2+, another NGF-stimulated kinase activity was detected in the fractions eluted by a lower concentration of NaCl than that which eluted the Mg(2+)-activated kinase. Other growth factors, namely, epidermal growth factor and basic fibroblast growth factor, also stimulated the activity of NGF-dependent MAP kinase. Possible involvement of the kinase in the outgrowth of neurites has been suggested. The NGF-induced activation of NGF-dependent MAP kinase was blocked by the presence of K-252a. In contrast, the activation of NGF-dependent MAP kinase by basic fibroblast growth factor and by epidermal growth factor was not blocked, but actually stimulated by K-252a, a result that correlates well with the analogous actions of the drug on the outgrowth of neurites that is induced by these growth factors. The latter observation strengthens the possibility of a close relationship between the outgrowth of neurites and the activation of NGF-dependent MAP kinase.     

Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.     

Nerve Growth Factor-Induced Down-Regulation of Calmodulin-Dependent Protein Kinase III in PC12 Cells Involves Cyclic AMP-Dependent Protein Kinase

Treatment of PC12 cells with nerve growth factor (NGF), epidermal growth factor (EGF), or agents that raise intracellular cyclic AMP (cAMP) levels (e.g., forskolin) reduces the activity of calmodulin-dependent protein kinase III (CaM-PK III) over a period of 8 h. The mechanism of this effect of NGF has now been examined in more detail, making use of a mutant PC12 cell line (A126-1B2) that is deficient in cAMP-dependent protein kinase activity. Control experiments showed that A126-1B2 cells retain other NGF-mediated responses (e.g., the induction of ornithine decarboxylase, a cAMP-independent event) and contain a complement of CaM-PK III and its substrate, elongation factor-2, comparable to that of wild-type cells. The ability of NGF or forskolin, but not of EGF, to down-regulate CaM-PK III was markedly attenuated in A126-1B2 compared to wild-type cells. Treatment of wild-type cells with the cAMP phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced the effects of NGF, but not of EGF. The possibility that NGF led to a stimulation of cAMP-dependent protein kinase activity in wild-type cells was assessed by measurement of the "activation ratio" (-cAMP/+cAMP) of this enzyme before and at various times after NGF addition. A small, but significant, increase in the activation ratio from 0.3 to 0.48 was observed, reaching a peak 5 min after NGF treatment. EGF had no effect on the activation ratio in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

Abstract: Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down-regulated by long-term treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Such prolonged exposure to PMA (1 µ M ) resulted in the inhibition of NGF-induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF-induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs α, δ, ε, and ζ. PMA down-regulation depleted PKCs α, δ, and ε, and partially depleted ζ. To see which isoform was involved in NGF-induced AA release, an isoform-specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs α and β specifically, blocked NGF-induced AA release. In addition, thymeleatoxin, a specific activator of PKCs α, β, and γ, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC α plays a role in NGF-induced AA release.     

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.     

Identification of an Activator of the Microtubule-Associated Protein 2 Kinases ERK1 and ERK2 in PC12 Cells Stimulated with Nerve Growth Factor or Bradykinin

Treatment of PC12 pheochromocytoma cells with nerve growth factor (NGF) or bradykinin leads to the activation of extracellular signal-regulated kinases ERK1 and ERK2, two isozymes of microtubule-associated protein 2 (MAP) kinase that are present in numerous cell lines and regulated by diverse extracellular signals. The activation of MAP kinase is associated with its phosphorylation on tyrosine and threonine residues, both of which are required for activity. In the present studies, we have identified a factor in extracts of PC12 cells treated with NGF or bradykinin, named MAP kinase activator, that, when reconstituted with inactive MAP kinase from untreated cells, dramatically increased MAP kinase activity. Activation of MAP kinase in vitro by this factor required MgATP and was associated with the phosphorylation of a 42- (ERK1) and 44-kDa (ERK2) polypeptide. Incorporation of 32P into ERK1 and ERK2 occurred primarily on tyrosine and threonine residues and was associated with a single tryptic peptide, which is identical to one whose phosphorylation is increased by treatment of intact PC12 cells with NGF. Thus, the MAP kinase activator identified in PC12 cells is likely to be a physiologically important intermediate in the signaling pathways activated by NGF and bradykinin. Moreover, stimulation of the activator by NGF and bradykinin suggests that tyrosine kinase receptors and guanine nucleotide-binding protein-coupled receptors are both capable of regulating these pathways.     

Nerve Growth Factor-Stimulated Nuclear S6 Kinase in PC12 Cells

Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85^S6K. These data indicate that p85^S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago.     

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

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).     

Blockage of Nerve Growth Factor Action in PC12h Cells by Staurosporine, a Potent Protein Kinase Inhibitor

Staurosporine, which has a structure similar to that of K-252a, a potent protein kinase inhibitor that blocks nerve growth factor (NGF) action in PC12 and PC12h cells, is also known as a potent inhibitor of several protein kinases. This study shows that in PC12h cells staurosporine has a dual action: at lower concentrations than that required by K-252a, it is an inhibitor of NGF induction of neurite formation and of changes in the phosphorylation of specific proteins, whereas at concentrations higher than that required to inhibit NGF-induced neurite outgrowth, it rapidly enhances outgrowth by itself.     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

A Purine Analog-Sensitive Protein Kinase Activity Associates with Trk Nerve Growth Factor Receptors

Abstract: Previous studies showed that purine analogs block with varying efficiency and specificity certain effects of nerve growth factor (NGF) on PC12 cells. These compounds also inhibit protein kinase activities. The analog 6-thioguanine has thus far been shown to inhibit only protein kinase N, an NGF-activated protein kinase, whereas 2-aminopurine also blocks other kinases. In the present study, immunoprecipitates of Trk NGF receptors from PC12 cells (NGF treatment) were assayed for protein kinase activity by using the substrates myelin basic protein and histone HF1 under phosphorylating conditions optimal for protein kinase N and in the presence or absence of purine analogs. Activity was detected and ∼50–80% was inhibited by these compounds. The purine analog-sensitive activity was maximally stimulated by NGF within 5 min, was partially decreased by 10 min, and still remained over basal levels after 15 h of NGF treatment. Analysis of myelin basic protein phosphorylated by anti-Trk immunoprecipitates revealed an NGF-stimulated increase in phosphothreonine and phosphotyrosine. Phosphorylation of threonine, but not of tyrosine residues, was inhibited by 6-thioguanine, which therefore inhibits a serine/threonine kinase associated with NGF receptor rather than the receptor kinase itself. Neither 2-aminopurine nor 6-thioguanine inhibited the NGF-dependent induction of Trk-associated kinase activity. Our findings thus indicate association of a purine analog-sensitive serine/threonine protein kinase activity with Trk NGF receptors.     

Glycosaminoglycan Composition of PC 12 Pheochromocytoma Cells: A Comparison with PC12D Cells, a New Subline of PC12 Cells

PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PC12D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of [35S]sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of [35S]sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.     

Mimicry and Inhibition of Nerve Growth Factor Effects: Interactions of Staurosporine, Forskolin, and K252a in PC12 Cells and Normal Rat Chromaffin Cells In Vitro

The structurally similar compounds staurosporine and K252a are potent inhibitors of protein kinases. K252a has previously been reported to inhibit most or all of the effects of nerve growth factor (NGF) on PC12 pheochromocytoma cells, and staurosporine has been reported both to inhibit and to mimic NGF-induced neurite outgrowth from a PC12 cell subclone in a dose-dependent manner. We have studied the interactions of these agents with each other, with NGF, and with forskolin, an activator of adenylate cyclase, on the parent PC12 cell line and on normal neonatal and adult rat chromaffin cells. Staurosporine alone or in conjunction with forskolin induces outgrowth of short neurites from PC12 cells but does not substitute for NGF in promoting cell survival. It does not abolish NGF-induced neurite outgrowth but does reverse the effects of NGF on catecholamine synthesis. K252a abolishes NGF-induced neurite outgrowth but only partially decreases outgrowth induced by NGF plus forskolin. It does not inhibit neurite outgrowth produced by staurosporine or staurosporine plus forskolin. These findings with PC12 cells suggest that staurosporine might act downstream from K252a and NGF on components of one or more signal transduction pathways by which NGF selectively affects the expression of certain traits. Both neonatal and adult rat chromaffin cells show dramatic flattening and extension of filopodia in response to staurosporine, an observation suggesting that some of the same pathways might remain active in cells that do not exhibit a typical NGF response. Only a small amount of neurite outgrowth is observed, however, and only in neonatal cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein Kinase Inhibitor H-7 Differentially Affects Early and Delayed Nerve Growth Factor Responses in PC12 Cells

Abstract: The effects of the protein kinase inhibitor H-7 on early and delayed responses to nerve growth factor (NGF) were investigated in PC12 cells. H-7 reduced the NGF-induced expression of c-Fos in a dose-dependent manner without affecting the time course of c-Fos appearance. Conversely, H-7 potentiated delayed NGF effects, i.e., neurite outgrowth and Ca²⁺/phospholipid-dependent protein kinase (PKC) induction, but not choline acetyltransferase induction. Long-term treatment with NGF resulted in an increase of at least four tyrosine-phosphorylated protein bands with molecular masses between 39 and 48 kDa, which was also potentiated by H-7. In the absence of NGF, H-7 had no significant effect on c-Fos expression, tyrosine phosphorylation of the 45 kDa protein, or choline acetyltransferase activity. However, 4 days of exposure to H-7 alone induced PKC activity and tyrosine phosphorylation of the 39-kDa protein. The action of H-7 derivatives on neurite outgrowth did not correlate with their inhibition profile of cyclic nucleotide-dependent protein kinases. Down-regulation of PKC activity by prolonged exposure to phorbol ester did not completely abolish the effects of NGF and H-7 on induction of c-Fos, choline acetyltransferase activity, and neurite outgrowth, indicating that PKC-independent pathways contribute to these actions. These results suggest that additional pathway(s) sensitive to H-7 may exist, which induce immediate early gene expression and suppress neuronal differentiation of PC12 cells.     

Wheat Germ Agglutinin Inhibits the Effects of Nerve Growth Factor on the Phosphorylation of Proteins in PC12h Cells

Treatment of PC12h cells in tissue culture with nerve growth factor (NGF) led to an increased incorporation of [32P]orthophosphoric acid into specific proteins. The increased phosphorylation of 60,000-dalton and 20,000-dalton proteins in the 0.2% Triton X-100 detergent-soluble fraction, of 35,000-dalton protein in the 0.2% Triton X-100 detergent-insoluble fraction, and of slow migrating protein (SMP) in the nonhistone nuclear fraction was observed upon NGF treatment. On the other hand, wheat germ agglutinin (WGA) treatment of PC12h cells induced a slightly decreased phosphorylation of these NGF-responsive proteins. Incubation of cell-free extracts from PC12h cells with [gamma-32P]ATP led to the phosphorylation of a 100,000-dalton protein. In extracts from cells treated with NGF, the labeling of the 100,000-dalton protein was substantially and selectively reduced. In contrast, treatment of PC12h cells with WGA led to an increased phosphorylation of the 100,000-dalton protein in cell-free extracts. Thus, NGF and WGA showed opposite effects on the phosphorylation of specific proteins in both intact cells and cell-free extracts. In addition, it was also observed in both systems that pre- and posttreatment of PC12h cells with WGA abolished the effects of NGF on the phosphorylation and produced a phosphorylation pattern similar to that from PC12h cells treated only with WGA. In parent PC12 cells, it has been reported that the treatment of cells with WGA inhibits NGF binding to its receptors and converts the rapidly dissociating receptors to slowly dissociating receptors. Thus, WGA in conjunction with NGF, results in the practical disappearance of rapidly dissociating receptors on cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple Pathways of N-Kinase Activation in PC12 Cells

Past work established a cell-free assay for a nerve growth factor (NGF)-activated protein kinase activity (designated N-kinase) that utilizes tyrosine hydroxylase and histone H1 as substrates and that is distinct from a variety of well-characterized kinases. This study explores the specificity and mechanistic pathway(s) by which N-kinase activity is regulated in PC12 rat pheochromocytoma cells. N-kinase is rapidly activated in these cells by treatment with NGF, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), phorbol ester, or dibutyryl cyclic AMP. Our data indicate that the stimulated activity is the same for each agent by several criteria: It exhibits the same characteristic biphasic elution pattern by Mono S fast protein liquid chromatography (FPLC), except for the case of dibutyryl cyclic AMP in which one of the activity peaks is somewhat shifted; it shows the same elution pattern by FPLC on a Superose 12 column; it possesses identical substrate specificity; and, except in the case of dibutyryl cyclic AMP, it does not show additivity when each agent is added simultaneously with NGF. The multiple forms of N-kinase are interconvertible in that rechromatography on a Mono S column yields a single peak of activity. Also, when NGF and dibutyryl cyclic AMP are simultaneously presented to cells, the chromatographic profile resembles that with NGF alone. Activation occurs through several independent initial pathways. Down-regulation of protein kinase C by phorbol ester pretreatment prevents N-kinase activation by phorbol ester, but not by the other agents. A PC12 cell-derived line deficient in cyclic AMP-dependent protein kinase II activity exhibits N-kinase activation by all treatments except dibutyryl cyclic AMP. The properties of N-kinase suggests that it is similar or identical to the ribosomal S6 protein kinase described by Blenis and Erikson. Additional experiments revealed that N-kinase activity can be stimulated in several cell lines in addition to PC12 cells. These findings indicate that the N-kinase can be activated via multiple second-messenger pathways and that it could therefore potentially play a significant role in mediating shared intracellular responses to various extracellular signals.     

Nerve Growth Factor Stimulation of Arachidonic Acid Release from PC12 Cells: Independence from Phosphoinositide Turnover

The effect of nerve growth factor on the metabolism of arachidonic acid and the hydrolysis of phosphatidylinositol in PC12 cells was examined. Addition of nerve growth factor to PC12 cells isotopically labeled with [3H]arachidonic acid caused an increased release of radioactivity. In a similar manner, treatment of PC12 cells prelabeled with [3H]inositol increased inositol monophosphate accumulation in the presence of LiCl. Stimulation of [3H]arachidonic acid release by nerve growth factor was concentration dependent, attaining a maximum at 0.5 nM. Concentrations of nerve growth factor above 0.5 nM caused less than maximal stimulation. In contrast, nerve growth factor-stimulated accumulation of [3H]inositol monophosphate exhibited a sigmoidal dose-response curve with an apparent maximum at 8 nM. Increased accumulation of [3H]inositol monophosphate could be detected as early as 60 s after nerve growth factor addition, whereas nerve growth factor-stimulated release of [3H]arachidonic acid was not observed until 5 min after nerve growth factor treatment. The nerve growth factor-stimulated release of [3H]arachidonic acid was independent of extracellular calcium concentration. Increased [3H]inositol monophosphate accumulation elicited by nerve growth factor was dependent on the presence of extracellular calcium. These results suggest that the increased metabolism of arachidonic acid and the enhanced hydrolysis of phosphatidylinositol are separately regulated by nerve growth factor.     

Nerve Growth Factor and Gangliosides Stimulate the Release of Glycoproteins from PC 12 Pheochromocytoma Cells

PC12 pheochromocytoma cells in monolayer cultures secrete increased amounts of glycoproteins into the medium following the addition of nerve growth factor (NGF) or of brain gangliosides. After a 48-h incubation with 50 ng/ml NGF there is approximately a twofold increase in the total [14C]glucosamine-labeled, ethanol-precipitable cellular material released into the medium. Between 30 and 50% of the radioactivity is associated with a glycoprotein (Gpl) of molecular weight of 52,000; the remaining radioactivity is distributed between five and six major bands. Only a small amount (10%) is associated with a glycoprotein of Mr greater than 200,000 which might correspond to the NGF-induced large external glycoprotein. A substantial increase in the release of the glycoproteins is also seen on the addition of a variety of gangliosides including asialo GMl. This increase is independent of the presence of NGF. GMl and GDlb/GTlb but not GDla stimulate release above the levels seen in the presence of NGF. Addition of GDla (2 micrograms/ml) enhances selectively the release of various glycoproteins between 2.6- and 8-fold. The pattern of glycoprotein secretion is similar to that seen with NGF, although Gp2 (Mr 78,000) is more abundant. Stimulation of release by GDla is not accompanied by neurite outgrowth, suggesting that the glycoproteins are not directly associated with neuritogenesis. The release of these glycoproteins following the addition of NGF or gangliosides may relate to the neurotrophic properties that these two entirely different ligands exert on PC12 cells.     

pp42/44MAP Kinase Is a Component of the Neurogenic Pathway Utilized by Nerve Growth Factor in PC12 Cells

Nerve growth factor-stimulated mitogen-activated protein kinase (pp42/44MAP) kinase was characterized by sequential column chromatography on DEAE-Sephacel, phenyl-Sepharose CL4B, and S-200. The kinase displayed an apparent molecular mass of 42 kDa and reacted with an antiphosphotyrosine antibody. Peptide mapping of myelin basic protein revealed the presence of one phosphopeptide that was phosphorylated on Thr-97. pp42/44MAP kinase activity was dependent on Mg2+ and inhibited by K252a both in vitro and in vivo. Nerve growth factor-stimulated kinase activation was diminished by down-regulation of protein kinase C with 200 nM 12-phorbol 13-myristate acetate or with staurosporine (1 nM), a protein kinase C inhibitor. Genistein, a protein tyrosine kinase inhibitor, blocked nerve growth factor-mediated neurite extension as well as diminished activation of pp42/44MAP kinase. Our data demonstrate that activation of this kinase system by nerve growth factor displays a requirement for both protein kinase C as well as protein tyrosine kinase. In addition, other agents that are capable of promoting neurite outgrowth in PC12 cells, such as fibroblast growth factor or dibutyryl cyclic AMP, do so independently of activating this kinase system.