Eun Joo Jung

Autophagy protects the rotenone-induced cell death in α-synuclein overexpressing SH-SY5Y cells

Loss of dopaminergic cells induced by alpha-synuclein accumulation in substantia nigra causes the development of Parkinsons disease (PD). To date, although autophagy has been implicated in the pathology of PD, the molecular mechanism is still unclear. To study the role of autophagy in PD pathogenesis, we established stable SH-SY5Y cell lines overexpressing wild-type or mutant alpha-synuclein proteins (A30P or A53T). Overexpression of mutant alpha-synuclein induced some protein aggregates and cell death in the absence of drug. LC3-II protein, a critical marker for autophagy, was produced in an autophagy-dependent manner. The rotenone-induced cell death was interrupted by autophagy stimulation. Autophagy activation also restored the mitochondrial membrane potential (MMP) impaired by rotenone in mutant alpha-synuclein expressing cells. Additionally, autophagy activation significantly relieved rotenone-induced ROS accumulation and HIF-1alpha expression in neuronal cells expressing mutant alpha-synuclein proteins. These findings indicate that autophagy plays an important scavenger role against harmful influence of toxic protein aggregates produced in rotenone-treated cells.

Cytosolic accumulation of γH2AX is associated with tropomyosin-related kinase A-induced cell death in U2OS cells

Tropomyosin-related kinase A (TrkA) plays an important role in cell survival, differentiation, and apoptosis in various neuronal and nonneuronal cell types. Here we show that TrkA overexpression by the Tet-On system mimics NGF-mediated activation pathways in the absence of nerve growth factor (NGF) stimulation in U2OS cells. In addition, p53 upregulation upon DNA damage was inhibited by TrkA, and p21 was upregulated by TrkA in a p53-independent manner. TrkA overexpression caused cell death by interrupting cell cycle progression, and TrkA-induced cell death was diminished in the presence of its specific inhibitor GW441756. Interestingly, TrkA-mediated cell death was strongly related to γH2AX production and poly (ADP-ribose) polymerase cleavage in the absence of DNA damage inducer. In this study, we also reveal that γH2AX production by TrkA is blocked by TrkA kinase inhibitors K-252a and GW441756, and it is also significantly inhibited by JNK inhibitor SP600125. Moreover, reduction of cell viability by TrkA was strongly suppressed by SP600125 treatment, suggesting a critical role of JNK in TrkA-induced cell death. We also found that γH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, and the nuclear localization of γH2AX induced by DNA damage was partly altered to cytosol by TrkA overexpression. Our results suggest that the abnormal cytosolic accumulation of γH2AX is implicated in TrkA-induced cell death in the absence of DNA damage.

Interplay between autophagy and apoptosis in TrkA-induced cell death

Autophagy is a self-eating process to eradicate damaged proteins or organelles in cells. This process begins with formation of a double-membrane structure, called an autophagosome, which can sequester soluble proteins and organelles eventually degraded by lysosomal proteases after fusion with the lysosome. Autophagy was initially identified as a cell survival mechanism under stress conditions such as nutrient deprivation. More recently, it is also considered as type-II programmed cell death. In our recent report, we observed that overexpression of TrkA caused massive cell death via both apoptosis and autophagy. Overexpression of TrkA abated catalase activity and subsequently resulted in the production of a large amount of reactive oxygen species in cells. These consequences led to autophagic cell death. The autophagic cell death in TrkA-overexpressing cells was validated by GFP-LC3 dot formation, production of autophagosomes or acidic vacuoles, LC3 lipidation, and depletion of autopahgy-related genes. In addition, we also observed some evidence for apoptosis in TrkA-expressing cells. Many cells expressing TrkA exhibited annexin V-positive staining, activation of caspase-7 and BAX. Moreover, TrkA activated the JNK pathway, leading to phosphorylation of H2AX. In this report, we suggest that two cell death mechanisms occur simultaneously and interlink with each other. The JNK-calpain pathway might be a central process to mediate the two processes in TrkA-overexpressing cells, although further study still remains to prove the interplay between autophagy and apoptosis.

Cloning and characterization of a chicken protein tyrosine phosphatase, CPTP1

Protein tyrosine phosphorylation and dephosphorylation are an important regulatory reactions in cell physiology. We have cloned a cDNA that encode a cytosolic protein tyrosine phosphatase (CPTP1) from chicken intestine cDNA library. Amino acid sequence identity between the CPTP1 and low molecular weight form of human placenta enzyme (HPTP1B) was 92%. CPTP1 lacked 13 amino acids in N-terminal region, while it had an additional 48 amino acids in the C-terminal region in comparison with the truncated form of HPTP1B of 321 amino acids. This C-terminal sequence was different from those of all known PTPs. The CPTP1 does not have a membrane targeting or nuclear localization sequences at its C-terminus like other PTPs such as HPTP1B and murine homolog of the human T-cell protein tyrosine phosphatase (MPTP) do. The cloned cDNA has been expressed in E. coli and purified by affinity chromatography. Dephosphorylation kinetics of this enzyme closely resembled those of the known PTPs. The dephosphorylation reaction required a reducing agent such as glutathione and dithiothreitol and was inhibited by sodium vanadate and formaldehyde. Deletion of 72 amino acids from C-terminal side of CPTP1 gene resulted in higher expression in E. coli and more potent phosphatase activity than wild type CPTP1 gene product. This result suggests that the C-terminal region of the CPTP1 protein negatively regulates phosphatase activity. These results also imply that CPTP1 might be a nontransmembrane-type enzyme with a structure and localization specificity distinct from other known cytosolic PTP1B type homolog.

Control of TrkA-induced cell death by JNK activation and differential expression of TrkA upon DNA damage

TrkA, a receptor for nerve growth factor, plays a crucial role in neuronal cell growth and differentiation. However, overactivation of TrkA signaling leads to cell death in various cell types. TrkA-mediated cell death shows some similarities to DNA damage-induced cell death. In this study, we examined how TrkA-induced cell death is regulated upon DNA damage. Cytoplasmic expression of TrkA protein was differentially modulated during the camptothecin-induced DNA damage response in TrkA-expressing U2OS cells. TrkA-induced cell death was synergistically increased by DNA damage, but it was blocked in the presence of the JNK inhibitor SP600125. Overexpression of a 54-kDa JNK isoform (JNK1α2) aggravated TrkA-induced cell death and was associated with TrkA functional activation. These results suggest that TrkA shares a functional connection with other mediators in the DNA damage response via JNK signaling.

Phosphorylation of chicken protein tyrosine phosphatase 1 by casein kinase II in vitro

The phosphorylation and dephosphorylation of proteins on tyrosyl residues are key regulatory mechanisms of cell growth and signal transduction and are controlled by opposing activities of protein tyrosine kinases and phosphotyrosyl phosphatases (PTPs). We have previously cloned and characterized a nontransmembrane chicken protein tyrosine phosphatase 1 (CPTP1) similar to human placental PTP1B (HPTP1B). CPTP1 contains several phosphorylation sequence motifs (S/T-X-X-D/E) for casein kinase II (CKII), [(I>E>V)-Y-(E>G)-(E>D>P>N)-(I/V>L)] for p56(lck), and (P-E-S-P) for MAP kinase. To examine whether phosphatase activity of CPTP1 could be controlled by phosphorylation, CPTP1 and HPTP1B fusion proteins purified from E. coli were subjected to the in vitro phosphorylation by CKII. Phosphoamino acid analysis revealed that CPTP1 was phosphorylated on both serine and threonine residues by CKII in vitro. In addition, the degree of the phosphorylation of CPTP1 by CKII was shown to be five times higher than that of HPTP1B. Phosphorylation on both serine and threonine residues of CPTP1 in vitro results in an inhibition of its phosphatase activity. This result suggests that phosphorylation of CPTP1 and HPTP1B by CKII might be implicated in the regulation of their catalytic activities in the cell.

Proteomic analysis of novel targets associated with TrkA-mediated tyrosine phosphorylation signaling pathways in SK-N-MC neuroblastoma cells

Tropomyosin‐related kinase A (TrkA) is a receptor‐type protein tyrosine kinase and exploits pleiotypic roles via nerve growth factor (NGF)‐dependent or NGF‐independent mechanisms in various cell types. Here, we showed that the inhibition of TrkA activity by GW441756 resulted in the suppression of tyrosine phosphorylation of cellular proteins including extracellular signal‐regulated protein kinase (ERK) and c‐Jun N‐terminal kinase (JNK). To find novel targets associated with TrkA‐mediated tyrosine phosphorylation signaling pathways, we investigated GW441756 effects on TrkA‐dependent targets in SK‐N‐MC neuroblastoma cells by proteomic analysis. The major TrkA‐dependent protein spots controlled by GW441756 were determined by PDQuest image analysis, identified by MALDI‐TOF MS and MALDI‐TOF/TOF MS/MS, and verified by 2DE/Western blot analysis. Thus, we found that most of the identified protein spots were modified forms in a normal condition, and their modifications were regulated by TrkA activity. Especially, our results demonstrated that the modifications of α‐tubulin and heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C1/C2) were significantly upregulated by TrkA, whereas α‐enolase modification was downregulated by TrkA, and it was suppressed by GW441756, indicating that TrkA activity is required for their modifications. Taken together, we suggest here that the major novel TrkA‐dependent targets such as α‐tubulin, hnRNP C1/C2, and α‐enolase could play an essential role in TrkA‐mediated tyrosine phosphorylation signaling pathways via regulation of their posttranslational modifications.

Caveolin-1 inhibits TrkA-induced cell death by influencing on TrkA modification associated with tyrosine-490 phosphorylation

Caveolin-1, a main structural protein constituent of caveolae, plays an important role in the signal transduction, endocytosis, and cholesterol transport. In addition, caveolin-1 has conflictive role in the regulation of cell survival and death depending on intracellular signaling pathways. The receptor tyrosine kinase TrkA has been known to interact with caveolin-1, and exploits multiple functions such as cell survival, death and differentiation. In this report, we investigated how TrkA-induced cell death signaling is regulated by caveolin-1 in both TrkA and caveolin-1 overexpressing stable U2OS cells. Here we show that TrkA co-localizes with caveolin-1 mostly as a large aggresome around nucleus by confocal immunofluorescence microscopy. Interestingly, TrkA-mediated Bak cleavage was suppressed by caveolin-1, indicating an inhibition of TrkA-induced cell death signaling by caveolin-1. Moreover, caveolin-1 altered TrkA modification including tyrosine-490 phosphorylation and unidentified cleavage(s), resulting in the inhibition of TrkA-induced apoptotic cell death. Our results suggest that caveolin-1 could suppress TrkA-mediated pleiotypic effects by altering TrkA modification via functional interaction.

Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

We previously reported that TrkA overexpression causes accumulation of γH2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we established TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and γH2AX in TrkA-induced cell death. γH2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.

Identification of simvastatin-regulated targets associated with JNK activation in DU145 human prostate cancer cell death signaling

The results of this study show that c-Jun N-terminal kinase (JNK) activation was associated with the enhancement of docetaxel-induced cytotoxicity by simvastatin in DU145 human prostate cancer cells. To better understand the basic molecular mechanisms, we investigated simvastatin-regulated targets during simvastatin-induced cell death in DU145 cells using two-dimensional (2D) proteomic analysis. Thus, vimentin, Ras-related protein Rab-1B (RAB1B), cytoplasmic hydroxymethylglutaryl-CoA synthase (cHMGCS), thioredoxin domain-containing protein 5 (TXNDC5), heterogeneous nuclear ribonucleoprotein K (hnRNP K), N-myc downstream-regulated gene 1 (NDRG1), and isopentenyl-diphosphate Delta-isomerase 1 (IDI1) protein spots were identified as simvastatin-regulated targets involved in DU145 cell death signaling pathways. Moreover, the JNK inhibitor SP600125 significantly inhibited the upregulation of NDRG1 and IDI protein levels by combination treatment of docetaxel and simvastatin. These results suggest that NDRG1 and IDI could at least play an important role in DU145 cell death signaling as simvastatin-regulated targets associated with JNK activation.