©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Neuroprotective Compound, Aurin Tricarboxylic Acid, Stimulates the Tyrosine Phosphorylation Cascade in PC12 Cells (*)

Noriko Okada , Shinichi Koizumi (§)

From the (1)Bio-Organic Research Department, International Research Laboratories, Ciba-Geigy (Japan) Limited, 10-66 Miyukicho, Takarazuka 665, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Aurin tricarboxylic acid (ATA), a general nuclease inhibitor, was reported to prevent PC12 cells from cell death caused by serum starvation(1) . In our study, ATA also protected PC12 cells, but not NIH3T3 cells, from serum-starved cell death. When we investigated the mechanism of action of ATA on these cells, ATA was found to increase tyrosine phosphorylation in PC12 cells, but not in NIH3T3 cells. Further investigation on tyrosine-phosphorylated proteins revealed that ATA, similar to nerve growth factor and epidermal growth factor, induced tyrosine phosphorylation of mitogen-activated protein kinases. Since the tyrosine phosphorylation of mitogen-activated protein kinases is thought to play an important role in growth factor-dependent signal pathways, this finding suggests that the action of ATA on PC12 cells is mediated by tyrosine phosphorylation cascade, similar to growth factor signaling. In addition, we found that Shc proteins, phosphatidylinositol 3-kinase, and phospholipase C- were also phosphorylated in ATA-treated PC12 cells. These key proteins in signal transduction pathways are known to associate with ligand-activated growth factor receptors and are phosphorylated on tyrosine. Thus, the phosphorylation of these three proteins by ATA stimulation supports the speculation that ATA activates a certain receptor tyrosine kinase.


INTRODUCTION

PC12 cells, derived from rat pheochromocytoma, are widely used as a model system to study the effects of neurotrophic factors, especially those of NGF.()Recent studies revealed that PC12 cells provide a good model system to study not only neuronal differentiation but also neuroprotection from cell death by NGF. Without trophic supports, PC12 cells will undergo apoptotic cell death.

The PC12 cell death under serum-free condition is accompanied by one of the apoptotic properties, DNA fragmentation(1, 2) . The protective effect of NGF is mediated by the activation of p140, a NGF receptor having protein tyrosine kinase activity, and the subsequent stimulation of cellular signal transduction pathways.

ATA can prevent the PC12 cell death caused by serum and NGF starvation (1). ATA, which is a general endonuclease inhibitor, has been thought to inhibit the internucleosomal DNA cleavage during the apoptosis(1) . ATA can also protect cultured sympathetic neurons from cell death caused by the absence of trophic supports(2) . In vivo, ATA protects hippocampal neurons from NMDA- and ischemia-induced death(3) . In addition, ATA has been reported to regulate the expression of -amyloid precursor protein in neuroblastoma(4) . Although a variety of mechanisms are proposed for the neuroprotective effect of ATA, all of this evidence indicates that ATA is a very interesting compound in terms of neuroprotective activity.

ATA is also known to inhibit macromolecule synthesis(5, 6, 7, 8) , nucleic acid-protein interaction(9) , and activities of other enzymes involved in cellular metabolism, for example glucose-6-phosphate dehydrogenase (5) and phosphofructokinase(10) . However, these inhibitory activities of ATA were defined in vitro. It is not clear whether ATA is able to penetrate the plasma membrane to exert these activities.

In this study, investigating the possibility that ATA affected the growth factor signaling pathway, we found that ATA could stimulate most of major signal transduction cascades which are stimulated by NGF and EGF in PC12 cells.


EXPERIMENTAL PROCEDURES

Materials

The MTT cell growth assay kit was purchased from Chemicon International Inc. (Temecula, CA). Monoclonal anti-MAPK (ERK1+2) antibody was from Zymed (San Francisco, SF), polyclonal anti-SHC antibody was from Transduction Laboratories (Lexington, KY), and monoclonal anti-phosphotyrosine antibody (4G10), polyclonal anti-PI 3-kinase antibody, and monoclonal anti-PLC- antibody were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-mouse Ig and anti-rabbit Ig horseradish peroxidase-linked F(ab`)2 fragments, and ECL Western blotting detection kit were from Amersham (Buckinghamshire, United Kingdom). Protein G-Sepharose beads were from Pharmacia LKB (Uppsala, Sweden). PC12 cells were generously provided by Dr. Gordon Guroff, National Institutes of Health. NIH3T3 cells were obtained from ATCC (Rockville, MD). ATA was from Sigma, EGF and bFGF were from Upstate Biotechnology, Inc. Recombinant human NGF was prepared with baculovirus expression system in insect cells and purified before use. DMEM and supplements were obtained from Life Technologies, Inc.

Cell Culture

PC12 cells were grown in 100- or 60-mm dishes in DMEM with 10% horse serum and 5% fetal bovine serum. NIH3T3 cells were grown in 60-mm dishes in DMEM with 10% fetal bovine serum. The cells were maintained at 37 °C in a humidified atmosphere with 5% CO.

Analysis of DNA Fragmentation

PC12 cells (approximately 5 10 cells) or NIH3T3 cells (approximately 1 10 cells) were incubated at 37 °C for indicated time under the experimental conditions. After the treatment, cells were harvested mechanically with a rubber policeman and collected by centrifugation at 650 g for 10 min at 4 °C. Cells were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5% Triton X-100) and kept on ice for 10 min. After centrifugation at 27,000 g for 10 min, the soluble DNA was isolated and extracted with TE-saturated phenol and phenol/chloroform (1:1), followed by ethanol-precipitation. The DNA was dissolved in TE buffer and incubated with RNase A at 37 °C overnight. All recovered soluble DNA per condition were subjected to 1.5% agarose gel electrophoresis and visualized by a UV transilluminator.

MTT Assay

The MTT assay was performed according to supplied procedures with some modification. The cells (approximately 5 10 cells/well) were cultured in 96-well culture plates to subconfluence in 50 µl of medium/well. After treatment of cells under experimental conditions, 10 µl of MTT solution was added to each well, and the plates were incubated at 37 °C for another hour in a CO incubator. Next, 100 µl of 0.04 N HCl/isopropanol was added to each well and the plates were shaken to dissolve the crystals of the tetrazolium salt. The results were quantified by measuring the absorbance at 570 nm with 650 nm as a reference.

Anti-phosphotyrosine Immunoblot of Whole Cell Lysates

Cells were grown in 60-mm dishes, and the medium was replaced with serum-free medium overnight. Unless otherwise stated, ATA, NGF, EGF, or bFGF was added directly to the medium and incubated for 5 min, to a final concentration of 100 µM, 100 ng/ml, 20 ng/ml, or 10 ng/ml, respectively. The medium was then removed, and cells were lysed with 200 µl of SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS w/v, 10% glycerol) containing 1 mM orthovanadate, a tyrosine phosphatase inhibitor. The lysates were sonicated for about 10 s to degrade the DNA. After determination of protein concentration in each preparation with BCA protein assay reagent (Pierce) and boiling with 5% of -mercaptoethanol, 25 µg of protein was applied on 8% SDS-PAGE. After transferring to a nitrocellulose membrane, blots were blocked with 5% bovine serum albumin, then probed with 0.5 µg/ml anti-phosphotyrosine antibody (4G10) for 1 h. After repeating the 5-min wash four times with washing buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% Nonidet P-40), blots were incubated with peroxidase-labeled anti-mouse Ig antibody at 1:2000 dilution for 1 h, followed by washing as described above. The resultants were visualized using ECL Western blotting detection kit by exposure to x-ray films (Kodak).

Immunoprecipitation

Cells grown in 100-mm dishes were replaced with serum-free medium overnight. Unless otherwise indicated, ATA, NGF, or EGF was directly added to the medium and incubated for 5 min, to a final concentration of 100 µM, 100 ng/ml, or 20 ng/ml, respectively. After removing the medium, cells were lysed with 500 µl of ice-cold TNE buffer (10 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, 10 µg/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 mM orthovanadate) on ice for 10 min and the lysates centrifuged at 12,000 g for 10 min. For immunoprecipitation with anti-phosphotyrosine antibody, the protein content in each supernatant was normalized in each experiment. The supernatants were rotated at 4 °C for 2 h with 20 µl of 50% (v/v) protein G-Sepharose beads. After the proteins nonspecifically bound to protein G were removed by centrifugation (1500 g, for 3 min), 5 µg of a primary antibody was added to supernatants and rotated at 4 °C for 2 h. Then 20 µl of protein G-Sepharose beads were added and incubated for 2 h at 4 °C while rotating. The immunocomplexes bound to the beads were washed 5 times with 500 µl of TNE buffer and collected by centrifugation. For immunoprecipitation with anti-phosphotyrosine antibody, the tyrosine-phosphorylated proteins bound to the antibody were eluted by rotating for 2 h at 4 °C with 20 µl of 100 µM phenylphosphate in TNE buffer, and 5 µl of 5 SDS-PAGE sample buffer containing 25% -mercaptoethanol (v/v) was added to the eluted solution. For immunoprecipitation with other antibodies, the proteins were solubilized in 20 µl of SDS-PAGE sample buffer. The samples were boiled after addition of -mercaptoethanol, to a final concentration of 5% (v/v), and loaded to SDS-polyacrylamide gel. The immunoblotting with the indicated antibody was performed as described as above. For reprobing the blots, the membrane was incubated for 30 min in 62.5 mM Tris-HCl buffer, pH 6.7, containing 2% of SDS and 10 mM -mercaptoethanol, blocked, and reprobed with the indicated antibody.


RESULTS

ATA Supports the Survival of PC12 Cells under Serum Starvation, but Not That of NIH3T3 Cells

ATA has been reported to prevent PC12 cells from death caused by serum deprivation(1) . We have confirmed the result by the MTT assay. Under serum-free conditions for 72 h, PC12 cells cultured in the presence of 100 ng/ml ATA showed a comparable survival rate to that in the presence of 100 ng/ml NGF (Fig. 1A). In the presence of 100 µM NGF, no sign of cell death was found by microscopic observation of cells under serum-free condition. The higher formation of MTT in the cells with serum than that with NGF or ATA is due to the serum-dependent proliferation of PC12 cells and not due to the partial cell death in the presence of NGF or ATA. Internucleosomal DNA fragmentation, which is a characteristic of apoptosis, was shown in the cell death of PC12 cells caused by serum starvation(2) . This DNA fragmentation was clearly blocked by ATA as well as NGF after 12 h incubation (Fig. 1B). On the other hand, ATA did not show any survival effect on mouse fibroblast NIH3T3 cells cultured in serum-free medium for 24 h (Fig. 2A). In addition, ATA did not block the DNA fragmentation in NIH3T3 cells which was also observed under serum-free conditions, whereas bFGF completely blocked it (Fig. 2B). These results indicated that the survival action of ATA has cell selectivity.


Figure 1: Effect of ATA on the cell death caused by serum starvation in PC12 cells. A, after cultured in indicated conditions for 72 h, the cell viabilities were measured by MTT assay as described under ``Experimental Procedures.'' The numbers of cell viability are expressed relative to those cultured in serum-supplemented medium (designated as 100%). Experimental data are mean ± S.D. (n = 6). B, PC12 cells were cultured in serum-supplemented medium (+serum), or serum-free medium (-) with 100 ng/ml NGF (+NGF) or 100 µM ATA (+ATA) for 12 h. The soluble DNA was isolated and analyzed as described under ``Experimental Procedures.'' Molecular size markers (in kilobase pairs) are indicated on the left.




Figure 2: Effect of ATA on the cell death caused by serum starvation in NIH3T3 cells. A, after cultured in indicated conditions for 24 h, the cell viabilities were measured by MTT assay as described under ``Experimental Procedures.'' The numbers of cell viability are expressed relative to those cultured in serum-supplemented medium (designated as 100%). Experimental data are means ± S.D. (n = 6). B, cells were cultured in serum-supplemented medium (+serum), or in serum-free medium (none) with 10 ng/ml bFGF (+bFGF), or 100 or 200 µM ATA (+ATA) for 12 h. The soluble DNA was isolated and analyzed as described under ``Experimental Procedures.'' Molecular size markers (in kilobase pairs) are indicated on the left.



ATA Increases Tyrosine Phosphorylation in PC12 Cells

It is known that tyrosine phosphorylation cascade plays an important role in the action of growth factors and trophic factors. Therefore, to determine whether ATA affects tyrosine phosphorylation of any protein in PC12 cells, we examined the profile of tyrosine-phosphorylated proteins in the lysate of PC12 cells treated with 100 µM ATA. After 5 min of treatment of cells, ATA clearly increased the tyrosine phosphorylation of several proteins, mainly 180-, 130-, 85-, and 60-kDa proteins, and the phosphorylation of each protein declined in 60 min (Fig. 3A). The profile of tyrosine-phosphorylated proteins of ATA-treated cells were different from that of NGF- or EGF-treated cells (Fig. 3B). In NIH3T3 cells, which ATA does not prevent from serum-starved cell death, bFGF increased the tyrosine phosphorylation of several proteins. However, ATA treatment did not increase tyrosine phosphorylation (Fig. 3C). The correlation between the survival response and the stimulation of tyrosine phosphorylation in PC12 cells and NIH3T3 cells suggests that the survival action of ATA on PC12 cells is mediated by the stimulation of tyrosine phosphorylation.


Figure 3: Effects of ATA on tyrosine phosphorylation in PC12 cells (A and B) and in NIH3T3 cells (C). Whole cell lysates were subjected to 8% SDS-PAGE and immunoblot with an anti-phosphotyrosine antibody as described under ``Experimental Procedures.'' A, PC12 cells treated with 100 µM ATA for indicated times; B, PC12 cells treated with 20 ng/ml EGF, 100 ng/ml NGF or ATA for 5 min; C, NIH3T3 cells treated with ATA or 10 ng/ml bFGF for 5 min. The arrowheads on the right indicate the major phosphorylated proteins by ATA (A) and by bFGF (C). Molecular mass markers (in kDa) are indicated on the left.



Tyrosine Phosphorylation of MAPK

If the increased tyrosine phosphorylation mediates the survival action of ATA on PC12 cells, it is quite possible that ATA stimulates the phosphorylation of proteins phosphorylated following treatment with other growth factors such as NGF or EGF. Therefore, we further investigated the phosphorylation of proteins that are involved in the signal transduction pathway of peptide growth factors.

Initially, we examined whether ATA induces tyrosine phosphorylation of MAPKs/ERKs, which proteins are known to be phosphorylated at their tyrosine and threonine residues in response to a variety of growth factors, including NGF and EGF(11, 12, 13, 14, 15) . The phosphorylated MAPKs were detected by anti-MAPK immunoblotting in anti-phosphotyrosine immunoprecipitates of cell lysate. The phosphorylated MAPKs, both of MAPK1 (44 kDa) and MAPK2 (42 kDa), were clearly increased in the immunoprecipitates prepared from the cells treated with ATA as well as NGF or EGF, as compared with those from non-treated cells (Fig. 4). This result shows that ATA induced the phosphorylation of tyrosine residues on MAPKs in PC12 cells.


Figure 4: Tyrosine phosphorylation of MAPKs in PC12 cells treated with ATA, EGF, and NGF. The cells were treated with 100 µM ATA, 20 ng/ml EGF, and 100 ng/ml NGF, for 5 min. Then the cell lysates after normalized protein content were immunoprecipitated with an anti-phosphotyrosine antibody. The immunocomplexes precipitated with protein G-Sepharose beads were subjected to 8% SDS-PAGE, followed by immunoblot with an anti-MAPK antibody as described under ``Experimental Procedures.'' The positions of MAPKs are indicated by arrowheads on the right. Molecular mass marker is indicated on the left.



Shc Proteins Are Phosphorylated by ATA as Well as by NGF and EGF

The activation of MAPKs by growth factors is considered to be mediated through p21-GAP activation. Recently, Shc proteins have been found to play a role upstream of p21 activation by NGF/EGF stimulation(16, 17, 18) . Shc proteins associate with the tyrosine-autophosphorylated receptors and are phosphorylated on their tyrosine residues(19) .

We investigated whether Shc proteins were also phosphorylated in the cells stimulated by ATA. Lysates from PC12 cells treated with ATA, NGF, and EGF, for 5 min, were immunoprecipitated with anti-Shc antibody. The immunoprecipitates were immunoblotted with anti-phosphotyrosine or anti-Shc antibody. The anti-Shc antibody immunoprecipitated three Shc proteins, p46, p52, and p66 (Fig. 5, rightpanel), and the Shc proteins were phosphorylated on tyrosine in response to each of ATA, NGF, and EGF (Fig. 5, leftpanel). The p66 was phosphorylated most by EGF treatment, and least by ATA treatment. The finding that the phosphorylation of Shc proteins is induced by ATA suggests that the phosphorylation of MAPKs in PC12 cells stimulated by ATA is mediated by a pathway similar to that activated by NGF or EGF.


Figure 5: Tyrosine phosphorylation of Shc proteins in PC12 cells treated with ATA, EGF, and NGF. The cells were treated with 100 µM ATA, 20 ng/ml EGF, and 100 ng/ml NGF, for 5 min. All lysates were immunoprecipitated with an anti-Shc antibody and analyzed by 8% SDS-PAGE, followed by immunoblot with an anti-phosphotyrosine antibody (leftpanel). The membrane after ECL detection was stripped of bound antibodies and blotted with an anti-Shc antibody (rightpanel). All procedures were performed as described under ``Experimental Procedures.'' The positions of Shc proteins are indicated with arrowheads.



ATA Stimulates the PI 3-Kinase Pathway

The PI 3-kinase is phosphorylated on tyrosine by a number of receptor tyrosine kinases (20, 21). Recent studies indicate that the 85-kDa subunit (p85) of PI 3-kinase, similar to Shc proteins, forms a stable association with the growth factor receptors and is phosphorylated on tyrosine(22, 23, 24, 25) .

We examined whether ATA induced the phosphorylation of PI 3-kinase. Lysates from cells treated with ATA, EGF, or NGF, was immunoprecipitated with anti-PI 3-kinase antibody and the immunoprecipitates were immunoblotted with anti-phosphotyrosine antibody. In cells treated with ATA for 1 min, similar to those treated with NGF or EGF, two tyrosine-phosphorylated proteins, whose molecular masses were 100 kDa and 110 kDa, were detected. In the cells treated with ATA for 5 min, these two proteins were more phosphorylated than in the 1-min treated cells (Fig. 6A, upper panel). It has been reported that the p100 and p110 were co-precipitated with p85 in anti-PI 3-kinase immunoprecipitates of NGF- or EGF- treated PC12 cells(26) . The p100 and p110 are thought to be p85-binding proteins and mediate the activation of PI 3-kinase by treatment with NGF and EGF(26) . In cells treated with ATA for 5 min, and in cells treated with NGF, or EGF for 1 min, two phosphorylated proteins were detected around 50-60 kDa. These may be pp52and pp58, which are thought to be associate with p85 via receptors(19) . The increase in the amount of tyrosine-phosphorylated p85 was directly detected in PC12 cells treated with ATA by anti-phosphotyrosine immunoprecipitation followed by anti-PI 3-kinase immunoblotting (Fig. 6B). As shown in Fig. 3B, a major tyrosine-phosphorylated protein was observed at a molecular mass position similar to that for p85 of PI 3-kinase when the cells had been stimulated by ATA. However, it was thought to be a different protein from p85 of PI 3-kinase, because the dominant phosphorylation of the 85-kDa protein was detected in only ATA-treated cells, but neither in NGF- nor in EGF-treated cells.


Figure 6: Tyrosine phosphorylation of p85 of PI 3-kinase and p85-binding proteins in PC12 cells treated with ATA, EGF, and NGF. A, the cells were treated with 100 µM ATA, 20 ng/ml EGF, and 100 ng/ml NGF, for indicated times. The cell lysates were immunoprecipitated with an anti-PI 3-kinase antibody. The immunocomplexes were analyzed by 8% SDS-PAGE followed by immunoblot with an anti-phosphotyrosine antibody (upperpanel). After stripping, the membrane was reblotted with an anti-PI 3-kinase antibody (lowerpanel). B, cells treated with 100 µM ATA or non-treated were lysed. After normalization of protein content, the cell lysates were immunoprecipitated with an anti-phosphotyrosine antibody. The immunocomplexes were subjected to 8% SDS-PAGE, followed by immunoblotting with anti-PI 3-kinase antibody (lowerpanel). After stripping the membrane was reblotted with an anti-phosphotyrosine antibody (upperpanel). All procedures were performed as described under ``Experimental Procedures.'' The position of p85 of PI 3-kinase is indicated by arrowheads in lower panels. Molecular mass markers are indicated on the right and left.



PLC- Was Also Tyrosine-phosphorylated by ATA

PLC- can also stably associate with receptors in a growth factor-dependent fashion via the SH2 domain and is phosphorylated on tyrosine by a number of receptor tyrosine kinases (27-31). We also investigated tyrosine phosphorylation of PLC- in response to ATA. The tyrosine phosphorylation of PLC- (148 kDa) in ATA-treated PC12 cells was detected by anti-phosphotyrosine immunoprecipitation of cell lysates followed by anti-PLC- immunoblotting (Fig. 7). The tyrosine phosphorylation of PLC- was increased by ATA treatment, though the phosphorylation was less than that induced by NGF and EGF treatment.


Figure 7: Tyrosine phosphorylation of PLC- in PC12 cells treated with ATA, EGF, and NGF. The cells were treated with 100 µM ATA, 20 ng/ml EGF, and 100 ng/ml NGF, for 5 min. The cell lysates after normalized protein content were immunoprecipitated with an anti-phosphotyrosine antibody. The immunocomplexes were analyzed by 8% SDS-PAGE followed by immunoblot with an anti-PLC- antibody as described under ``Experimental Procedures.'' The position of PLC- is indicated by an arrowhead on the right. Molecular mass markers are indicated on the left.




DISCUSSION

As already reported by Batistatou and Greene(1) , ATA promotes survival of serum-starved PC12 cells and inhibits the DNA fragmentation which occurs during serum-starved cell death. ATA has been thought to suppress the internucleosomal cleavage occurring in apoptosis by inhibiting endonuclease activity(2, 32) . In NIH3T3 cells, however, ATA did not prevent either the internucleosomal cleavage or the cell death caused by serum starvation. These facts suggest that the action of ATA have a selectivity between these two types of cells. Batistatou and Greene have reported that ATA rescued sympathetic neurons as well as PC12 cells from serum-starved cell death(1) . This suggested to us that the survival action of ATA may be selective to PC12 cells or neuronal cells. However, to conclude the neuronal specificity of the survival action of ATA, it will be necessary to investigate the effect on other varieties of cell types.

The correlation between the induction of tyrosine phosphorylation and the survival action of ATA in PC12 and NIH3T3 cells indicates that tyrosine phosphorylation is the signal mediating the survival action of ATA. This is further supported by the fact that the survival action of neurotrophic factors is dependent on the activation of a member of Trk family. However, we found that ATA did not stimulate NGF or EGF receptor autophosphorylation in PC12 cells (data not shown). In addition, the profile of tyrosine-phosphorylated proteins in PC12 cells stimulated by ATA was different from that by NGF or EGF (Fig. 3B). Furthermore, ATA was not able to stimulate differentiation or proliferation of PC12 cells (data not shown). These three observations indicate that the survival action of ATA may share some parts of signals related to the survival with the pathway that is also stimulated by growth factors. However, ATA-induced survival is not directly mediated by the stimulation of receptors for NGF or EGF.

MAPKs are important components of the signal transduction pathway in the differentiation, proliferation, and trophic response of cells and are known to be phosphorylated on tyrosine residues in PC12 cells in response to NGF or EGF. The observation that ATA also stimulates the tyrosine phosphorylation of MAPKs in PC12 cells indicates the presence of a common pathway between ATA and growth factors and suggests that the pathway may be responsible for the survival activity of ATA.

In signal transduction stimulated by growth factors, the activation of p21 takes place between the activations of receptor and MAPKs. The Shc proteins reside in the signal pathway right after the receptor tyrosine kinase and upstream of the activation p21(33). Shc proteins associate with the tyrosine-phosphorylated receptors via their SH2 domains and are themselves phosphorylated on tyrosine residues. The Shc proteins can also associate with growth factor receptor-bound protein 2 (Grb2), which contains SH2 and SH3 domains (34). This association of Shc proteins with Grb2 mediates the activation of p21 in response to growth factors via Son of Sevenless (Sos), the Ras nucleotide exchange factor(19, 35, 36, 37, 38, 39) . The observation that tyrosine phosphorylation of Shc proteins is induced by ATA, as well as NGF and EGF, indicates that the same pathway is stimulated by both ATA and the growth factors and results in the tyrosine phosphorylation of MAPKs. This also suggests that ATA activates a protein tyrosine kinase that behaves like a receptor for growth factors. In addition, the increase in the tyrosine phosphorylation of p85 subunit of PI 3-kinase and PLC- by ATA treatment further supports the existence of such a tyrosine kinase, because both of them are known to associate with activated growth factor receptors through their SH2 domains. Since it is unclear whether ATA penetrates the plasma membrane or not, we cannot assert whether the activation of the signal cascade is triggered by the binding of ATA to a receptor or by action of intracellular ATA on a tyrosine kinase. However, the other actions of ATA on cells, which are antagonizing both NMDA binding on NMDA receptor (40) and human immunodeficiency virus binding on CD4(41, 42) , suggest that the site of the action of ATA may be at the surface of cells rather than on endonuclease in nucleus.

ATA protects cultured sympathetic neurons from cell death induced by the deprivation of serum and NGF(1) . The mechanisms of this action of ATA on neuronal cells are still unclear; however, there is a possibility that the receptor that can be activated by ATA in PC12 cells is expressed on sympathetic neurons.

It has been reported that ATA protects hippocampal neurons from NMDA- and ischemia-induced toxicity in vivo(3) . The NMDA antagonistic activity of ATA may account for the protective effect of ATA on this ischemia-induced toxicity(40) . However, growth factors, such as brain-derived neurotrophic factor, are also reported to protect neurons from NMDA- or ischemia-induced cell death(43) . Therefore, the effect of ATA in vivo may also explained by stimulation of tyrosine kinase pathways. Further experiments are necessary to correlate the tyrosine kinase activation and neuroprotection by ATA in vivo.

ATA can form heterogeneous polymers, and some of the effects of ATA, such as anti-human immunodeficiency virus activity (44) and inhibition of protein-nucleic acid interaction(45) , are reported to increase with the molecular mass. It remains to be determined which molecular mass species of ATA can act on PC12 cells.

Here, we showed that the MAPKs, Shc proteins, PI 3-kinase, and PLC- were phosphorylated on tyrosine by ATA treatment, each of which was also phosphorylated by growth factor stimulation. However, the major proteins phosphorylated by ATA treatment (180, 130, 85, and 60 kDa) have not been identified yet. This suggests that the survival action of ATA on PC12 cells is mediated by tyrosine phosphorylation cascade, similar to the action of growth factors. Our results are not clear enough to demonstrate that the survival action of ATA on PC12 cells depends on the ATA-induced tyrosine phosphorylation that is shown here. However, it supports the idea that ATA activates a certain growth factor receptor tyrosine kinase and triggers the same signal pathway.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-797-74-2627; Fax: 81-797-74-2455.

The abbreviations used are: NGF, nerve growth factor; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; MAPK, microtubule-associated protein kinase; PI, phosphoinositide; PLC, phospholipase C; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; NMDA, N-methyl-D-aspartate 2.


ACKNOWLEDGEMENTS

We are grateful to Dr. B. Goldsmith for reading this manuscript. We also thank K. Bessho for excellent technical assistance.


REFERENCES
  1. Batistatou, A., and Greene, L. A. (1991) J. Cell Biol.115, 461-471 [Abstract]
  2. Batistatou, A., and Greene, L. A. (1993) J. Cell Biol.122, 523-532 [Abstract]
  3. Roberts-Lewis, J. M., Marcy, V. R., Zhao, Y., Vaught, J. F., Siman, R., and Lewis, M. E. (1993) J. Neurochem.61, 378-381 [Medline] [Order article via Infotrieve]
  4. Joseph, R., Tsang, W., Han, E., and Saed, G. M. (1993) Brain Res.625, 244-255 [Medline] [Order article via Infotrieve]
  5. Bina-Stein, M., and Tritton, T. R. (1975) Mol. Pharmacol.12, 191-193 [Abstract]
  6. Grollman, A. P., and Stewart, M. L. (1968) Proc. Natl. Acad. Sci. U. S. A.61, 719-725 [Medline] [Order article via Infotrieve]
  7. Liao, L. L., Horwits, S. B., Huang, M. T., Grollman, A. P., Steward, L., and Martin, J. (1975) J. Med. Chem.18, 117-120 [Medline] [Order article via Infotrieve]
  8. Givens, J. F., and Manly, K. F. (1976) Nucleic Acids Res.3, 405-418 [Abstract]
  9. Blumenthal, T., and Landers, T. A. (1973) Biochem. Biophys. Res. Commun.55, 680-688 [Medline] [Order article via Infotrieve]
  10. McCune, S A., Foe, L. G., Kemp, R. G., and Jurin, R. R. (1989) Biochem. J.259, 925-927 [Medline] [Order article via Infotrieve]
  11. Miyasaka, T., Chao, M. V., Sherline, P., and Saltiel, A. R. (1990) J. Biol. Chem.265, 4730-4735 [Abstract/Free Full Text]
  12. Tsao, H., Aletta, J. M., and Greene, L. A. (1990) J. Biol. Chem.265, 15471-15480 [Abstract/Free Full Text]
  13. Ahn, N., and Krebs, E. (1990) J. Biol. Chem.265, 11495-11501 [Abstract/Free Full Text]
  14. Boulton, T. G., Nye, S. H., Robbins, D. J., Ip, N. Y., Radziejewska, E., Morgenbesser, S. D., DePinho, R. A., Panayotatos, N., Cobb, M. H., and Yancopoulos, G. D. (1991) Cell65, 663-675 [Medline] [Order article via Infotrieve]
  15. Hoshi, M., Nishida, E., and Sakai, H. (1988) J. Biol. Chem.263, 5396-5401 [Abstract/Free Full Text]
  16. Ohmichi, M., Matuoka, K., Takenaka, T., and Saltiel, A. R. (1994) J. Biol. Chem.269, 1143-1148 [Abstract/Free Full Text]
  17. Pronk, G. J., Vries-Smits, A. M. M., Buday, L. Downward, J., Maassen, J. A., Medema, R. H., and Bos, J. L. (1994) Mol. Cell. Biol.14, 1575-1581 [Abstract]
  18. Stephens, R. M., Loeb, D. M., Copeland, T. D., Pawson, T., Greene, L. A., and Kaplan, D. R. (1994) Neuron12, 691-705 [Medline] [Order article via Infotrieve]
  19. Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Nicoletti, I., Grignani, F., Pawson, T., and Pelicci, P. G. (1992) Cell70, 93-104 [Medline] [Order article via Infotrieve]
  20. Bjorge, J. D., Chan, T., Antczak, M., Kung, H., and Fugita, D. J. (1990) Proc. Natl. Acad. Sci. U. S. A.87, 3816-3820 [Abstract]
  21. Soltoff, S. P., Rabin, S. L., Cantley, L. C., and Kaplan, D. R. (1992) J. Biol. Chem.267, 17472-17477 [Abstract/Free Full Text]
  22. Coughlin, S. R., Escobedo, J. A., and Williams, L. T. (1989) Science243, 1191-1194 [Medline] [Order article via Infotrieve]
  23. Escobedo, J. A., Kaplan, D. R., Kavanaugh, W. M., Turck, C. W., and Williams, L. T. (1991) Mol. Cell. Biol.11, 1125-1132 [Medline] [Order article via Infotrieve]
  24. Escobedo, J. A., Navankasattusas, S., Kavanaugh, W. M., Milfay, D., Fried, V. A., and Williams, L. T. (1991) Cell65, 75-82 [Medline] [Order article via Infotrieve]
  25. Otsu, M., Hiles, I., Gout, I, Fry, M. J., Ruiz-Larrea, F., Panayotou, G., Thompson, A., Dhand, R., Hsuan, J., Totty, N., Smith, A. D., Morgan, S. J., Courtneidge, S. A., Parker, P. J., and Waterfield, M. D. (1991) Cell65, 91-104 [Medline] [Order article via Infotrieve]
  26. Ohmichi, M., Decker, S. J., and Saltiel, A. R. (1992) Neuron9, 769-777 [Medline] [Order article via Infotrieve]
  27. Wahl, M. I., Daniel, T. O., and Carpenter, G. (1988) Science241, 968-970 [Medline] [Order article via Infotrieve]
  28. Margolis, B., Rhee, S. G., Felder, S., Mervic, M., Lyall, R., Levitzki, A., Ullrich, A., Zilberstein, A., and Schlessinger, J. (1989) Cell57, 1101-1107 [Medline] [Order article via Infotrieve]
  29. Wahl, M. I., Olashaw, N. E., Nishibe, S., Rhee, S. G., Pledger, W. J., and Carpenter, G. (1989) Mol. Cell. Biol.9, 2934-2943 [Medline] [Order article via Infotrieve]
  30. Anderson, D., Koch, C. A., Grey, L., Ellis, C., Moran, M. F., and Pawson, T. (1990) Science250, 979-982 [Medline] [Order article via Infotrieve]
  31. Margolis, B., Li, N., Koch, A., Mohammadi, M., Hurwitz, D. R., Zilberstein, A., Ullrich, A., Pawson, T., and Schlessinger, J. (1990) EMBO J.9, 4375-4380 [Abstract]
  32. Mogil, R. J., Shi, Y., Bissonnette, R. P., Bromley, P., Yamaguchi, I., and Green, D. R., (1994) J. Immunol.152, 1674-1683 [Abstract/Free Full Text]
  33. Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R., Li, W., Batzer, A., Thomas, S., Brugge, J., Pelicci, P. G., Schlessinger, J., and Pawson, T. (1992) Nature360, 689-692 [CrossRef][Medline] [Order article via Infotrieve]
  34. Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D., and Schlessinger, J. (1992) Cell70, 431-442 [Medline] [Order article via Infotrieve]
  35. Oliver, J. P., Raabe, T., Henkemeyer, M., Dickson, B., Mbamalu, G., Margolis, B., Schlessinger, J., Hafen, E., and Pawson, T. (1993) Cell73, 179-191 [Medline] [Order article via Infotrieve]
  36. Egan, S. E., Giddings, B. W., Brooks, M. W., Buday, L., Sizeland, A. M., and Weiberg, R. A. (1993) Nature363, 45-51 [CrossRef][Medline] [Order article via Infotrieve]
  37. Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T., and Bowtell, D. (1993) Nature363, 83-85 [CrossRef][Medline] [Order article via Infotrieve]
  38. Li, N., Batzer, A., Daly, R., Yajnik, V., Skolnik, E., Chardin, P., Bar-Sagi, D., Margolis, B., and Schlessinger, J. (1993) Nature363, 85-88 [CrossRef][Medline] [Order article via Infotrieve]
  39. Gale, N. W., Kaplan, S., Lowenstein, E. J., Schlessinger, J., and Bar-Sagi, D. (1993) Nature363, 88-92 [CrossRef][Medline] [Order article via Infotrieve]
  40. Zeevalk, G. D., Schoepp, D., and Nicklas, W. J., (1993) J. Neurochem.61, 386-389 [Medline] [Order article via Infotrieve]
  41. Weaver, J. L., Gergely, P., Pine, P. S., Patzer, E., and Aszalos, A. (1990) AIDS Res. Hum. Retroviruses6, 1125-1130 [Medline] [Order article via Infotrieve]
  42. Cushman, M., Wang, P., Chang, S. H., Wild, C., De Clercp, E., Schols, D., Goldman, M. E., and Bowen, J. A. (1991) J. Med. Chem.34, 329-337 [Medline] [Order article via Infotrieve]
  43. Tsukahara, T., Yonekawa, Y., Tanaka, K., Ohara, O., Watanabe, S., Kimura, T., Nishijima, T., and Taniguchi, T. (1994) Neurosurgery34, 323-331 [Medline] [Order article via Infotrieve]
  44. Gan, Y., Weaver, J. L., Pine, P. S., Zoon, K. C., and Aszalos, A. (1990) Biochem. Biophys. Res. Commun.172, 1298-1303 [Medline] [Order article via Infotrieve]
  45. Gonzalez, R. G., Blackburn, B. J., and Schleich, T. (1980) Biochemistry19, 4299-4303 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.