Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells

Treatment of PC12 cells with either nerve growth factor (NGF), a differentiating factor, or epidermal growth factor (EGF), a mitogen, resulted in 7-15-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein (MAP) 2 on serine and threonine residues in vitro. Both the NGF-activated kinase and the EGF-activated kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxylapatite, and were identical with each other in their chromatographic behavior, apparent molecular mass (approximately 40 kDa) on gel filtration, substrate specificity, and phosphopeptide-mapping pattern of MAP2 phosphorylated by each kinase. Moreover, both kinases were found to be indistinguishable from a mitogen-activated MAP kinase previously described in growth-factor-stimulated or phorbol-ester-stimulated fibroblastic cells, based on the same criteria. Kinase assays in gels after SDS/polyacrylamide gel electrophoresis revealed further that the NGF- or EGF-activated MAP kinase in PC12 cells, as well as the EGF-activated MAP kinase in fibroblastic 3Y1 cells resided in two closely spaced polypeptides with an apparent molecular mass of approximately 40 kDa. In addition, these MAP kinases were inactivated by either acid phosphatase treatment or protein phosphatase 2A treatment. These results indicate that MAP kinase may be activated through phosphorylation by a differentiating factor as well as by a mitogen. MAP kinase activation by EGF was protein kinase C independent; it reached an almost maximal level 1 min after EGF treatment and subsided rapidly within 30-60 min. On the other hand, NGF-induced activation of MAP kinase was partly protein kinase C dependent and continued for at least 2-3 h.     

The roles of macromolecular synthesis and phosphorylation in the regulation of a protein kinase activity transiently stimulated by nerve growth factor

Past work identified and characterized an apparently novel protein kinase activity (designated HMK) that is highly and transiently stimulated in PC12 pheochromocytoma cells by nerve growth factor (NGF). In vitro, HMK phosphorylates both high molecular weight microtubule-associated proteins and myelin basic protein. This study investigates the potential mechanisms of HMK regulation in intact PC12 cells and reveals the following. 1) HMK activation is independent of macromolecular synthesis while the subsequent post-induction suppression requires both RNA and protein synthesis. 2) Neither cAMP-dependent nor Ca2+/phospholipid-dependent protein kinases appear to play a role in regulation of HMK activity by NGF. 3) In vitro, HMK activity is inactivated by protein phosphatase 2A. 4) In vivo, HMK activation by NGF is inhibited by the kinase inhibitor, K-252a. (5) Vanadate, a tyrosine phosphatase inhibitor, induces HMK activity in intact cells, while okadaic acid, a serine/threonine phosphatase inhibitor, is much less efficacious. 6) Application of okadaic acid to vanadate-pretreated cells synergistically stimulates HMK activity to a level comparable to that achieved with NGF. (7) Activation of HMK by NGF is not significantly affected when cells are pretreated with okadaic acid. However, the subsequent NGF-promoted deactivation of HMK is greatly accelerated by okadaic acid. (8) NGF down-regulated HMK activity can be heterologously restimulated by exposure to vanadate and okadaic acid. These data suggest that phosphorylation plays a critical role in both the up- and down-regulation of HMK activity in NGF-treated cells. Moreover, suppression of HMK activity requires ongoing macromolecular synthesis and appears to occur by inactivation rather than degradation.     

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.     

The beta-PDGF receptor induces neuronal differentiation of PC12 cells.

Expression of the mouse beta-PDGF receptor by gene transfer confers PDGF-dependent and reversible neuronal differentiation of PC12 pheochromocytoma cells similar to that observed in response to NGF and basic FGF. A common property of the PDGF, NGF, and basic FGF-induced differentiation response is the requirement for constant exposure of cells to the growth factor. To test the hypothesis that a persistent level of growth factor receptor signaling is required for the maintenance of the neuronal phenotype, we examined the regulation of the serine/threonine-specific MAP kinases after either short- (10 min) or long-term (24 h) stimulation with growth factors. Mono Q FPLC resolved two peaks of growth factor-stimulated MAP kinase activity that coeluted with tyrosine phosphorylated 41- and 43-kDa polypeptides. MAP kinase activity was markedly stimulated (approximately 30-fold) within 5 min of exposure to several growth factors (PDGF, NGF, basic FGF, EGF, and IGF-I), but was persistently maintained at 10-fold above basal activity after 24 h only by the growth factors that also induce PC12 cell differentiation (PDGF, NGF, and basic FGF). Thus the beta-PDGF receptor is in a subset of tyrosine kinase-encoded growth factor receptors that are capable of maintaining continuous signals required for differentiation of PC12 cells. These signals include the constitutive activation of cytoplasmic serine/threonine protein kinases.     

Epidermal growth factor, but not nerve growth factor, stimulates tyrosine-specific protein-kinase activity in pheochromocytoma (PC12) plasma membranes

Rat pheochromocytoma (PC12) cells contain specific plasma membrane receptors for both epidermal growth factor (EGF) and nerve growth factor (NGF). Whereas EGF addition to PC12 cells causes a persistent enhancement of proliferation. NGF addition induces a transient stimulation of growth, followed by growth arrest and neuronal differentiation. Despite these differences in biological response, EGF and NGF share a number of early receptor-mediated responses, which are likely te be related to their effect on cell proliferation. In this paper we show that EGF, but not NGF, is able to stimulate the phosphorylation of membrane proteins. In addition, EGF was able to stimulate phosphorylation of a synthetic peptide (RR-SRC) by PC12 membranes in a concentration-dependent manner. Kinetic analysis of the phosphorylation reaction indicated that EGF increased the Vmax from 13 to 70 pmoles/min/mg protein, while no change was observed in Km. Furthermore, EGF was able to stimulate tyrosine phosphorylation of angiotensin I and II, to the same extent as RR-SRC. In contrast no effects of NGF on peptide phosphorylation by PC12 membranes were observed. Cross-linking experiments demonstrated the presence of receptors for both NGF and EGF in PC12 membranes. These different effects of NGF and EGF on activation of membrane-associated protein-kinase activity demonstrate that NGF might be able to stimulate growth transiently without stimulating protein kinase activity.     

Cross talk among tyrosine kinase receptors in PC12 cells: desensitization of mitogenic epidermal growth factor receptors by the neurotrophic factors, nerve growth factor and basic fibroblast growth factor.

We have studied the effects of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) on epidermal growth factor (EGF) binding to PC12 cells. We show that NGF and bFGF rapidly induce a reduction in 125I-EGF binding to PC12 cells in a dose-dependent manner. This decrease amounts to 50% for NGF and 35% for bFGF. Both factors appear to act through a protein kinase C(PKC)-independent pathway, because their effect persists in PKC-downregulated PC12 cells. Scatchard analysis indicates that NGF and bFGF decrease the number of high affinity EGF binding sites. In addition to their effect on EGF binding, NGF and bFGF activate in intact PC12 cells one or several serine/threonine kinases leading to EGF receptor threonine phosphorylation. Using an in vitro phosphorylation system, we show that NGF- or bFGF-activated extracellular regulated kinase 1 (ERK1) is able to phosphorylate a kinase-deficient EGF receptor. Phosphoamino acid analysis indicates that this phosphorylation occurs mainly on threonine residues. Furthermore, two comparable phosphopeptides are observed in the EGF receptor, phosphorylated either in vivo after NGF treatment or in a cell-free system by NGF-activated ERK1. Finally, a good correlation was found between the time courses of ERK1 activation and 125I-EGF binding inhibition after NGF or bFGF treatment. In conclusion, in PC12 cells the NGF- and bFGF-stimulated ERK1 appears to be involved in the induction of the threonine phosphorylation of the EGF receptor and the decrease in the number of high affinity EGF binding sites.     

Differentiation factors, including nerve growth factor, fibroblast growth factor, and interleukin-6, induce an accumulation of an active Ras.GTP complex in rat pheochromocytoma PC12 cells.

Ras has been thought to be involved in neuronal differentiation of rat pheochromocytoma PC12 cells. PC12 cells are immature adrenal chromaffin-like cells which undergo differentiation to sympathetic neuron-like cells in response to nerve growth factor (NGF). Fibroblast growth factor (FGF) and interleukin (IL)-6 can also induce differentiation of PC12 cells. In this paper, we report that NGF, FGF, and IL-6 induce an accumulation of an active Ras.GTP complex. In the serum-starved culture of PC12 cells, 6% of the Ras protein was complexed with GTP. Upon stimulation with NGF, the percentage of Ras.GTP increased to 24% after 2 min, and the high level of Ras.GTP was maintained for at least 16 h. On the other hand, the activation of Ras by FGF and IL-6 showed distinct kinetics; about 3-fold increase of Ras.GTP was detected at 10 min, and afterward, the level returned to the basal level within 60 min. These observations provide direct evidence that activation of Ras is involved in signal transduction from these differentiation factors. In addition, it was found that growth factors, including epidermal growth factor, insulin, and insulin-like growth factor-I, and a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), can also activate Ras under the same conditions. A tyrosine kinase-specific inhibitor, genistein, inhibited the increase of Ras.GTP induced by NGF and other factors. On the other hand, down-regulation of protein kinase C (PKC) by prolonged treatment with TPA, which sufficiently blocked TPA-induced Ras activation, did not abolish the formation of Ras.GTP by NGF. These results suggest that tyrosine kinases rather than PKC play a major role in the NGF-induced activation of Ras in PC12 cells.     

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.     

Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule-associated protein 1.2 (MAP1.2)

This study characterizes effects of nerve growth factor (NGF) on the steady-state level and phosphorylation of a high molecular mass microtubule-associated protein in PC12 rat pheochromocytoma cells. Past work showed that NGF significantly raises the relative levels of this phosphoprotein, designated MAP1.2, with a time course similar to that of neurite outgrowth. To study this in greater detail, MAP1.2 in PC12 cell lysates was resolved by SDS-PAGE in gels containing 3.25% acrylamide/4 M urea and identified by comigration with material immunoprecipitated from the lysates by MAP1 antibodies. Quantification by metabolic radiolabeling with [35S]methionine or by silver staining revealed a 3.0-3.5-fold increase in MAP1.2 levels relative to total cell protein after NGF treatment for 2 wk or longer. A partial increase was detectable after 3 d, but not after 2 h of NGF exposure. As measured by incorporation of [32P]phosphate, NGF had a dual effect on MAP1.2. Within 15 min to 2 h, NGF enhanced the incorporation of phosphate into MAP1.2 by two- to threefold relative to total cell phosphoproteins. This value slowly increased thereafter so that by 2 wk or more of NGF exposure, the average enhancement of phosphate incorporation per MAP1.2 molecule was over fourfold. The rapid action of NGF on MAP1.2 could not be mimicked by either epidermal growth factor, a permeant cAMP derivative, phorbol ester, or elevated K+, each of which alters phosphorylation of other PC12 cell proteins. SDS-PAGE revealed multiple forms of MAP1.2 which, based on the effects of alkaline phosphatase on their electrophoretic mobilities, differ, at least in part, in extent of phosphorylation. Before NGF treatment, most PC12 cell MAP1.2 is in more rapidly migrating, relatively poorly phosphorylated forms. After long-term NGF exposure, most is in more slowly migrating, more highly phosphorylated forms. The effects of NGF on the rapid phosphorylation of MAP1.2 and on the long-term large increase in highly phosphorylated MAP1.2 forms could play major functional roles in NGF-mediated neuronal differentiation. Such roles may include effects on microtubule assembly, stability, and cross-linking and, possibly for the rapid effects, nuclear signaling.     

Regulation of protein kinase C by nerve growth factor, epidermal growth factor, and phorbol esters in PC12 pheochromocytoma cells

We have used a permeabilized cell assay and a synthetic peptide substrate (KRTLRR) to specifically monitor the activity of protein kinase C in PC12 cells preincubated with nerve growth factor (NGF), epidermal growth factor (EGF), or phorbol esters. Pretreatment of PC12 cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate or 1 microM phorbol dibutyrate stimulated the rate of KRTLRR peptide phosphorylation 4.8- and 2.6-fold, respectively. Furthermore, pretreatment of cells with NGF or EGF transiently increased the KRTLRR peptide kinase activity. Peak stimulations of KRTLRR peptide kinase (1.3-2-fold) were observed after 1-5 min of growth factor treatment and returned to control levels within 15-20 min. The KRTLRR peptide kinase activity fulfilled two criteria of protein kinase C. A synthetic peptide inhibitor of protein kinase C inhibited both growth factor- and phorbol ester-stimulated KRTLRR peptide kinase activity. In addition, growth factors and phorbol esters failed to stimulate KRTLRR peptide kinase activity in cells rendered protein kinase C-deficient by long-term treatment with 1 microM 12-O-tetradecanoylphorbol 13-acetate. In contrast to the transient activation of protein kinase C, ribosomal S6 kinase, assayed with the synthetic peptide RRLSSLRA, was persistently activated by NGF and EGF. The findings indicate that protein kinase C serves an early and transient role in the molecular actions of NGF and EGF in PC12 cells.     

The phosphorylation of stathmin by MAP kinase

Stathmin, a ubiquitous cytosolic phosphoprotei which may play a role in integrating the effects of diverse signals regulating proliferation, differentiation and other cell functions, was found to be phosphorylated rapidly and stoichiometrically by mitogen-activated protein (MAP) kinasein vitro. Ser-25 was identified as the major site and Ser-38 as a minor site of phosphorylation, while the p42 and p44 isoforms of MAP kinase were the only significant stathmin kinases detected in PC12 cells after stimulation by nerve growth factor (NGF). The results suggest that MAP kinases are the enzymes responsible for increasing the level of phosphorylation of Ser-25, which has been observed previously in PC12 cells following stimulation by NGF.Submitted February 1993.     

Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.     

SNT, a differentiation-specific target of neurotrophic factor-induced tyrosine kinase activity in neurons and PC12 cells.

To elucidate the signal transduction mechanisms used by ligands that induce differentiation and the cessation of cell division, we utilized p13suc1-agarose, a reagent that binds p34cdc2/cdk2. By using this reagent, we identified a 78- to 90-kDa species in PC12 pheochromocytoma cells that is rapidly phosphorylated on tyrosine following treatment with the differentiation factors nerve growth factor (NGF) and fibroblast growth factor but not by the mitogens epidermal growth factor or insulin. This species, called SNT (suc-associated neurotrophic factor-induced tyrosine-phosphorylated target), was also phosphorylated on tyrosine in primary rat cortical neurons treated with the neurotrophic factors neurotrophin-3, brain-derived neurotrophic factor, and fibroblast growth factor but not in those treated with epidermal growth factor. In neuronal and fibroblast cells, where NGF can also act as a mitogen, SNT was tyrosine phosphorylated to a much greater extent during NGF-induced differentiation than during NGF-induced proliferation. SNT was phosphorylated in vitro on serine, threonine, and tyrosine in p13suc1-agarose precipitates from NGF-treated PC12 cells, indicating that this protein may be a substrate of kinase activities associated with p13suc1-p34cdc2/cdk2 complexes. In addition, SNT was associated predominantly with nuclear fractions following subcellular fractionation of NGF-treated PC12 cells. Finally, in PC12 cells, NGF-stimulated tyrosine phosphorylation of SNT was dependent on the levels of Trk tyrosine kinase activity and was constitutively induced by expression of pp60v-src. However, Ras was not required for constitutive SNT tyrosine phosphorylation, suggesting that this protein functions distally to Trk and pp60v-src but in a pathway parallel to that of Ras. SNT is the first identified specific target of differentiation factor-induced tyrosine kinase activity in neuronal cells.     

Neurotrophin signal transduction by the Trk receptor

The initial event in the neuronal differentiation of PC12 cells is the binding of the neurotrophin nerve growth factor (NGF) to the Trk receptor. This interaction stimulates the intrinsic tyrosine kinase activity of TRk, initiating a signalling cascade involving the phosphorylation of intracellular proteins on tyrosine, serine, and threonine residues. These signals are then in turn propagated to other messengers, ultimately leading to differentiation, neurotrophin-dependent survival and the loss of proliferative capacity. To transmit NGF signals, NGF-activated Trk rapidly associated with the cytoplasmic proteins, SHC, PI-3 kinase, and PLC-γ1. These proteins are involved in stimulating the formation of various second messenger molecules and activating the Ras signal transduction pathway. Studies with Trk mutants indicate that the acivation of the Ras pathway is necessary for complete differentiation of PC12-derived cells and for the maintenance of the differentiated phenotype. Trk also induces the tyrosine phosphorylation of SNT, a specific target of neurotrophic factor activity in neuronal cells. This review will discuss the potential roles of Trk and the proteins of the Trk signalling pathways in NGF function, and summarize our attempts to understand the mechanisms used by Trk to generate dthe many phenotypic responses of PC12 cells to NGF. 1994 John Wiley & Sons, Inc.     

Overexpression of the protein phosphatase 2A regulatory subunit Bgamma promotes neuronal differentiation by activating the MAP kinase (MAPK) cascade

Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional regulator of cellular signaling. Variable regulatory subunits associate with a core dimer of scaffolding and catalytic subunits and are postulated to dictate substrate specificity and subcellular location of the heterotrimeric PP2A holoenzyme. The role of brain-specific regulatory subunits in neuronal differentiation and signaling was investigated in the PC6-3 subline of PC12 cells. Endogenous Bbeta, Bgamma, and B'beta protein expression was induced during nerve growth factor (NGF)-mediated neuronal differentiation. Transient expression of Bgamma, but not other PP2A regulatory subunits, facilitated neurite outgrowth in the absence and presence of NGF. Tetracycline-inducible expression of Bgamma caused growth arrest and neurofilament expression, further evidence that PP2A/Bgamma can promote differentiation. In PC6-3 cells, but not non-neuronal cell lines, Bgamma specifically promoted long lasting activation of the mitogen-activated protein (MAP) kinase cascade, a key mediator of neuronal differentiation. Pharmacological and dominant-negative inhibition and kinase assays indicate that Bgamma promotes neuritogenesis by stimulating the MAP kinase cascade downstream of the TrkA NGF receptor but upstream or at the level of the B-Raf kinase. Mutational analyses demonstrate that the divergent N terminus is critical for Bgamma activity. These studies implicate PP2A/Bgamma as a positive regulator of MAP kinase signaling in neurons.     

Nerve growth factor activates kinases that phosphorylate atypical protein kinase C

Activation of atypical protein kinase C by nerve growth factor (NGF) involves phosphorylation. In order to identify kinases that regulate atypical PKC (aPKC), we surveyed PC12 cell lysates for protein kinases that are activated by NGF and which could phosphorylate aPKC. Employing an in-gel kinase assay where aPKC-zeta was copolymerized within the gel matrix as a substrate, three kinases, pp175, pp87 and pp60, were identified as enzymes that phosphorylated aPKC. Phosphorylation of aPKC by these three kinases coincided with NGF-induced activation of the enzyme. Each kinase possessed a unique subcellular distribution pattern and could be activated by either ceramide or H(2)0(2), second messengers that mimic NGF signaling events. Upstream, pp175 and pp60 lie in a ras pathway, whereas pp87 lies in a pathway dependent upon src. Altogether, these findings reveal that the aPKCs are subject to regulation by a novel group of kinases.     

Activation of the Bcl-2 promoter by nerve growth factor is mediated by the p42/p44 MAPK cascade

The Bcl-2 protein has an anti-apoptotic effect in neuronal and other cell types. We show for the first time that the Bcl-2 promoter is activated by the neuronal survival factor nerve growth factor (NGF) and that this effect is dependent on a region of the promoter from -1472 to -1414. This activation requires the Rap-1 G protein and the MEK-1 and p42/p44 MAPK enzymes but is independent of other NGF-activated signalling pathways involving protein kinase A or protein kinase C.