Characterization of nitroproteome in neuron-like PC12 cells differentiated with nerve growth factor: identification of two nitration sites in alpha-tubulin

Nitric oxide (NO) is a precursor of reactive nitrating species, peroxynitrite and nitrogen dioxide, which modify proteins to generate oxidized species such as 3-nitrotyrosine that has been used as a hallmark of peroxynitrite-mediated oxidative stress on proteins. In the last few years however, a growing body of evidence indicates that NO also regulates a myriad of physiologic responses by modifying tyrosine residues. Looking for the molecular event triggered by NO in nerve growth factor (NGF)-induced neuronal differentiation, we recently reported that in differentiating PC12 cells, the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and alpha-tubulin is the major target. In the present work, we focus on the investigation of the sites of tyrosine nitration in alpha-tubulin purified by two-dimensional gel electrophoresis following anti-alpha-tubulin immunoprecipitation of protein extract from NGF-treated PC12 cells. Using Western blotting and matrix-assisted laser desorption/ionization-time of flight analysis, we show for the first time, both in vivo and in vitro, that nitration can occur on alpha-tubulin at sites other than the C-terminus and we positively identify Tyr 161 and Tyr 357 as two specific amino acids endogenously nitrated.     

Up-regulation of protein tyrosine nitration in methamphetamine-induced neurotoxicity through DDAH/ADMA/NOS pathway

Protein tyrosine nitration is an important post-translational modification mediated by nitric oxide (NO) associated oxidative stress, occurring in a variety of neurodegenerative diseases. In our previous study, an elevated level of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protein was observed in different brain regions of acute methamphetamine (METH) treated rats, indicating the possibility of an enhanced expression of protein nitration that is mediated by excess NO through the DDAH1/ADMA (Asymmetric Dimethylated l-arginine)/NOS (Nitric Oxide Synthase) pathway. In the present study, proteomic methods, including stable isotope labeling with amino acids in cell culture (SILAC) and two dimensional electrophoresis, were used to determine the relationship between protein nitration and METH induced neurotoxicity in acute METH treated rats and PC12 cells. We found that acute METH administration evokes a positive activation of DDAH1/ADMA/NOS pathway and results in an over-production of NO in different brain regions of rat and PC12 cells, whereas the whole signaling could be repressed by DDAH1 inhibitor N^ω-(2-methoxyethyl)-arginine (l-257). In addition, enhanced expressions of 3 nitroproteins were identified in rat striatum and increased levels of 27 nitroproteins were observed in PC12 cells. These nitrated proteins are key factors for Cdk5 activation, cytoskeletal structure, ribosomes function, etc. l-257 also displayed significant protective effects against METH-induced protein nitration, apoptosis and cell death. The overall results illustrate that protein nitration plays a significant role in the acute METH induced neurotoxicity via the activation of DDAH1/ADMA/NOS pathway.     

Tyrosine Nitration is a Novel Post-translational Modification Occurring on the Neural Intermediate Filament Protein Peripherin

The biological implication of protein tyrosine nitration in signaling pathways triggered by nitric oxide is recently emerging. Here we report for the first time that nitrotyrosination occurs in the neural intermediate filament protein peripherin. In neuron-like PC12 cells, nitrated peripherin is associated with the cytoskeleton fraction, its level increases during the progression of NGF-induced differentiation and the nitrated protein remains closely associated with stable microtubules. Tyr 17 and Tyr 376 were identified by MALDI-TOF analyses as two specific residues endogenously nitrated. Finally, peripherin nitration is not restricted to PC12 cells but it is also present in vivo in rat brain. Gabriella Tedeschi and Graziella Cappelletti contributed equally to this work.     

Redox regulation of nerve growth factor-induced neuronal differentiation of PC12 cells through modulation of the nerve growth factor receptor, TrkA

We investigated the effects of the cellular redox state on nerve growth factor (NGF)-induced neuronal differentiation and its signaling pathways. Treatment of PC12 cells with buthionine sulfoximine (BSO) reduced the levels of GSH, a major cellular reductant, and enhanced NGF-induced neuronal differentiation, activation of AP-1 and the NGF receptor tyrosine kinase, TrkA. Conversely, incubation of the cells with a reductant, N-acetyl-L-cysteine (NAC), inhibited NGF-induced neuronal differentiation and AP-1 activation. Consistent with the suppression, NAC inhibited NGF-induced activation of TrkA, formation of receptor complexes comprising TrkA, Shc, Grb2, and Sos, and activation of phospholipase Cgamma and phosphatidylinositol 3-kinase. Biochemical analysis suggested that the cellular redox state regulates TrkA activity through modulation of protein tyrosine phosphatases (PTPs). Thus, cellular redox state regulates signaling pathway of NGF through PTPs, and then modulates neuronal differentiation.     

Rapid auxin-induced nitric oxide accumulation and subsequent tyrosine nitration of proteins during adventitious root formation in sunflower hypocotyls

Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25–80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development.     

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.     

The nitration of tau protein in neurone-like PC12 cells

Tyrosine nitration of proteins is emerging as a post-translational modification playing a role in physiological conditions. Looking for the molecular events triggered by nitric oxide in nerve growth factor-induced neuronal differentiation, we now find that nitration occurs on the microtubule-associated protein tau. In differentiated PC12 cells, we have identified as tau a nitrated protein that co-immunoprecipitates with alpha-tubulin and indicated that the modified protein is associated with the cytoskeleton but it is confined to a restricted cell region. This paper supplies the first evidence that nitration of tau occurs in a physiological process and suggests that it could play a role in neuronal differentiation.     

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.     

Immunolocalization of S-nitrosoglutathione, S-nitrosoglutathione reductase and tyrosine nitration in pea leaf organelles

S-Nitrosoglutathione (GSNO) is a nitrosothiol which plays a major role in the metabolism of NO in higher plants mediating signaling processes. Protein tyrosine nitration (NO₂–Tyr) is a post-translational modification which contributes to protein regulation. The subcellular localization of GSNO, S-nitrosoglutathione reductase (GSNOR), an enzyme which catalyzes its decomposition and protein tyrosine nitration was studied in pea (Pisum sativum L.) leaf plants with the aid of the electron microscopy immunogold-labeling technique. Our findings show that GSNO, GSNOR and nitrated proteins are present in the different subcellular compartments of leaf cells which include chloroplasts, cytosol, mitochondria, and peroxisomes. Given that pea peroxisomes are one of the cell compartments where nitric oxide (NO) has been thoroughly studied, our results provide additional insights into the metabolism of NO in this organelle where NO and GSNO could function as signal molecules in cross talk between the different cell compartments.     

Borna disease virus persistent infection activates mitogen-activated protein kinase and blocks neuronal differentiation of PC12 cells

Persistence of Borna disease virus (BDV) in the central nervous system causes damage to specific neuronal populations. BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal differentiation in response to NGF. We report that persistence of BDV led to a progressive change of phenotype of PC12 cells and blocked neurite outgrowth in response to NGF. Infection down-regulated the expression of synaptophysin and growth-associated protein-43, two molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the block in response to NGF was due in part to the down-regulation of NGF receptors. Moreover, although BDV caused constitutive activation of the ERK1/2 pathway, activated ERKs were not translocated to the nucleus efficiently. These observations may account for the absence of neuronal differentiation of persistently infected PC12 cells treated with NGF.     

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.     

Transient Association of the Phosphotyrosine Phosphatase SHP-2 with TrkA Is Induced by Nerve Growth Factor

Abstract: Nerve growth factor (NGF) treatment of rat PC12 pheochromocytoma cells results in an increase in the tyrosine phosphorylation of the NGF receptor, TrkA, leading to differentiation to a neuronal phenotype. Dephosphorylation by protein tyrosine phosphatases (PTPases) is thought to play an important role in regulating this signaling pathway. To identify PTPases that are recruited to the activated TrkA receptor, we used an ingel PTPase assay to examine the presence of PTPases in TrkA immunoprecipitates. The Src homology 2 domain containing PTPase SHP-2 was found to associate transiently with TrkA following receptor activation, reaching a peak after 1 min of NGF treatment and then decreasing rapidly. The association of SHP-2 with TrkA was accompanied by the tyrosine phosphorylation of SHP-2 and an association of SHP-2 with multiple tyrosine-phosphorylated proteins. In addition, the PTPase activity in SHP-2 immunoprecipitates increased greater than twofold after 1 min of NGF treatment. This is the first demonstration that the association of SHP-2 with TrkA is induced by NGF and that this association leads to SHP-2 activation and tyrosine phosphorylation. We conclude that SHP-2 plays a significant role in early biochemical events in TrkA-mediated signal transduction.     

Neuronal differentiation signals are controlled by nerve growth factor receptor/Trk binding sites for SHC and PLC gamma.

Differentiation and survival of neuronal cell types requires the action of neurotrophic polypeptides such as nerve growth factor (NGF). In the central and peripheral nervous system and the phaeochromocytoma cell model PC12, NGF exerts its effects through the activation of the signalling capacity of Trk, a receptor tyrosine kinase (RTK) which upon interaction with NGF becomes phosphorylated on tyrosines and thereby acquires the potential to interact with signal-transducing proteins such as phospholipase C-gamma (PLC gamma), phosphatidylinositol-3'-kinase (PI3'-K) and SHC. Mutagenesis of the specific binding sites for these src homology 2 (SH2) domain-containing substrates within the Trk cytoplasmic domain suggests a non-essential function of PI3'-K and reveals a major role for the signal controlled by the SHC binding site at tyrosine 490 and a co-operative function of the PLC gamma-mediated pathway for neuronal differentiation of PC12 cells.     

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.     

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.