K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity.

The rat pheochromocytoma PC12 cell line differentiates into a sympathetic neuronal phenotype upon treatment with either nerve growth factor (NGF) or basic fibroblast growth factor. The alkaloid-like compound K-252a has been demonstrated to be a specific inhibitor of NGF-induced biological responses in PC12 cells (Koizumi, S., Contreras, M. L., Matsuda, Y., Hama, T., Lazarovici, P., and Guroff, G. (1988) J. Neurosci. Res. 8, 715-721). NGF interacts with the protein product of the proto-oncogene trk and rapidly stimulates the tyrosine phosphorylation of both p140prototrk and a number of cellular substrates. Here we show that these phosphorylation events are directly inhibited in PC12 cells by K252a in a dose-dependent manner, indicating that the site of action of this inhibitor is at the NGF receptor level. K-252a inhibits p140prototrk activity in vitro, demonstrating that K-252a has a direct effect on the p140prototrk tyrosine kinase. Though many of the biochemical responses to NGF in PC12 cells are mimicked by basic fibroblast growth factor and epidermal growth factor, K-252a has no effect on the action of these growth factors in PC12 cells, demonstrating that the initial biological events initiated by NGF are distinctive during neuronal differentiation.     

The trk tyrosine protein kinase mediates the mitogenic properties of nerve growth factor and neurotrophin-3.

The product of the trk proto-oncogene encodes a receptor for nerve growth factor (NGF). Here we show that NGF is a powerful mitogen that can induce resting NIH 3T3 cells to enter S phase, grow in semisolid medium, and become morphologically transformed. These mitogenic effects are absolutely dependent on expression of gp140trk receptors, but do not require the presence of the previously described low affinity NGF receptor. gp140trk also serves as a receptor for the related factor neurotrophin-3 (NT-3), but not for brain-derived neurotrophic factor. Both NGF and NT-3 induce the rapid phosphorylation of gp140trk receptors and the transient expression of c-Fos proteins. However, NT-3 appears to elicit more limited mitogenic responses than NGF. These results indicate that the product of the trk proto-oncogene is sufficient to mediate signal transduction processes induced by NGF and NT-3, at least in proliferating cells.     

Protein tyrosine phosphatase activation during nerve growth factor-induced neuronal differentiation of PC12 cells.

We have studied the role of protein tyrosine phosphatases (PTPases) during neuronal differentiation of PC12 cells. Nerve growth factor (NGF), a well-characterized differentiating agent for these cells, led to a decrease in DNA synthesis within 24 h. This was accompanied by a 2- to 3-fold increase in the activity of PTPases, measured as the dephosphorylation of polyacidic or polybasic substrates phosphorylated on tyrosine. PTPase activation was independent of cell density and proportional to NGF concentration, with a half-maximal effect occurring at 0.35 nM. High-performance liquid chromatography size exclusion chromatography revealed that PTPases with molecular masses of 550, 300, and 60 kilodaltons were activated in response to NGF. Additional studies showed that the presence of NGF made PC12 cells refractory to the mitogenic effect of epidermal growth factor. Our data indicate that NGF-induced neuronal differentiation and growth arrest in PC12 cells are associated with activation of several PTPases. We speculate that PTPase activation in response to NGF may inhibit the mitogenic actions of other growth factors.     

Characterization of the tyrosine kinases RAFTK/Pyk2 and FAK in nerve growth factor-induced neuronal differentiation

The related adhesion focal tyrosine kinase (RAFTK), a member of the focal adhesion kinase (FAK) family and highly expressed in brain, is a key mediator of various extracellular signals that elevate intracellular Ca(2+) concentration. We investigated RAFTK and FAK signaling upon nerve growth factor (NGF) stimulation of PC12 cells. NGF induced the tyrosine phosphorylation of RAFTK in a time- and dose-dependent manner, whereas no change in the tyrosine phosphorylation of FAK was observed. Chemical inhibition showed that RAFTK phosphorylation was inhibited by blocking phospholipase Cgamma activity or intracellular Ca(2+). Blocking of extracellular Ca(2+) or phosphatidylinositol 3-kinase activity partially reduced the phosphorylation of RAFTK. In addition, disruption of actin polymerization abolished RAFTK phosphorylation, indicating that an intact actin-based cytoskeletal organization is required for RAFTK phosphorylation. The focal adhesion molecule paxillin was co-immunoprecipitated with RAFTK, and its tyrosine phosphorylation was increased in a Ca(2+)-dependent manner upon NGF stimulation. Confocal microscopic analysis demonstrated that RAFTK translocated from the cytoplasm to potential neurite initiation sites at the cell periphery, where RAFTK co-localized with paxillin and bundled actin in the early phase (within 5 min) of NGF stimulation, whereas FAK co-localized with paxillin at "point contacts," which are the primary cell adhesion sites in neuronal cells. Significant distribution of RAFTK was observed in the neurites and growth cones of differentiated PC12 cells. Furthermore, potassium depolarization induced the tyrosine phosphorylation of both RAFTK and paxillin in an intracellular Ca(2+)-dependent manner in the differentiated PC12 cells. Taken together, these results demonstrate that RAFTK is involved in NGF-induced cytoskeletal organization and may play a role in neurite and growth cone function(s).     

Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors

We prepared methyl 2,5-dihydroxycinnamate as a stable analogue of erbstatin, a tyrosine kinase inhibitor. This analogue was about 4 times more stable than erbstatin in calf serum. It inhibited epidermal growth factor receptor-associated tyrosine kinase in vitro with an IC₅₀ of 0.15 μg/ml. It also inhibited in situ autophosphorylation of epidermal growth factor receptor in A431 cells. Methyl 2,5-dihydroxycinnamate was shown to delay the S-phase induction by epidermal growth factor in quiescent normal rat kidney cells, without affecting the total amount ofDNA synthesis. The effect of erbstatin on S-phase induction was smaller, possibly because of its shorter life time.     

Thrombopoietin inhibits nerve growth factor-induced neuronal differentiation and ERK signalling

Thrombopoietin (TPO), a hematopoietic growth factor regulating platelet production, and its receptor (TPOR) were recently shown to be expressed in the brain where they exert proapoptotic activity. Here we used PC12 cells, an established model of neuronal differentiation, to investigate the effects of TPO on neuronal survival and differentiation. These cells expressed TPOR mRNA. TPO increased cell death in neuronally differentiated PC12 cells but had no effect in undifferentiated cells. Surprisingly, TPO inhibited nerve growth factor (NGF)-induced differentiation of PC12 cells in a dose- and time-dependent manner. This inhibition was dependent on the activity of Janus kinase-2 (JAK2). Using phospho-kinase arrays and Western blot we found downregulation of the NGF-stimulated phosphorylation of the extracellular signal-regulated kinase p42ERK by TPO with no effect on phosphorylation of Akt or stress kinases. NGF-induced phosphorylation of ERK-activating kinases, MEK1/2 and C-RAF was also reduced by TPO while NGF-induced RAS activation was not attenuated by TPO treatment. In contrast to its inhibitory effects on NGF signalling, TPO had no effect on epidermal growth factor (EGF)-stimulated ERK phosphorylation or proliferation of PC12 cells. Our data indicate that TPO via activation of its receptor-bound JAK2 delays the NGF-dependent acquisition of neuronal phenotype and decreases neuronal survival by suppressing NGF-induced ERK activity.     

Nerve growth factor promotes the activation of phosphatidylinositol 3-kinase and its association with the trk tyrosine kinase.

We investigated the involvement of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in the initiation of signal transduction by nerve growth factor (NGF) in the rat pheochromocytoma PC12 cell line. PtdIns 3-kinase catalyzes the formation of phosphoinositides with phosphate in the D-3 position of the inositol ring and previously has been found to associate with other activated protein tyrosine kinases, including growth factor receptor tyrosine kinases. Anti-phosphotyrosine immunoprecipitates had PtdIns 3-kinase activity that reached a maximum (9 times the basal activity) after a 5-min exposure of PC12 cells to NGF (100 ng/ml). Since NGF activates the tyrosine kinase activity of gp140trk, the protein product of the trk proto-oncogene, we also examined the association of PtdIns 3-kinase with gp140trk. Anti-gp140trk immunoprecipitates from NGF-stimulated PC12 cells had increased PtdIns 3-kinase activity compared to that of unstimulated cells, and larger increases were detected in cells overexpressing gp140trk, indicating that PtdIns 3-kinase associates with gp140trk. NGF produced large increases in [32P]phosphatidylinositol 3,4-bisphosphate and [32P]phosphatidylinositol 3,4,5-trisphosphate in PC12 cells labeled with [32P]orthophosphate, indicating an increase in PtdIns 3-kinase activity in intact cells. Using an anti-85-kDa PtdIns 3-kinase subunit antibody, we found that NGF promoted the tyrosine phosphorylation of an 85-kDa protein and two proteins close to 110 kDa. These studies demonstrate that NGF activates PtdIns 3-kinase and promotes its association with gp140trk and also show that NGF promotes the tyrosine phosphorylation of the 85-kDa subunit of PtdIns 3-kinase. Thus, PtdIns 3-kinase activation appears to be involved in differentiation as well as mitogenic responses.     

Fibroblast growth factor receptors participate in the control of mitogen-activated protein kinase activity during nerve growth factor-induced neuronal differentiation of PC12 cells.

The current paradigm for the role of nerve growth factor (NGF) or FGF-2 in the differentiation of neuronal cells implies their binding to specific receptors and activation of kinase cascades leading to the expression of differentiation specific genes. We examined herein the hypothesis that FGF receptors (FGFRs) are involved in NGF-induced neuritogenesis of pheochromocytoma-derived PC12 cells. We demonstrate that in PC12 cells, FGFR expression and activity are modulated upon NGF treatment and that a dominant negative FGFR-2 reduces NGF-induced neuritogenesis. Moreover, FGF-2 expression is modulated by NGF, and FGF-2 is detected at the cell surface. Oligonucleotides that specifically inhibit FGF-2 binding to its receptors are able to significantly reduce NGF-induced neurite outgrowth. Finally, the duration of mitogen-activated protein kinase (MAPK) activity upon FGF or NGF stimulation is shortened in FGFR-2 dominant negative cells through inactivation of signaling from the receptor to the Ras/MAPK pathway. In conclusion, these results demonstrate that FGFR activation is involved in neuritogenesis induced by NGF where it contributes to a sustained MAPK activity in response to NGF.     

Trafficking of TrkA-green fluorescent protein chimerae during nerve growth factor-induced differentiation

A chimera of the nerve growth factor (NGF) receptor, TrkA, and green fluorescent protein (GFP) was engineered by expressing GFP in phase with the carboxyl terminus of TrkA. TrkA-GFP becomes phosphorylated on tyrosine residues in response to NGF and is capable of initiating signaling cascades leading to prolonged MAPK activation and differentiation in PC12 nnr5 cells. TrkA constructs, progressively truncated in the carboxyl-terminal domain, were prepared as GFP chimerae in order to identify which part of the receptor intracellular domain is involved in its trafficking. Immunofluorescence observations show that TrkA-GFP is found mainly in cell surface membrane ruffles and in endosomes. Biochemical analysis indicated that the cytoplasmic domain of TrkA is not necessary for correct maturation and cell surface translocation of the receptor. An antibody against the extracellular domain of TrkA (RTA) was used as ligand to stimulate internalization and phosphorylation of TrkA. Co-localization studies with anti-phosphorylated TrkA antibodies support a role for such complexes in the propagation of signaling from the cell surface, resulting in the activation of TrkA in areas of the endosome devoid of receptor-ligand complexes. Confocal time-lapse analysis reveals that the TrkA-GFP chimera shows highly dynamic trafficking between the cell surface and internal locations. TrkA-positive vesicles were estimated to move 0.46 +/- 0.09 microm/s anterograde and 0.48 +/- 0.07 microm/s retrograde. This approach and the fidelity of the biochemical properties of the TrkA-GFP demonstrate that real-time visualization of trafficking of tyrosine kinase receptors in the presence or absence of the ligand is feasible.     

Protein kinase C mediates platelet-derived growth factor-induced tyrosine phosphorylation of p42

One of the early events after stimulation of Swiss 3T3 cells with either platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), diacylglycerol, or several other mitogens is the near stoichiometric phosphorylation at tyrosine and serine of a scarce cytoplasmic protein (p42). TPA and diacylglycerol are known to directly stimulate the activity of a protein-serine/threonine kinase, protein kinase C (PKC). PDGF and several other mitogens stimulate tyrosine kinases directly and PKC indirectly. We have therefore examined the involvement of PKC in p42 tyrosine phosphorylation in Swiss 3T3 cells. Firstly, six agents which stimulated phosphorylation of p42 also stimulated phosphorylation of a known PKC substrate, an 80,000-Mr protein (p80). Secondly, in PKC-deficient cells (cells in which PKC activity was reduced to undetectable levels by prolonged exposure to TPA), PDGF-induced p42 phosphorylation was reduced three- to fourfold. Phosphoamino acid analysis of phosphorylated p42 from PDGF-stimulated PKC-deficient cells revealed primarily phosphoserine and only a trace of phosphotyrosine, suggesting that the reduction in PDGF-stimulated tyrosine phosphorylation of p42 resulting from PKC deficiency is greater than three- to fourfold. Finally, comparison of antiphosphotyrosine immunoprecipitates of PKC-deficient versus naive cells revealed that most other PDGF-induced tyrosine phosphorylation events were quite similar. These data suggest that mitogens such as PDGF, which directly stimulate phosphorylation of some proteins at tyrosine, induce p42 tyrosine phosphorylation via a cascade of events involving PKC.     

Rit contributes to nerve growth factor-induced neuronal differentiation via activation of B-Raf-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.     

Inhibition of the cellular actions of nerve growth factor by staurosporine and K252A results from the attenuation of the activity of the trk tyrosine kinase.

The protein kinase inhibitors staurosporine and K252A inhibit some of the cellular actions of nerve growth factor (NGF). To explore the molecular mechanisms involved, we test the ability of these agents to block one of the earliest cellular responses to NGF, protein tyrosine phosphorylation. Concentrations of 10-100 nM staurosporine and K252A inhibit NGF-dependent tyrosine phosphorylation in PC12 cells and inhibit trk oncogene-dependent tyrosine phosphorylation in trk-transformed NIH3T3 (trk-3T3 cells). In contrast, these compounds are without effect on epidermal growth factor (EGF)-stimulated tyrosine phosphorylation in PC12 cells. NGF-stimulated tyrosine phosphorylation of the pp140c-trk NGF receptor and tyrosine phosphorylation of pp70trk are also inhibited by similar concentrations of staurosporine and K252A, whereas tyrosine phosphorylation of the EGF receptor, insulin receptor, and v-src is not affected. Both staurosporine and K252A inhibit the autophosphorylation of pp70trk on tyrosine residues in an in vitro immune complex kinase reaction. Incubation of trk-3T3 cells with 10 nM staurosporine causes rounded transformed cells to revert to a normal flattened phenotype, whereas src-transformed cells are unaffected by this agent. These data suggest that staurosporine and K252A specifically inhibit the trk tyrosine kinase activity through a direct mechanism, probably accounting for the attenuation by these agents of the cellular actions of NGF.     

Modifications of potassium currents at the nerve growth factor-induced differentiation of pheochromocytoma cells

Potassium currents were studied in non-differentiated and neuron type-differentiated (induction by nerve growth factor) cells of P12 pheochromocytoma. In the differentiated cells, an increase in the density of non-inactivating potassium current was observed. Fast and slow inactivating potassium currents underwent no changes.     

SH2-B is required for nerve growth factor-induced neuronal differentiation

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cgamma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bbeta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bbeta, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bbeta(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bbeta co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bbeta as well as exogenously expressed GFP-SH2-Bbeta but not GFP-SH2-Bbeta(R555E). Overexpression of SH2-Bbeta(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bbeta enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bbeta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cgamma in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bbeta may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bbeta is a novel signaling molecule required for NGF-induced neuronal differentiation.     

Constitutively activated neu oncoprotein tyrosine kinase interferes with growth factor-induced signals for gene activation.

The neu receptor oncoprotein tyrosine kinase, capable of transforming cultured fibroblasts and causing mammary carcinomas in transgenic mice, carries a point mutation in its transmembrane domain and shows a constitutive tyrosine kinase activity. We analyzed the neu tyrosine kinase and its substrates in transfected NIH 3T3 fibroblasts by phosphotyrosine immunoblotting. Tyrosine phosphorylated proteins were similar but not identical in epidermal growth factor (EGF)-stimulated cells expressing the human EGF receptor (EGFR) or a chimeric EGFR/neu receptor but differed from phosphotyrosyl proteins constitutively expressed in neu oncogene-transformed cells. The neu oncoprotein in the latter cells was phosphorylated in tyrosine in a ligand-independent manner and had a shortened half-life in comparison with the normal neu protein. Tumor promoter pretreatment inhibited ligand-induced receptor tyrosine phosphorylation and decreased tyrosine phosphorylated neu oncoprotein. Prolonged pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also prevented the induction of immediate early growth factor-regulated genes in response to neu activation. Expression of the neu oncogene but not the protooncogene in NIH 3T3 cells was associated with enhanced levels of the jun and fos oncoproteins and loss of serum growth factor induction of immediate early mRNA responses. The constitutively activated neu oncoprotein tyrosine kinase thus deregulates cellular genomic responses to growth factors.     

Induction of neurite outgrowth by v-src mimics critical aspects of nerve growth factor-induced differentiation.

PC12 cells treated with nerve growth factor (NGF) or infected with Rous sarcoma virus differentiate into sympathetic, neuronlike cells. To compare the differentiation programs induced by NGF and v-src, we have established a PC12 cell line expressing a temperature-sensitive v-src protein. The v-src-expressing PC12 cell line was shown to elaborate neuritic processes in a temperature-inducible manner, indicating that the differentiation process was dependent on the activity of the v-src protein. Further characterization of this cell line, in comparison with NGF-treated PC12 cells, indicated that the events associated with neurite outgrowth induced by these two agents shared features but could be distinguished by others. Both NGF- and v-src-induced neurite outgrowths were reversible. In addition, NGF and v-src could prime PC12 cells for NGF-induced neurite outgrowth, and representative early and late NGF-responsive genes were also induced by v-src. However, unlike NGF-induced neurite growth, v-src-induced neurite outgrowth was not blocked at high cell density. A comparison of phosphotyrosine containing-protein profiles showed that v-src and NGF each increase tyrosine phosphorylation of multiple cellular proteins. There was overlap in substrates; however, both NGF-specific and v-src-specific tyrosine phosphorylations were observed. One protein which was found to be phosphorylated in both the NGF- and v-src-induced PC12 cells was phospholipase C-gamma 1. Taken together, these results suggest that v-src's ability to function as an inducing agent may be a consequence of its ability to mimic critical aspects of the NGF differentiation program and raise the possibility that Src-like tyrosine kinases are involved in mediating some of the events triggered by NGF.