Test-retest reliability of psychological and neurobehavioral tests self-administered by computer

Adult rat chromaffin cells may proliferate or extend neurites when stimulated by nerve growth factor (NGF) but their response is predominantly proliferative, making them a unique model for studying how mitogenic specificity is achieved. We examined contributions of the NGF receptors trk and p75 and of the major NGF signaling pathways to proliferation versus neurite outgrowth. The type of initial NGF response does not correlate with intensity of immunoreactivity for trk or p75. However, proliferation is initiated at lower NGF concentrations than neurite outgrowth, suggesting that it requires a less intense signal. Mitogenic cooperativity between receptors at low NGF concentrations is suggested by inhibitory effects of p75-blocking antibodies, but responses to trk-agonist antibody indicate that trk activation alone can induce proliferation. NGF-induced phosphorylation of ras-mediated mitogen-activated protein kinases (MAPK) Erk1 and Erk2 is as prolonged in normal chromaffin cells as in PC12 cells, where NGF is neuritogenic. Trk-agonist antibody, which is as mitogenic as NGF but less neuritogenic, causes equally prolonged but less intense ERK phosphorylation. The MAPK kinase(MEK-1) inhibitor PD98059 partially inhibits Erk phosphorylation and does not inhibit chromaffin cell proliferation, while depolarization selectively inhibits proliferation without blocking Erk phosphorylation. Proliferation is markedly reduced by the phosphoinositol-3 (PI-3) kinase inhibitor LY294002 while downregulation of protein kinase C (PKC) causes no change. These findings suggest that low-level, rather than short-duration, stimulation of NGF signaling pathways causes NGF to be mitogenic. Ras-mediated MAPK activation may be more critical in neurite outgrowth than in proliferation and PI-3 kinase may be the major mitogenic determinant.     

p75 and TrkA receptor signaling independently regulate amyloid precursor protein mRNA expression, isoform composition, and protein secretion in PC12 cells

The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose-dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain-derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75NTR-activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross-linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors--in contrast to p75NTR-associated signaling--stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.     

High affinity nerve growth factor binding displays a faster rate of association than p140trk binding. Implications for multi-subunit polypeptide receptors

Nerve growth factor (NGF) binds to two cell surface receptors, p140trk and p75NGFR, which are both expressed in responsive sensory, sympathetic, and basal forebrain cholinergic neurons. While p140trk belongs to the family of receptor tyrosine kinases, p75NGFR is a member of the TNF/Fas/CD40/CD30 family of receptors. Current views of neurotrophin receptor function have tended to interpret p140trk as the high affinity NGF-binding site. To assess if the binding of NGF to p140trk was distinguishable from binding to high affinity sites on neuronal cells, PC12 cell sublines were generated which expressed p140trk alone, or coexpressed both p140trk and p75NGFR. Kinetic analysis of 125I-NGF binding indicates that it has an unusually slow rate of association with p140trk (k + 1 = 8 x 10(5) M-1 s-1). When both p140trk and p75NGFR receptors are coexpressed, the rate of association of NGF is increased 25-fold to produce a higher affinity binding site. An increase in the rate of internalization was also observed. Since high affinity binding and internalization are believed to be prerequisite for the biological activities of NGF, these results suggest that the biological effects by NGF are derived from a novel kinetic binding site that requires the expression of both receptors. The implications of these results with respect to multisubunit polypeptide receptors are discussed.     

Staurosporine induces neurite outgrowth in neuronal hybrids (PC12EN) lacking NGF receptors

A novel neuronal model (PC12EN cells), obtained by somatic hybridization of rat adrenal medullary pheochromocytoma (PC12) and bovine adrenal medullary endothelial (BAME) cells, was developed. PC12EN cells maintained numerous neuronal characteristics: they expressed neuronal glycolipid conjugates, synthesized and secreted catecholamines, and responded to differentiative agents with neurite outgrowth. PC12EN lacked receptors for EGF and both the p75 and trk NGF receptors, while FGF receptor expression was maintained. Staurosporine (5-50 nM), but not other members of the K252a family of protein kinase inhibitors, rapidly induced neurite outgrowth in PC12EN, as also found in the parental PC12 cells, but not in BAME cells. Similarly, both acidic and basic FGF (1-100 ng/ml) were neurotropic in PC12EN. In contrast to the mechanism by which FGF promoted neurite outgrowth in PC12EN, the neurotropic effect of staurosporine did not involve activation of established signalling pathways, such as tyrosine phosphorylation of erk (ras pathway) or SNT (a specific target of neuronal differentiation). In addition, staurosporine induced the tyrosine phosphorylation of the focal adhesion kinase p125FAK. However, since the latter effect was also observed with other protein kinase inhibitors of the K252a family, which induced PC12EN cells flattening but no neurite extension, we propose that FAK tyrosine phosphorylation may be related to ubiquitous changes in cell shape. We anticipate that PC12EN neuronal hybrids will become useful models in neuroscience research for evaluating unique cellular signalling mechanisms of novel neurotropic compounds.     

Nerve growth factor treatment prevents the increase in superoxide produced by epidermal growth factor in PC12 cells

Stimulation of pheochromocytoma (PC12) cells with the mitogen epidermal growth factor (EGF) produced a rapid and robust accumulation of intracellular reactive oxygen species (ROS), an accumulation which, in other systems, has been shown to be essential for mitogenesis. Brief pretreatment of the cells with nerve growth factor (NGF) suppressed the EGF-mediated ROS increase. EGF failed to produce elevations in ROS in a PC12 variant stably expressing a dominant-negative p21(ras) construct (PC12-N17) or in cells pretreated with the MEK inhibitor PD098059. NGF failed to suppress the increase in ROS in the PC12 variant nnr5, which lacks p140(trk) receptors. The suppression of the increase in ROS by NGF was restored in nnr5 cells stably expressing p140(trk) (nnr5-trk), but NGF failed to prevent the increase in ROS in nnr cells expressing mutant p140(trk) receptors that lack binding sites for Shc and phospholipase Cgamma. Among several inhibitors of superoxide-generating enzymes, only the lipoxygenase inhibitor, nordihydroguaiaretic acid reduced EGF-mediated ROS accumulation. The inhibitory action of NGF on ROS production was mimicked by the nitric oxide donor, sodium nitroprusside, and was blocked by an inhibitor of nitric-oxide synthetase, L-nitroarginine methyl ester. These results suggest a novel mechanism for the rapid interruption of mitogenic signaling by the neurotrophin NGF.     

Overexpression of protein kinase C-delta and -epsilon in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity

We have determined the patterns of mRNA and protein expression of 7 protein kinase C (PKC) isozymes in NIH 3T3 cells. Only PKC-alpha is expressed abundantly in NIH 3T3 cells; endogenous levels of the other 6 PKC isozymes are low or undetectable. We have overexpressed PKC-delta and -epsilon in these cells to observe activation/translocation of these two isozymes and the biological consequences of overexpression. Both PKC-delta and -epsilon, but not PKC-alpha, are partially associated with the insoluble fraction even in the absence of phorbol 12-myristate 13-acetate (PMA). Upon PMA stimulation, both PKC-delta and -epsilon translocate to the insoluble fraction of cell homogenates, as can be observed with the endogenous PKC-alpha. Overexpression of PKC-delta induces significant changes in morphology and causes the cells to grow more slowly and to a decreased cell density in confluent cultures. These changes are accentuated by treatment with PMA. Overexpression of PKC-epsilon does not lead to morphological changes, but causes increased growth rates and higher cell densities in monolayers. None of the PKC-delta overexpressers grow in soft agar with or without PMA, but all the cell lines that overexpress PKC-epsilon grow in soft agar in the absence of PMA, but not in its presence. NIH 3T3 cells that overexpress PKC-epsilon also form tumors in nude mice with 100% incidence. This indicates that high expression of PKC-epsilon contributes to neoplastic transformation.     

BCR-ABL induces neurite-like structures and BCR lacking the SH2-binding domain induces cell rounding in PC12 cells

The activated tyrosine kinase oncoprotein BCR-ABL is responsible for pathogenesis of Philadelphia chromosome-positive human leukemias. Because BCR carries a GAP (GTPase-activating protein) activity toward cytoskeleton-related small GTP-binding proteins, we utilized a neuronal PC12 cell system to test morphogenic potentials of BCR-ABL or BCR. We report here unique morphological phenotypes of PC12 cells expressing either BCR-ABL or a BCR mutant which lacks the SH2-binding domain (BCR Delta162-413). Although MAP kinase was not activated in PC12 cells expressing BCR-ABL, they showed incomplete neurite extensions even in the absence of the nerve growth factor (NGF). Overproduction of BCR Delta162-413 in PC12 cells, on the other hand, induced cell rounding in the absence of NGF. Interestingly, those cells could hardly make terminal differentiation in the presence of NGF and continued to grow without changing their round shape, although NGF receptor as well as MAP kinase appeared to be activated. Interestingly, the botulinum C3 toxin induced neurite-like structures in PC12 cells overexpressing BCR Delta162-413 without NGF.     

Functional analysis of trk proto-oncogene product in medulloblastoma cells

MED-L cells with the 75 kd low-affinity nerve growth factor receptor (p75NGFR) and MED-H cells with the proto-oncogene tropomyosin receptor kinase product (p140trk) were isolated selectively from a parent MED-3 cell line derived from cerebellar medulloblastoma by panning, and the interaction of nerve growth factor (NGF) with these cell lines was analyzed. NGF treatment induced neuronal differentiation, growth inhibition, and tyrosine phosphorylation of p140trk in MED-H cells, but not in MED-L cells. Medulloblastoma cells express the functional NGFR, p140trk, which regulates their differentiation and growth.     

Association of protein kinases ERK1 and ERK2 with p75 nerve growth factor receptors

Extracellular signal-regulated protein kinases (ERKs) constitute a family of protein serine-threonine kinases implicated in a variety of cell-signaling pathways. In cultured rat pheochromocytoma PC12 cells, ERK1 and ERK2 are activated by nerve growth factor (NGF), which also induces rapid association between ERK1 and the high affinity gp140prototrk tyrosine kinase NGF receptor. In the present work, we investigated the possible association between ERKs and the low affinity NGF receptor, p75. Extracts of PC12 cells (before and after NGF treatment) were subjected to immunoprecipitation with anti-p75 antibodies or antiserum; the immune complexes were then assessed for the presence of ERK proteins and tyrosine phosphorylation or for ERK activity using a specific substrate peptide. ERK1 and, to a lesser extent, ERK2 were found to be constitutively associated with p75. NGF did not modulate the total amount of ERK proteins coimmunoprecipitated with p75 but did markedly stimulate the level of p75-associated ERK catalytic activity. NGF treatment also enhanced the tyrosine phosphorylation of a p75-associated species that co-migrates with ERK1 in Western blots. Finally, K-252a, a compound that specifically inhibits activation by NGF of gp140prototrk, abolished the latter effect. These findings indicate that NGF, via activation of gp140prototrk, leads to association of enzymatically active ERKs with p75 and raise the possibility that this interaction may play a role in the NGF mechanism of action.     

Plasmalopsychosine of human brain mimics the effect of nerve growth factor by activating its receptor kinase and mitogen-activated protein kinase in PC12 cells. Induction of neurite outgrowth and prevention of apoptosis

Plasmalopsychosine, a characteristic fatty aldehyde conjugate of beta-galactosylsphingosine (psychosine) found in brain white matter, enhances p140trk (Trk A) phosphorylation and mitogen-activated protein kinase (MAPK) activity and as a consequence induces neurite outgrowth in PC12 cells. The effect of plasmalopsychosine on neurite outgrowth and its prolonged activation of MAPK was similar to that of nerve growth factor (NGF), and the effect was specific to neuronal cells. Plasmalopsychosine was not capable of competing with cold chase-stable, high affinity binding of NGF to Trk A, indicating that plasmalopsychosine and NGF differ in terms of Trk A-activating mechanism. Tyrosine kinase inhibitors K-252a and staurosporine, known to inhibit the neurotrophic effect of NGF, also inhibited these effects of plasmalopsychosine, suggesting that plasmalopsychosine and NGF share a common signaling cascade. Plasmalopsychosine prevents apoptosis of PC12 cells caused by serum deprivation, indicating that it has "neurotrophic factor-like" activity. Taken together, these findings indicate that plasmalopsychosine may play an important role in development and maintenance of the vertebrate nervous system.     

bcl-2 inhibits wild-type p53-triggered apoptosis but not G1 cell cycle arrest and transactivation of WAF1 and bax

We have earlier shown that wild-type (wt) p53 expressed from a temperature-sensitive construct (ts p53) triggers apoptosis in the v-myc retrovirus-induced, p53-negative T-cell lymphoma line J3D (Y. Wang et al., Cell Growth & Differ., 4: 467-473, 1993). We also found that constitutive bcl-2 expression inhibits wt p53-triggered apoptosis in these cells (Y. Wang et al., Oncogene, 8: 3427-3431, 1993). Here we demonstrate that more than 90% of the ts p53-transfected J3D cells were arrested in G1 at 18 h after induction of wt p53 expression by temperature shift to 32 degrees C. At this time, at least 80% of the cells remained viable. After 30 h at 32 degrees C, around 50% of the cells had died by apoptosis, while most of the remaining cells were still alive in G1, indicating that p53-induced apoptosis occurred following G1 arrest. The G1 cell cycle arrest at 18 h after temperature shift to 32 degrees C was reversible, as shown by the fact that the cells readily resumed exponential growth following temperature shift back to 37 degrees C, although viability dropped from around 80 to 65%. Expression of both WAF1 and bax mRNA was induced by wt p53 in both the ts p53 and ts p53/bcl-2 transfected cells. The kinetics of G1 cell cycle arrest at 32 degrees C was similar in both the ts p53 and the ts p53/bcl-2 double transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding-induced activation of overexpressed p185HER2 is essential in triggering neuronal differentiation of PC12 cells

To determine whether p185HER2 overexpression per se triggers p185HER2 cellular signaling or whether an extracellular signal is required, we transfected PC12 cells with the human erbB-2 proto-oncogene, and established a cell line that overexpresses p185HER2. PC12-HER2 cells, maintained in suspension culture or plated on a collagen layer, showed the same morphology and growth rate as PC12 and PC12 mock-transfected control cells. When treated with monoclonal antibody (MAb) MGr6 or other anti-p185HER2 MAbs, PC12-HER2 cells specifically underwent neuronal differentiation comparable to that induced by nerve growth factor (NGF), and the differentiation-inducing effect of the MAb was dramatically enhanced by the addition of a second anti-mouse IgG. MAb-induced cell differentiation correlated with p185HER2 phosphorylation, recruitment of Shc and Grb-2 transducer molecules into complexes, and MAPK phosphorylation. These data indicate the requirement for a specific binding-induced activation of the overexpressed p185HER2 receptor in inducing PC12 cell differentiation. PC12-HER2 cells represent a suitable system for selection of p185HER2-activating ligands (peptides, phage-displayed peptides or proteins) or specific inhibitors of its tyrosine kinase activity.     

Ectopic expression of a chimeric colony-stimulating factor-1/TrkB-receptor promotes CSF-1-dependent survival of cultured sympathetic neurons

The regulation of the density of innervation and the promotion of survival of neurons are the original effects depending on neurotrophins. Here we analyse such effects evoked by trkB tyrosine kinase in transfected PC12 cells and transfected sympathetic neurons. In order to exclude the previously described modulation of trk kinase activity by the extracellular activation of the low-affinity p75 neurotrophin receptor, we applied a chimeric receptor approach: The extracellular domain of colony-stimulating factor-1 (CSF-1) receptor was fused to the transmembrane and cytoplasmic domain of the trkB tyrosine kinase receptor, allowing its selective activation by the heterologous ligand. Protein expression and CSF-1-induced tyrosine phosphorylation of the chimeric receptor protein was demonstrated in transfected COS cells. After stable transfection into nerve growth factor (NGF)-responsive PC12 cells, CSF-1 mediated the K252a-sensitive induction of fiber outgrowth. Furthermore, we were able to show by heterologous expression of the chimeric receptor, that activation of trkB tyrosine kinase activity is sufficient to promote survival of neurotrophin deprived sympathetic neurons.     

Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines

Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene-transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT-29, human kidney cancer cell 293, and human hepatocellular carcinoma Hep G2 cells, but not in primary culture of mouse embryonic fibroblast C3H 10T1/2, rat embryonic fibroblast, and human foreskin fibroblast cells in a concentration- and time-dependent manner. Many cellular and biochemical effects of curcumin in mouse fibroblast cells have been reported, such as inhibition of protein kinase C (PKC) activity induced by phorbol 12-myristate 13-acetate treatment, inhibition of tyrosine protein kinase activity, and inhibition of arachidonic acid (AA) metabolism. Treatment of NIH 3T3 cells with the PKC inhibitor staurosporine, the tyrosine kinase inhibitor herbimycin A, and the AA metabolism inhibitor quinacrine induces apoptotic cell death. These results suggest that, in some immortalized and transformed cells, blocking the cellular signal transduction might trigger the induction of apoptosis.     

Growth hormone stimulates the formation of a multiprotein signaling complex involving p130(Cas) and CrkII. Resultant activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK)

We have demonstrated previously that growth hormone (GH) activates focal adhesion kinase (FAK), and this activation results in the tyrosine phosphorylation of two FAK substrates, namely paxillin and tensin. We now show here in Chinese hamster ovary cells stably transfected with rat GH receptor cDNA that human (h)GH induces the formation of a large multiprotein signaling complex centered around another FAK-associated protein, p130(Cas) and the adaptor protein CrkII. hGH stimulates the tyrosine phosphorylation of both p130(Cas) and CrkII, their association, and the association of multiple other tyrosine-phosphorylated proteins to the complex. Both the c-Src and c-Fyn tyrosine kinases are tyrosine phosphorylated and activated by cellular hGH stimulation and form part of the multiprotein signaling complex as does tensin, paxillin, IRS-1, the p85 subunit of phosphatidylinositol 3-kinase, C3G, SHC, Grb-2, and Sos-1. c-Cbl and Nck are also tyrosine-phosphorylated by cellular stimulation with hGH and associate with the p130(Cas)-CrkII complex. c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is activated in response to hGH in accordance with the formation of the abovementioned signaling complex, and hGH stimulated JNK/SAPK activity is increased in CrkII overexpressing NIH3T3 cells compared with vector transfected NIH3T3 cells. The formation of such a large multiprotein signaling complex by GH, with the resultant activation of multiple downstream effector molecules, may be central to many of the pleiotropic effects of GH.     

Reciprocal modulation of TrkA and p75NTR affinity states is mediated by direct receptor interactions

Equilibrium binding of 125I-nerve growth factor (125I-NGF) to cells coexpressing the tyrosine kinase receptor A (TrkA) and common neurotrophin receptor (p75NTR), cells coexpressing both receptors where p75NTR is occupied, and cells expressing only p75NTR, revealed reciprocal modulation of receptor affinity states. Analysis of receptor affinity states in PC12 cells, PC12 cells in the presence of brain-derived neurotrophic factor (BDNF), and PC12nnr5 cells suggested that liganded and unliganded p75NTR induce a higher affinity state within TrkA, while TrkA induces a lower affinity state within p75NTR. These data are consistent with receptor allosterism, and prompted a search for TrkA/p75NTR complexes in the absence of NGF. Chemical crosslinking studies revealed high molecular weight receptor complexes that specifically bound 125I-NGF, and were immunoprecipitated by antibodies to both receptors. The heteroreceptor complex of TrkA and p75NTR alters conformation and/or dissociates in the presence of NGF, as indicated by the ability of low concentrations of NGF to prevent heteroreceptor crosslinking. These data suggest a new model of receptor interaction, whereby structural changes within a heteroreceptor complex are induced by ligand binding.     

Caveolin interacts with Trk A and p75(NTR) and regulates neurotrophin signaling pathways

Neurotrophins signal through Trk tyrosine kinase receptors and the low-affinity neurotrophin receptor p75(NTR). We have shown previously that activation of Trk A tyrosine kinase activity can inhibit p75(NTR)-dependent sphingomyelin hydrolysis, that caveolae are a localized site for p75(NTR) signaling, and that caveolin can directly interact with p75(NTR). The ability of caveolin to also interact with tyrosine kinase receptors and inhibit their activity led us to hypothesize that caveolin expression may modulate interactions between neurotrophin signaling pathways. PC12 cells were transfected with caveolin that was expressed efficiently and targeted to the appropriate membrane domains. Upon exposure to nerve growth factor (NGF), caveolin-PC12 cells were unable to develop extensive neuritic processes. Caveolin expression in PC12 cells was found to diminish the magnitude and duration of Trk A activation in vivo. This inhibition may be due to a direct interaction of caveolin with Trk A, because Trk A co-immunoprecipitated with caveolin from Cav-Trk A-PC12 cells, and a glutathione S-transferase-caveolin fusion protein bound to Trk A and inhibited NGF-induced autophosphorylation in vitro. Furthermore, the in vivo kinetics of the inhibition of Trk A tyrosine kinase activity by caveolin expression correlated with an increased ability of NGF to induce sphingomyelin hydrolysis through p75(NTR). In summary, our results suggest that the interaction of caveolin with neurotrophin receptors may have functional consequences in regulating signaling through p75(NTR) and Trk A in neuronal and glial cell populations.     

A novel differentiation factor for PC12 cells from culture supernatant of mouse hepatocyte cell line MLE-15A2

We have found a factor that induces neurite outgrowth of rat PC12 cells in the culture supernatant of the cell line MLE-15A2. This factor was designated as MDDF. The factor was sensitive to protease, dithiothreitol, and high-temperature treatments. The apparent molecular mass was 80 kDa on Superdex 200 gel filtration. No significant tyrosine phosphorylation was detected after MDDF stimulation in Western blotting analysis with anti-phosphotyrosine antibody, suggesting that the signal transduction may not be mediated by a tyrosine kinase cascade that is involved in signaling of most of the known factors. Activation of MAP kinase was very weak and was seen only 5 min after stimulation, suggesting that prolonged activation of MAP kinase was not required for neurite outgrowth induced by MDDF. Because the biochemical characteristics of MDDF are different from those of any known peptide factors that induce neurite outgrowth of PC12 cells, MDDF may be a novel differentiation factor for PC12 cells.     

Signalling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein kinases (JNKS): evidence for a divergence of the ERKs and JNKs pathways induced by Ret

The RET proto-oncogene encodes a functional receptor tyrosine kinase (Ret) for the Glial cell line Derived Neurotrophic Factor (GDNF). RET is involved in several neoplastic and non-neoplastic human diseases. Oncogenic activation of RET is detected in human papillary thyroid tumours and in multiple endocrine neoplasia type 2 syndromes. Inactivating mutations of RET have been associated to the congenital megacolon, i.e. Hirschprung's disease. In order to identify pathways that are relevant for Ret signalling to the nucleus, we have investigated its ability to induce the c-Jun NH2-terminal protein kinases (JNK). Here we show that triggering the endogenous Ret, expressed in PC12 cells, induces JNK activity; moreover, Ret is able to activate JNK either when transiently transfected in COS-1 cells or when stably expressed in NIH3T3 fibroblasts or in PC Cl 3 epithelial thyroid cells. JNK activation is dependent on the Ret kinase function, as a kinase-deficient RET mutant, associated with Hirschsprung's disease, fails to activate JNK. The pathway leading to the activation of JNK by RET is clearly divergent from that leading to the activation of ERK: substitution of the tyrosine 1062 of Ret, the Shc binding site, for phenylalanine abrogates ERK but not JNK activation. Experiments conducted with dominant negative mutants or with negative regulators demonstrate that JNK activation by Ret is mediated by Rho/Rac related small GTPases and, particularly, by Cdc42.     

Functional expression of TrkA receptors in hippocampal neurons

Nerve growth factor (NGF) initiates its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. The requirements for NGF signaling through the TrkA receptor have been defined extensively from studies in immortalized cells, involving transfection of NIH 3T3, COS, and PC12 cells. In the present study, we tested the effects of extracellular and intracellular mutations of TrkA after DNA-mediated transfection in primary cultures of embryonic day 17 hippocampal neurons. We found that the action of the TrkA receptor on neuronal differentiation depends on specific motifs in the extracellular domain and on tyrosine 490 (Y490), the site for SHC protein binding. In contrast with previous observations in a PC12 background, a mutation in the SHC Y490 binding site in TrkA resulted in a loss of NGF-dependent process formation. These results indicate that tyrosine 490 is necessary for neurite outgrowth in hippocampal neurons. Moreover, a constitutively active form of TrkA did not give enhanced responsiveness in hippocampal neurons, indicating that the behavior of TrkA receptors in primary neuronal cells is distinct from that of other cell types.