Active p21Ras is sufficient for rescue of NGF-dependent rat sympathetic neurons

Nobes, Catherine D.; Reppas, John B.; Markus, Annette; Tolkovsky, A M

doi:10.1016/0306-4522(95)00420-3

Cited by 46 publications

(34 citation statements)

References 43 publications

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“…Abolition of NAC-promoted survival with PD98059 f urther indicates a necessary role for M EK activity and is consistent with a role for ERK activation as well (X ia et al, 1995). The implication of Ras activation in NAC -promoted neuronal survival is consistent with reports that a constitutively active form of Ras rescues NGFdeprived sympathetic neurons and PC12 cells (Rukenstein et al, 1991;Nobes et al, 1996).…”

Section: Mechanism By Which Nac Protects Neurons From Loss Of Trophicsupporting

confidence: 90%

Prevention of PC12 Cell Death byN-Acetylcysteine Requires Activation of the Ras Pathway

1998

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We have shown that N-acetylcysteine (NAC) promotes survival of sympathetic neurons and pheochromocytoma (PC12) cells in the absence of trophic factors. This action of NAC was not related to its antioxidant properties or ability to increase intracellular glutathione levels but was instead dependent on ongoing transcription and seemed attributable to the action of NAC as a reducing agent. Here, we investigate the mechanism by which NAC promotes neuronal survival. We show that NAC activates the Ras-extracellular signal-regulated kinase (ERK) pathway in PC12 cells. Ras activation by NAC seems necessary for survival in that it is unable to sustain serum-deprived PC12 MM17-26 cells constitutively expressing a dominantnegative form of Ras. Promotion of PC12 cell survival by NAC is totally blocked by PD98059, an inhibitor of the ERKactivating MAP kinase/ERK kinase, suggesting a required role for ERK activation in the NAC mechanism. In contrast, LY294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI3K) that partially block NGF-promoted PC12 cell survival, have no effect on prevention of death by NAC. We hypothesized previously that the ability of NAC to promote survival correlates with its antiproliferative properties. However, although NAC does not protect PC12 MM17-26 cells from loss of trophic support, it does inhibit their capacity to synthesize DNA. Thus, the antiproliferative effect of NAC does not require Ras activation, and inhibition of DNA synthesis is insufficient to mediate NAC-promoted survival. These findings highlight the role of Ras-ERK activation in the mechanism by which NAC prevents neuronal death after loss of trophic support.

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Section: Mechanism By Which Nac Protects Neurons From Loss Of Trophicsupporting

confidence: 90%

Prevention of PC12 Cell Death byN-Acetylcysteine Requires Activation of the Ras Pathway

1998

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“…The MAPK pathway has been also implicated in the control of nerve growth factor (NGF)- and IGF-1-induced survival in rat sympathetic neuron cells [34]. In the present study, PD98059, a MEK1 inhibitor, had no effect on the anti-apoptotic effects of either serum or troglitazone.…”

Section: Discussioncontrasting

confidence: 44%

“…In the present study, we showed that wortmannin partially inhibited the anti-apoptotic effect of troglitazone, which suggested existence of the PI3K-dependent pathway. Alteration of the PI3K-dependent pathway, due to serum deprivation upstream of PI3K, may be caused by targeting of IGF-1 receptor [34]and IRS by PPARγ.…”

Section: Discussionmentioning

confidence: 99%

Activation of Peroxisome Proliferator-Activated Receptor-g Inhibits Apoptosis Induced by Serum Deprivation in LLC-PK1 Cells

Haraguchi

Shimura²,

Onaya³

2002

Nephron Exp Nephrol

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Peroxisome proliferator-activated receptor-γ (PPARγ) belongs to a superfamily of nuclear receptors, which plays important roles in lipid and glucose metabolism. However, expression of PPARγ in extra-adipose tissues and stimulation of apoptosis by PPARγ activators has been previously reported. We investigated the functions of PPARγ using a clonal kidney cell line (LLC-PK1). RT-PCR revealed the expression of PPARγ in LLC-PK1 cells. The cells accumulated fat droplets and increased β-oxidation of free fatty acids in response to troglitazone, a ligand for PPARγ. At physiological concentrations, ligands for PPARγ including troglitazone, BRL49653, and 15-deoxy-δ-12,14-prostaglandin J₂ inhibited serum-deprivation-induced apoptosis of the cells. On the other hand, PPARα activators did not inhibit the apoptosis. Apoptosis of LLC-PK1 cells was determined by a cell viability assay, condensation of the nucleus on fluorescent and electron microscopy, and DNA fragmentation as indicated by the appearance of nucleosomal ladders on an agarose gel. Troglitazone also suppressed serum-deprivation-induced activation of Caspase 3. However, troglitazone did not suppress apoptosis induced by ATP deprivation. Anti-apoptotic effects of troglitazone were partially blocked by a phosphatidylinositol-3-kinase (PI3K) inhibitor, wortmannin, but not by other kinase inhibitors such as PD98059 and AG490. These results suggest that PPARγ is functionally expressed in LLC-PK1 cells, and its activation inhibits apoptosis induced by serum deprivation, at least in part, through the PI3K pathway.

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“…Radiolabeling, Inhibition of MEK, and NGF Stimulation-For 2D gel work, SCG neurons (about 200,000/dish) were plated for 1 h in phosphate-free RPMI 1640 medium containing 3% dialyzed rat serum (to remove any inorganic phosphate) and 0.5 mM CPTcAMP (to enable neuronal attachment to the substrate in the absence of any MEK/ERK stimulation (10,12,15)). Neurons were then labeled in the same medium for 3 h with 32 P (ϳ200 Ci/dish) or 33 P (ϳ500 Ci/dish) in the presence of 1 mM araC after which the MEK inhibitor U0126 (10 M final concentration) or an equivalent amount of DMSO (0.5%) (control) was added.…”

Section: Materials-mentioning

confidence: 99%

Differential Phosphoprotein Labeling (DIPPL), a Method for Comparing Live Cell Phosphoproteomes Using Simultaneous Analysis of 33P- and 32P-Labeled Proteins

Wyttenbach

Tolkovsky

2006

Molecular & Cellular Proteomics

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We developed a differential method to reveal kinase-specific phosphorylation events in live cells. In this method, cells in which the specified kinase is inactive are labeled with 32 Pi, whereas cells in which the kinase is active are labeled with 33 Pi. The two cell extracts are then mixed, and proteins are separated on a single two-dimensional gel. The dried gel is exposed twice. The first exposure reveals both 32 P-and 33 P-labeled proteins; the kinasespecific spots are revealed because of 33 P labeling. The second exposure is conducted with two acetate sheets intervening between the gel and the detection plate. This maneuver screens out the less energetic 33 P-labeled proteins while allowing the more energetic 32 P-labeled proteins to be detected, thus leaving only those spots that were phosphorylated independently of the specified kinase. We demonstrate the utility of this method for detecting kinase substrates in rare tissue by focusing on extracellular signal-regulated kinase-specific phosphorylation of stathmin/OP18 in primary rat sympathetic neurons. Molecular & Cellular Proteomics 5:553-559, 2006.Phosphorylation is a major reversible protein modification. It regulates a myriad of protein functions including enzyme activity, protein-protein interaction, cellular localization, and protein degradation. It is estimated that there are about 100,000 potential phosphorylation sites in the human proteome of which fewer than 2000 are currently known (1). In addition to identification of phosphorylation sites, there is a need for quantitation of phosphorylation events especially with regard to understanding the regulation of signal transduction. Although novel and sophisticated methods have been developed to enrich for phosphoproteins or phosphopeptides prior to analysis by mass spectrometry, this approach requires large amounts of protein and so is not always feasible when using rare tissue. Moreover from the standpoint of systems biology, it may be useful to acquire an image of protein phosphorylation of the entire proteome prior to homing in on specific proteins.Labeling cells with phosphorus-32 ( 32 P) has long been used as a means for identifying phosphoproteins. Principle ␤ emission energy for 32 P is 1.709 MeV, making it highly sensitive, and its rapid uptake into cellular ATP makes it very versatile. Although it is possible to run simultaneous gels to compare changes in phosphoprotein profiles between differentially treated samples, it would be an advantage to be able to discriminate changes in phosphorylation between two samples on a single 2D 1 gel just as DIGE is used to highlight changes in protein expression between two samples while eliminating the variability of protein separation patterns (2). We previously noted that it is possible to prelabel cellular proteins metabolically with [35 S]methionine (principle ␤ emission, 0.167 MeV) and then label the same cells with 32 P to detect which of these proteins are phosphorylated; as 35 S emission has lower energy it is possible to screen out the lower...

show abstract

Active p21Ras is sufficient for rescue of NGF-dependent rat sympathetic neurons

Cited by 46 publications

References 43 publications

Prevention of PC12 Cell Death byN-Acetylcysteine Requires Activation of the Ras Pathway

Prevention of PC12 Cell Death byN-Acetylcysteine Requires Activation of the Ras Pathway

Activation of Peroxisome Proliferator-Activated Receptor-g Inhibits Apoptosis Induced by Serum Deprivation in LLC-PK1 Cells

Differential Phosphoprotein Labeling (DIPPL), a Method for Comparing Live Cell Phosphoproteomes Using Simultaneous Analysis of 33P- and 32P-Labeled Proteins

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