EGF-Induced sustained tyrosine phosphorylation and decreased rate of down-regulation of EGF receptor in PC12h-R cells which show neuronal differentiation in response to EGF

PC12h-R cell, a subclone of PC12 cells, exhibited a neuron-like phenotype, including neurite outgrowth and increased acetylcholinesterase activity, in response to epidermal growth factor (EGF) as well as nerve growth factor (NGF). We examined the mechanism by which EGF induced the neuronal differentiation in PC12h-R cells. The EGF-induced neuronal differentiation of PC12h-R cells was not blocked by K252a, whereas that induced by NGF was. EGF induced sustained tyrosine phosphorylation of the EGF receptor in PC12h-R cells, but not in the parent PC12h cells, which do not show neuronal differentiation in response to EGF. In addition, the rate of EGF-induced down-regulation of the EGF receptor in PC12h-R cells was decreased compared with that in PC12h cells. Furthermore, we found that the duration of EGF-induced tyrosine phosphorylation of the EGF receptor in PC12h-R cells was similar to that of NGF-induced tyrosine phosphorylation of p140 ^trkA in PC12h cells. The EGF-induced phosphorylation of the EGF receptor in PC12h cells was less sustained than that of p140 ^trkA by NGF in PC12h cells. These findings suggested that the EGF-induced neuronal differentiation of PC12h-R cells is due to the sustained activation of the EGF receptor, resulting from the decreased down-regulation of the EGF receptor and that the duration of the receptor tyrosine kinase activity determines the cellular responses of PC12 cells. We concluded that sustained activation of the receptor tyrosine kinase induces neuronal differentiation, although transient activation promotes proliferation of PC12 cells. Special issue dedicated to Dr. Hans Thoenen.     

NGF and EGF rapidly activate p21ras in PC12 cells by distinct, convergent pathways involving tyrosine phosphorylation.

Activation of p21ras, demonstrated directly as an increase in p21ras-associated GTP, was induced rapidly but transiently by both nerve growth factor (NGF) and epidermal growth factor (EGF) in PC12 cells. The factors activate p21ras to equal extents and with virtually identical time courses. Growth factor-induced p21ras activation and tyrosine phosphorylation have similar time courses and sensitivities to genistein inhibition, indicating that p21ras activation is a result of tyrosine kinase activity. Furthermore, PC12 mutants lacking the Trk NGF receptor tyrosine kinase also lack NGF-inducible p21ras activation. The protein kinase inhibitor K252a and the methyltransferase inhibitor MTA abolish NGF-induced, but not EGF-induced, p21ras activation--effects correlated with inhibition only of NGF-induced tyrosine phosphorylation. In spite of differences in sensitivity to genistein, MTA, and K252a, EGF- and NGF-stimulated p21ras activation are not additive, implying that they do share at least one step in common.     

Specific inhibition of NGF receptor tyrosine kinase activity by K-252a.

An involvement of protein tyrosine kinase in the transduction of the signals initiated by nerve growth factor (NGF) was investigated. A tyrosine kinase inhibitor, herbimycin, inhibited neurite outgrowth of rat pheochromocytoma PC12 cells induced by NGF but not that by dibutyryl-cAMP. Herbimycin and genistein blocked NGF-dependent activation of ras p21 whose essential function in neuronal differentiation has been reported. These observations suggested that tyrosine kinase activity is involved in the signaling pathways. K-252a, by contrast, inhibited NGF-induced but not EGF-dependent activation of ras p21. Tyrosine kinase activity of gp140trk, a constituent of NGF receptor, is activated by NGF for much a longer period compared to the activation of EGF receptor autokinase activity by EGF. We further demonstrated that autophosphorylation of gp140trk is selectively inhibited by K-252a.     

Localizing a control region in the pathway to leukotriene C(4) secretion following stimulation of human basophils with anti-IgE antibody.

Mediator release from human basophils is a self-limited process, but down-regulation of the signaling cascades leading to secretion of leukotriene C(4) (LTC(4)) is controlled independently of the pathway leading to IL-4 secretion. In the current studies, we have explored the regulation of upstream signaling events leading to activation of extracellular signal-related kinases (ERKs; previously shown to be required for LTC(4) generation) in human basophils. IgE-, but not FMLP-mediated activation, induced sustained tyrosine phosphorylation of syk, of shc, and an association of shc to the Grb2/son of sevenless 2 complex. In contrast, IgE-mediated activation resulted in transient activation of p21(ras) and mitogen-activated protein/ERK kinase 1, which were kinetically associated with phosphorylation of ERKs. The canonical Shc/Grb2/son of sevenless pathway to activation of p21(ras) is therefore sustained, while p21(ras) activity is not. We have previously shown that phosphatidylinositol 3 kinase activity is required for p21(ras) activity and, in the current studies, we show that of the p85-sensitive forms of p110 possible, basophils express only p110 delta and that there are no changes in association between p21(ras) and p110 delta in stimulated basophils. We used the generation of phospho-Akt as a marker of the presence of phosphatidylinositol-3,4,5-trisphosphate and found that phospho-Akt is transient on a time scale consistent with p21(ras) activity. On the basis of information obtained in these and other studies, we localize down-regulation of IgE-mediated LTC(4) secretion to a region of the signaling cascade antecedent to p21(ras) activation, downstream of phosphatidylinositol 3 kinase activity and probably involving regulation of phosphatidylinositol-3,4,5-trisphosphate levels.     

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.     

Multiple effector domains within SNT1 coordinate ERK activation and neuronal differentiation of PC12 cells

Differentiation of neuronal precursor cells in response to neurotrophic differentiation factors is accompanied by the activation of membrane-anchored SNT signaling adaptor proteins. Two classes of differentiation factors, the neurotrophins and fibroblast growth factors, induce rapid tyrosine phosphorylation of SNT1(FRS2alpha), which in turn enables SNT1 to recruit Shp2 tyrosine phosphatase and Grb2 adaptor protein in complex with the Ras GDP/GTP exchange factor Sos. To determine effector functions of SNT that promote neuronal differentiation of PC12 pheochromocytoma cells, we engineered a chimeric protein, SNT1(IRS)CX, bearing the effector region of SNT1 and the insulin receptor recognition domains of IRS2. Insulin promoted tyrosine phosphorylation of SNT1(IRS)CX in transfected PC12 cells accompanied by sustained activation of ERK1/2 mitogen-activated protein kinases and neuronal differentiation. The SNT1(IRS)CX-mediated response was dependent on endogenous Ras, MEK, and Shp2 activities. Mutagenesis of SNT1(IRS)CX identified three classes of effector motifs within SNT critical for both sustained ERK activation and neuronal differentiation: 1) four phosphotyrosine motifs that mediate recruitment of Grb2, 2) two phosphotyrosine motifs that mediate recruitment of Shp2, and 3) a C-terminal motif that functions by helping to recruit Sos. We discuss possible mechanisms by which three functionally distinct SNT effector motifs collaborate to promote a downstream biochemical and biological response.     

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.     

The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells

The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X₅)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and implicate this pseudophosphatase as a regulator of neuronal differentiation.     

Engagement of endogenous ganglioside GM1a induces tyrosine phosphorylation involved in neuron-like differentiation of PC12 cells

Using the cholera toxin B subunit (CTB) that specifically binds to ganglioside GM1a on the plasma membrane, we investigated intracellular signaling mediated by endogenous GM1a involved in neuronal differentiation of PC12 cells. The treatment with CTB induced morphological alternations of PC12 cells, such as augmentation of the cell body, neurite extension, and branched spikes of tips of neurites. The neurite extension induced with CTB was strongly suppressed by the pretreatment of tyrosine kinase inhibitors in a dose-dependent manner. Western blotting analysis showed that CTB induced tyrosine phosphorylation of several cellular proteins with molecular masses around 120, 70, and 45-40 kDa in PC12 cells. Some of the proteins identified were extracellular-signal regulated kinase (ERKs) (ERK1 and ERK2). The peak activation of ERKs lasted for 60-90 min and gradually decreased thereafter. Immunoprecipitation analysis demonstrated that the intracellular events induced with CTB are not related with the activation of Trk proteins, suggesting that signals evoked by ligation of endogenous GM1a are unique and distinct from those induced with exogenous GM1a. Although the presence of a tyrosine kinase inhibitor, genistein, at a concentration of 10 microM diminished the neurite extension of PC12 cells induced with CTB, ERK activation was still observed. However, pretreatment with a MEK inhibitor, PD98059, abolished the activation of ERKs induced with CTB in a dose-dependent manner and only attenuated the morphological alternations of PC12 cells. Considered together, we concluded that tyrosine phosphorylation induced with CTB was responsible for neuron-like differentiation of PC12 cells and that the MEK-ERK cascade is part of the biological signals mediated by endogenous ganglioside GM1a on PC12 cells.     

Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor,thereby modulating activation of MAP kinase,Akt, and neurite outgrowth in PC12 cells

In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk(A). cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.     

Apigenin and LY294002 prolong EGF-stimulated ERK1/2 activation in PC12 cells but are unable to induce full differentiation

In rat pheochromocytoma cell line (PC12) cells, initial epidermal growth factor (EGF)-stimulated extracellular signal-regulated protein kinases 1/2 (ERK1/2) phosphorylation was similar to that promoted by nerve growth factor (NGF), but declined rapidly. Pre-treatment with apigenin or LY294002 sustained EGF-stimulated ERK1/2 phosphorylation whereas wortmannin partially blocked initial ERK1/2 phosphorylation. Changes in ERK1/2 phosphorylation correlated with alterations in p90 ribosomal S6 kinase activity. Wortmannin, LY294002 and apigenin totally blocked growth factor-induced protein kinase B phosphorylation. However, none of them potentiated Raf activation, which was in fact decreased by LY290042 and wortmannin. The sustained EGF-induced ERK1/2 activation promoted by apigenin was not sufficient to commit PC12 cells to differentiate, which was achieved by stimulation with NGF, either alone or in the presence of apigenin.     

Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase.

Endothelin-1 (ET-1) induces cell proliferation and differentiation through multiple G-protein-linked signaling systems, including p21ras activation. Whereas p21ras activation and desensitization by receptor tyrosine kinases have been extensively investigated, the kinetics of p21ras activation induced by engagement of G-protein-coupled receptors remains to be fully elucidated. In the present study we show that ET-1 induces a biphasic activation of p21ras in rat glomerular mesangial cells. The first peak of activation of p21ras, at 2-5 min, is mediated by immediate association of phosphorylated Shc with the guanosine exchange factor Sos1 via the adaptor protein Grb2. This initial activation of p21ras results in activation of the extracellular signal-regulated kinase (ERK) cascade. We demonstrate that ET-1 signaling elicits a negative feedback mechanism, modulating p21ras activity through ERK-dependent Sos1 phosphorylation, findings which were confirmed using an adenovirus MEK construct. Subsequent to p21ras and ERK deactivation, Sos1 reverts to the non-phosphorylated condition, enabling it to bind again to the Grb2/Shc complex, which is stabilized by persistent Shc phosphorylation. However, the resulting secondary activation of p21ras at 30 min does not lead to ERK activation, correlating with intensive, ET-1-induced expression of MAP kinase phosphatase-1, but does result in increased p21ras-associated phosphatidylinositol 3-kinase activity. Our data provide evidence that ET-1-induced biphasic p21ras activation causes sequential stimulation of divergent downstream signaling pathways.     

Epidermal growth factor induces phosphorylation of extracellular signal-regulated kinase 2 via multiple pathways.

Expression of p21rasAsn-17, a dominant negative mutant of p21ras that blocks p21ras activation by growth factors, inhibits activation of extracellular signal-regulated kinase 2 (ERK2) by insulin and platelet-derived growth factor in rat-1 cells [A. M. M. de Vries-Smits, B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos, Nature (London) 357:602-604, 1992]. Here we report that expression of p21rasAsn-17 does not abolish epidermal growth factor (EGF)-induced phosphorylation of ERK2 in fibroblasts. Since EGF activates p21ras in these cells, this indicates that EGF induces a p21ras-independent pathway for the phosphorylation of ERK2 as well. We investigated whether activation of protein kinase C (PKC) or increase in intracellular calcium could be involved in p21ras-independent signaling. In rat-1 cells, inhibition of either PKC, by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment, or calcium influx, by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) pretreatment, did not abolish EGF-induced ERK2 phosphorylation. However, a combined inhibition of both p21ras and calcium influx, but not PKC, resulted in a complete inhibition of EGF-induced ERK2 phosphorylation. In contrast, in Swiss 3T3 cells, inhibition of both p21ras activation and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation. These results demonstrate that in fibroblasts, EGF induces alternative pathways of ERK2 phosphorylation in a cell-type-specific manner.     

Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.