COMMUNICATION:
Activation and Involvement of p38 Mitogen-activated Protein Kinase in Glutamate-induced Apoptosis in Rat Cerebellar Granule Cells*

(Received for publication, November 19, 1996, and in revised form, April 14, 1997)

Hiroshi Kawasaki Dagger §, Takaya Morooka Dagger , Shun Shimohama §, Jun Kimura §, Tomoo Hirano par , Yukiko Gotoh Dagger ** and Eisuke Nishida Dagger

From the Dagger  Department of Genetics and Molecular Biology, Institute for Virus Research, the  Department of Biophysics, Graduate School of Science, and the Departments of § Neurology and par  Physiology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

In the mammalian central nervous system glutamate is the major excitatory neurotransmitter and plays a crucial role in plasticity and toxicity of certain neural cells. We found that glutamate stimulated activation of p38 and stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase (JNK)), two subgroup members of the mitogen-activated protein kinase superfamily in matured cerebellar granule cells. The p38 activation was largely mediated by N-methyl-D-aspartate receptors. Furthermore, we have revealed a novel signaling pathway, that is, Ca2+-mediated activation of p38 in glutamate-treated granule cells. The glutamate concentration effective for inducing apoptosis correlated with that for inducing p38 activation. SB203580, a specific inhibitor for p38, inhibited glutamate-induced apoptosis. Thus p38 might be involved in glutamate-induced apoptosis in cerebellar granule cells.


INTRODUCTION

L-Glutamic acid (glutamate) is the principal excitatory neurotransmitter in the mammalian central nervous system. Glutamate not only mediates excitatory neurotransmission but also is involved in other phenomena such as neuronal plasticity and cell death (neuroexcitotoxicity) (1-5). Cell death induced by glutamate is believed to be involved in neuronal loss associated with both acute (e.g. stroke) and chronic (e.g. Alzheimer's disease) neurodegenerative insults (6-8); thus dissection of glutamate signal transduction may have clinical significance for neuroprotection. Glutamate receptors are classified into metabotropic and ionotropic receptors, and ionotropic glutamate receptors are further categorized into N-methyl-D-aspartate (NMDA)1 receptors and non-NMDA receptors. In many cases, glutamate toxicity (especially later phases of neuronal degeneration) can be attributed to excessive stimulation of NMDA subtype of glutamate receptor (9-13).

The integral channel of the NMDA receptor is highly permeable to Ca2+, and the increase in intracellular Ca2+ concentration is thought to be the key event in evoking NMDA receptor-mediated cell death (7, 14). Several molecules have been shown to be activated by glutamate through Ca2+ influx including CaM kinase II, protein kinase C, nitric-oxide synthetase, NFkappa B (15), and mitogen-activated protein kinase (MAPK). However, the glutamate-induced signal transduction pathways leading to cell death are largely unknown.

Members of the MAPK superfamily are thought to be important mediators of signal transduction from the cell surface to the nucleus (16-21). In addition to classical MAPKs (also referred to as ERKs), the recent studies revealed two other members of the MAPK superfamily, p38 (also known as CSBP/RK/MPK2) (22-24) and stress-activated protein kinase (SAPK, also referred to as c-Jun N-terminal kinase (JNK)) (25, 26). p38 and SAPK are activated by inflammatory cytokines and cellular stresses such as ultraviolet light and high osmolarity (26-29). p38 and SAPK have recently been shown to be involved in cell death induced by nerve growth factor deprivation in PC12 cells, by ceramide in U937 and BAE cells, and by anti-IgM antibody in human B lymphocytes (30-32). In cultured chick fetal forebrain neurons, p38 activity was down-regulated by insulin, which can support survival of these cells (33).

These previous reports prompted us to examine whether the activities of p38 and SAPK are regulated by glutamate in cultured granule cells and whether they are correlated with the death-inducing effects of glutamate. In this study we have shown, for the first time, that p38 is activated by glutamate and NMDA. In addition, we have shown evidence suggesting an essential role of p38 in mediating death-promoting activity of glutamate.


MATERIALS AND METHODS

Cell Cultures and Preparation of Cell Extracts

Primary cell cultures of cerebellar granule cells were prepared from 6-7-day-old rats as described previously (34). Eight days after plating, matured granule cells were stimulated. In some experiments, the medium was replaced with modified Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 1.3 mM CaCl2, 5.6 mM D-glucose, 5 mM Hepes, pH 7.4) 5 min before glutamate treatment (35). Modified Locke's solution prepared by substituting 154 mM Na+ with 154 mM N-methyl-D-glucamine (NMDG) was also used. The cell extracts were prepared as described (36).

Antibodies

We produced anti-SAPK antiserum by immunizing mice with recombinant His-tagged rat SAPKalpha . The antiserum specifically recognized 46-kDa and 54-kDa SAPK (36). Anti-p38 antibody was purchased from Santa Cruz Biotechnology, Inc.

Preparation of Recombinant Proteins

Human c-Jun was expressed and purified as described (37). ATF2 is a kind gift from Drs. Suzanne J. Baker and Tom Curran (St. Jude Children's Research Hospital).

Immunoprecipitation and Immune Complex Kinase Assay

For immunoprecipitation, anti-p38 antibody (5 µg) and protein A-Sepharose CL-4B beads (20 µl) were added to an aliquot (200 µl) of the cell extract and incubated for 2 h at 4 °C. The precipitate was washed three times with the buffer containing 0.5 M NaCl. The immune complex was suspended in 15 µl of a solution consisting of 20 mM Tris, pH 7.5, 2 mM EGTA, 10 mM MgCl2, 100 µM [gamma -32P] ATP (3 µCi), and 1.6 µg of recombinant ATF2 and then incubated for 30 min at 30 °C. The reactions were stopped by addition of Laemmli's sample buffer. After SDS-PAGE, phosphorylation of ATF2 was quantified by an image analyzer (FUJIX BAS2000). In-gel kinase assay was performed as described (36).

Quantitation of Apoptosis

Coverslips (15 × 15 mm No. 1; Matsunami Glass, Ltd.) were rinsed in ethanol and placed at the bottom of 35-mm tissue culture plates, and granule cells were plated directly on the coverslips. Eight days after plating, the granule cells were exposed to indicated concentrations of glutamate and SB203580 to examine glutamate-induced cell death. The coverslips were removed from the plates, and the cells were fixed in fresh 3.7% formaldehyde in phosphate-buffered saline for 10 min at 37 °C. The cells were stained with 10 µg/ml DAPI for 30 min at 37 °C. The cells were washed three times in phosphate-buffered saline, mounted on slides, and examined by fluorescence microscopy. Cells were scored as apoptotic if they exhibited margination and condensation of the chromatin and cell shrinkage. For the quantitation of apoptotic cells, more than 500 nuclei from 20 fields were evaluated for each point.


RESULTS AND DISCUSSION

We utilized primary cultures of cerebellar granule cells from neonatal rats as a model system to investigate the intracellular signal transductions of glutamate stimulation, since previous studies have shown that glutamate can induce cell death of these cells (38, 39). We added triiodothyronine to the medium to reduce apoptotic cells under unstimulated conditions. In this medium, granule cells were well differentiated 8 days after plating as described in the previous report (40). Since recent reports have shown that p38 and SAPK, two members of the MAPK superfamily, may be involved in apoptosis in some cases (see introduction), it is an intriguing question if p38 and/or SAPK is activated downstream of glutamate. Immunoblotting with either anti-p38 antibody or anti-SAPK antiserum revealed that p38 and 54-kDa and 46-kDa SAPKs were expressed in the granule cells (Fig. 1A). Then we tested whether p38 and SAPK can be activated by glutamate in the granule cells. The activity of p38 was detected by an immune complex assay using ATF2 as a substrate, and the activity of SAPK was detected by an in-gel assay using c-Jun as a substrate. p38 was markedly activated within 5 min after the addition of 500 µM glutamate in granule cells, and the activity remained higher than basal levels for more than 1 h (Fig. 1B). SAPK was activated slightly but reproducibly (about 1.5-fold) by glutamate treatment (Fig. 1C). This relatively weak activation of SAPK may be partially due to the high basal activity of SAPK in granule cells in the absence of glutamate (see Fig. 1C, inset). Since the medium may contain a low level (~50 µM) of glutamate, it is possible that the endogenous glutamate contributes to the basal activation of SAPK.


Fig. 1. Expression and glutamate-induced activation of p38 and SAPK. A, cell extracts obtained from rat 3Y1 fibroblastic cells (12 µg of total protein), rat PC12 cells (15 µg of total protein), and rat cerebellar granule cells (6 µg of total protein) were immunoblotted with anti-p38 antibody (left panel) or anti-SAPK antiserum (right panel). Arrows indicate p38, 46-kDa SAPK, and 54-kDa SAPK. B, cerebellar granule cells, 8 days after plating, were treated with 500 µM glutamate for the indicated times. p38 was immunoprecipitated from the lysates, and its activity was measured by phosphorylation of ATF2 as described under "Materials and Methods." C, the lysates obtained from 500 µM glutamate-stimulated granule cells for the indicated times were subjected to an in-gel kinase assay containing c-Jun as a substrate (inset). Upper bands show the activity of 54-kDa SAPK, and the lower bands show that of 46-kDa SAPK. The 54-kDa SAPK activity is shown as fold increase. Values in B and C are the means ± S.D. of at least five independent experiments.
[View Larger Version of this Image (26K GIF file)]

Since previous reports showed that Ca2+ influx triggered by glutamate treatment plays a major role in glutamate toxicity (7, 41), we examined the role of Ca2+ in glutamate-induced activation of p38. An influx of Ca2+ from extracellular medium was required for the glutamate-induced activation of p38, because addition of 5 mM EGTA to the medium inhibited the activation (Fig. 2A). Moreover, addition of Ca2+ ionophore A23187 induced activation of p38 in the absence of glutamate (Fig. 2B). Thus Ca2+ influx may be sufficient for activating p38 in granule cells. The slight activation of SAPK induced by glutamate was also blocked by the addition of EGTA and thus dependent on Ca2+ influx (Fig. 2A).


Fig. 2. Role of Ca2+ influx in glutamate-induced activation of p38 and SAPK. A, effect of EGTA on glutamate-induced activation of p38 and SAPK in cerebellar granule cells. Eight days after plating, granule cells were pretreated with (right panel) or without (left panel) 5 mM EGTA for 5 min, followed by treatment with 500 µM glutamate for 5 min. p38 activity was measured by an immune complex kinase assay with anti-p38 antibody. SAPK activity was measured by in-gel kinase assay containing c-Jun as a substrate, and the 54-kDa SAPK activity is shown. B, activation of p38 by Ca2+ ionophore. Granule cells were treated with Ca2+ ionophore A23187 (5 µM) for 5 min, and p38 activity was measured.
[View Larger Version of this Image (21K GIF file)]

Ca2+ influx is mediated by several proteins such as NMDA receptor and voltage-sensitive Ca2+ channel (VSCC) (42). To investigate the role of VSCC in glutamate-induced p38 activation, an impermeant cation NMDG was used to substitute for Na+ to prevent membrane depolarization. In this experiment, the medium was replaced by the modified Locke's solution with either NaCl or NMDG (35). The level of p38 activation by glutamate in modified Locke's solution with NMDG was almost the same as that in modified Locke's solution with Na+ (Fig. 3A). In addition, pretreatment of granule cells with L-type VSCC inhibitors, nifedipine or verapamil, did not inhibit the glutamate-induced p38 activation (Fig. 3B). These results suggest that VSCC contributes little, if any, to the glutamate-induced p38 activation in cerebellar granule cells.


Fig. 3. Role of voltage-sensitive Ca2+ channel in glutamate-induced activation of p38. A, the medium was replaced by modified Locke's solution with NaCl (left panel) or modified Locke's solution with NMDG (right panel). In modified Locke's solution with NMDG, NMDG substituted for Na+ to prevent membrane depolarization by glutamate treatment. After 5 min, granule cells were treated with 500 µM glutamate for 5 min, and p38 activity was measured by an immune complex kinase assay. B, granule cells were pretreated with 30 µM nifedipine or 10 µM verapamil for 10 min and treated with 500 µM glutamate for 5 min. The p38 activity was measured as in A.
[View Larger Version of this Image (39K GIF file)]

The NMDA type of glutamate receptor is thought to play a crucial role in neurotoxicity (9-12). Especially glutamate-induced apoptosis was completely blocked by NMDA antagonist in cerebellar granule cells (13). When granule cells were pretreated with increasing concentrations of an NMDA antagonist DL-2-amino-5-phosphonopentanoic acid (DL-AP5), glutamate-induced p38 activation was inhibited in a dose-dependent fashion (Fig. 4A). Moreover, addition of NMDA to granule cells induced activation of p38 (Fig. 4B). This NMDA-induced activation of p38 was also inhibited by DL-AP5 dose dependently (Fig. 4C) and was completely inhibited by addition of EGTA to the medium (data not shown). Taken together, Ca2+ influx through NMDA type of glutamate receptor might be important for glutamate-induced p38 activation.


Fig. 4. Effect of NMDA antagonist on glutamate- or NMDA-induced activation of p38. A, granule cells were pretreated with indicated concentrations of NMDA antagonist DL-AP5 for 10 min and followed by treatment with 500 µM glutamate. p38 activity was measured by an immune complex kinase assay. B, granule cells were exposed to 100 µM or 500 µM NMDA for 5 min, and p38 activity was measured. C, granule cells were pretreated with indicated concentrations of DL-AP5 for 10 min and followed by treatment with 500 µM NMDA. p38 activity was measured.
[View Larger Version of this Image (47K GIF file)]

We then investigated the correlation between the glutamate toxicity and the activity of p38 in granule cells. Glutamate can induce either early necrosis or delayed apoptosis in cultures of cerebellar granule cells (13 and references therein). In this study we counted the number of neurons displaying cell shrinkage, chromatin condensation, and formation of typical apoptotic nuclei which are characteristics of apoptotic cell death. Under unstimulated conditions, the number of granule cells on the 9th day after plating was 97 ± 4.39% of that on the 8th day, suggesting that the cells were not dying at the time the experiments were being done. The number of apoptotic cells increased by glutamate treatment in a dose-dependent fashion (Fig. 5A) although the maximal level of population of cells that underwent apoptosis varied among experiments (from 30% to 95%). In all experiments EC50 (50% effective concentration) of glutamate to induce apoptosis was about 500 µM, and 100 µM glutamate had a marginal effect on the viability of the granule cells. Interestingly, the extent of p38 activation was correlated with the degree of apoptosis (Fig. 5A), suggesting possible involvement of p38 in the glutamate-induced apoptosis.


Fig. 5. Inhibition of p38 activation attenuates glutamate-induced apoptosis. A, correlation between glutamate-induced apoptosis and activation of p38 in cerebellar granule cells. Eight days after plating, granule cells were treated with various concentrations of glutamate. Five minutes after the addition of glutamate, the cells were extracted and subjected to an immune complex kinase assay with anti-p38 antibody. Twenty-four hours after the addition of glutamate, the cells were fixed and stained with DAPI for quantitation of apoptotic cells as described under "Materials and Methods." B, effect of SB203580 on glutamate-induced apoptosis of granule cells. Eight days after plating, granule cells were pretreated with or without 1 µM or 10 µM SB203580 for 1 h. Then the cells were treated with 500 µM glutamate for 24 h and stained with DAPI for quantitation of apoptotic cells. At least 500 cells were counted at each point. We here showed four typical independent experiments in which glutamate effectively induced apoptosis. Values represent the average of data from 10 independent experiments. SB203580 had little effect on untreated granule cells.
[View Larger Version of this Image (22K GIF file)]

To evaluate the significance of p38 activation in the glutamate-induced apoptosis, we tested the effect of SB203580, a p38 inhibitor (24, 43). SB203580 inhibited p38 with an IC50 of 0.6 µM, and even at 100 µM had no effect on the activities of 12 other protein kinases tested, including MAPK or SAPK (43). We also confirmed that 10 µM SB203580 did not inhibit MAPK activity and SAPK activity (data not shown). Granule cells were pretreated for 1 h with or without 1 µM or 10 µM SB203580, followed by incubation with 500 µM glutamate for 24 h. The apoptosis induced by glutamate was partially inhibited by the treatment with SB203580 (Fig. 5B). The extent of the inhibition appeared to depend on the degree of apoptosis induced by glutamate; SB203580 tended to inhibit apoptosis more effectively when glutamate induced apoptosis efficiently. On the average of 10 independent experiments SB203580 at 10 µM inhibited the glutamate-induced apoptosis by about 70%. In contrast, PD98059, a MAPKK inhibitor, did not inhibit the neurotoxic effect of glutamate at all (data not shown). These data suggest that p38 may mediate the glutamate-induced cell death.

Taken together, we assume that p38 is involved in glutamate neurotoxicity in cerebellar granule cells based on the following reasons. First, glutamate was a potent activator for p38 in cerebellar granule cells which are sensitive to glutamate toxicity. Second, not only glutamate but also NMDA could activate p38 efficiently. Third, glutamate-induced activation of p38 was largely Ca2+-dependent. Fourth, the level of p38 activation correlated with the degree of apoptosis in matured granule cells treated with glutamate. While 1-100 µM glutamate weakly activated p38, it did not induce apoptosis. It is possible that there is a threshold of p38 activity required for glutamate-induced apoptosis. Fifth, SB203580 partially inhibited the glutamate toxicity, although the level of inhibition varied among experiments. Apparently SB203580 tended to inhibit glutamate toxicity more efficiently when granule cells were sensitive to glutamate toxicity. The sensitivity might depend on both the cell density and the population of contaminated glial cells which may support neuronal survival. The fold increase of SAPK activation by glutamate or by NMDA was not remarkable. But it is possible that the high basal activity of SAPK is important for glutamate toxicity.

Our results have clearly shown that p38 and SAPK can be activated through the elevation of intracellular Ca2+, but the molecular mechanisms of Ca2+-induced activation of SAPK and p38 are largely unknown. It has previously been shown that several signaling molecules including PYK2, Src, and RasGRF could transduce Ca2+ signaling to the Ras-MAPK cascade (44-47). Most recently, ultraviolet- and high osmolarity-induced activation of SAPK was reported to depend partially on Ca2+ and PYK2 (48). These molecules might be involved in glutamate-induced activation of p38 and/or SAPK.

There are several molecules that have been implicated in mediating apoptosis-promoting effects of glutamate. For example, nitric-oxide synthetase and its product NO, and other reactive oxygen species are implicated as important downstream mediators of NMDA-induced toxicity (49-52). Here we have provided evidence that two members of the MAPK superfamily could also be involved in the glutamate-triggered signal transduction. It is possible that p38 is activated downstream of reactive oxygen species, since NO and H2O2 are good activators for p38 (53, 54). Interactions among these molecules will be elucidated in future studies.


FOOTNOTES

*   This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
**   To whom correspondence should be addressed. Tel.: 81-75-751-3992; Fax: 81-75-751-4019; E-mail: ygotoh{at}virus.kyoto-u.ac.jp.
1   The abbreviations used are: NMDA, N-methyl-D-aspartate; DL-AP5, DL-2-amino-5-phosphonopentanoic acid; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; PAGE, polyacrylamide gel electrophoresis; NMDG, N-methyl-D-glucamine; DMEM, Dulbecco's modified Eagle's medium; DAPI, 4,6-diamidino-2-phenylindole; VSCC, voltage-sensitive Ca2+ channel.

ACKNOWLEDGEMENT

We thank Dr. Yasumasa Bessho for critical reading of the manuscript.


REFERENCES

  1. Gasic, G. P., and Hollmann, M. (1992) Annu. Rev. Physiol. 54, 507-536 [CrossRef][Medline] [Order article via Infotrieve]
  2. Lipton, S. A., and Kater, S. B. (1989) Trends Neurosci. 12, 265-270 [CrossRef][Medline] [Order article via Infotrieve]
  3. Nakanishi, S. (1992) Science 258, 597-603 [Medline] [Order article via Infotrieve]
  4. Madison, D. V., Malenka, R. C., and Nicoll, R. A. (1991) Annu. Rev. Neurosci. 14, 379-397 [CrossRef][Medline] [Order article via Infotrieve]
  5. McDonald, J. W., and Johnston, M. V. (1990) Brain Res. Rev. 15, 41-70 [Medline] [Order article via Infotrieve]
  6. Olney, J. W. (1978) in Kainic Acid as a Tool in Neurobiology (McGeer, E. G., ed), pp. 95-121, Raven Press, New York
  7. Choi, D. W., and Rothman, S. M. (1990) Annu. Rev. Neurosci. 13, 171-182 [CrossRef][Medline] [Order article via Infotrieve]
  8. Lipton, S. A., and Rosenberg, P. A. (1994) N. Engl. J. Med. 330, 613-622 [Free Full Text]
  9. Choi, D. W., Koh, J. Y., and Peters, S. (1988) J. Neurosci. 8, 185-196 [Abstract]
  10. Finkbeiner, S., and Stevens, C. F. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4071-4074 [Abstract]
  11. Hahn, J. S., Aizenman, E., and Lipton, S. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6556-6560 [Abstract]
  12. Schramm, M., Eimerl, S., and Costa, E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1193-1197 [Abstract]
  13. Ankarcrona, M., Dypbukt, J. M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S. A., and Nicotera, P. (1995) Neuron 15, 961-973 [Medline] [Order article via Infotrieve]
  14. Balazs, R., Jorgensen, O. S., and Hack, N. (1988) Neuroscience 27, 437-451 [CrossRef][Medline] [Order article via Infotrieve]
  15. Grilli, M., Pizzi, M., Memo, M., and Spano, P. (1996) Science 274, 1383-1385 [Abstract/Free Full Text]
  16. Sturgill, T. W., and Wu, J. (1991) Biochim. Biophys. Acta 1092, 350-357 [Medline] [Order article via Infotrieve]
  17. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556 [Free Full Text]
  18. Nishida, E., and Gotoh, Y. (1993) Trends Biochem. Sci. 18, 128-131 [CrossRef][Medline] [Order article via Infotrieve]
  19. Marshall, C. J. (1994) Curr. Opin. Genet. Dev. 4, 82-89 [Medline] [Order article via Infotrieve]
  20. Gotoh, Y., and Nishida, E. (1995) Progress Cell Cycle Res. 1, 287-297
  21. Cobb, M. H., and Goldsmith, E. J. (1995) J. Biol. Chem. 270, 14843-14846 [Free Full Text]
  22. Han, J., Lee, J. D., Bibbs, L., and Ulevitch, R. J. (1994) Science 265, 808-811 [Medline] [Order article via Infotrieve]
  23. Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso, L. A., Zamanillo, D., Hunt, T., and Nebreda, A. R. (1994) Cell 78, 1027-1037 [Medline] [Order article via Infotrieve]
  24. Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., Strickler, J. E., McLaughlin, M. M., Siemens, I. R., Fisher, S. M., Livi, G. P., White, J. R., Adams, J. L., and Young, P. R. (1994) Nature 372, 739-746 [CrossRef][Medline] [Order article via Infotrieve]
  25. Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1025-1037 [Medline] [Order article via Infotrieve]
  26. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156-160 [CrossRef][Medline] [Order article via Infotrieve]
  27. Galcheva-Gargova, Z., Derijard, B., Wu, I.-H., and Davis, R. J. (1994) Science 265, 806-808 [Medline] [Order article via Infotrieve]
  28. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) J. Biol. Chem. 270, 7420-7426 [Abstract/Free Full Text]
  29. Matsuda, S., Kawasaki, H., Moriguchi, T., Gotoh, Y., and Nishida, E. (1995) J. Biol. Chem. 270, 12781-12786 [Abstract/Free Full Text]
  30. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) Science 270, 1326-1331 [Abstract]
  31. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., and Kolesnick, R. N. (1996) Nature 380, 75-79 [CrossRef][Medline] [Order article via Infotrieve]
  32. Graves, J. D., Draves, K. E., Craxton, A., Saklatvala, J., Krebs, E. G., and Clark, E. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13814-13818 [Abstract/Free Full Text]
  33. Heidenreich, K. A., and Kummer, J. L. (1996) J. Biol. Chem. 271, 9891-9894 [Abstract/Free Full Text]
  34. Bessho, Y., Nawa, H., and Nakanishi, S. (1993) J. Neurochem. 60, 253-259 [Medline] [Order article via Infotrieve]
  35. Resink, A., Hack, N., Boer, G. J., and Balazs, R. (1994) Brain Res. 655, 222-232 [Medline] [Order article via Infotrieve]
  36. Kawasaki, H., Moriguchi, T., Matsuda, S., Zen, L. H., Nakamura, S., Shimohama, S., Kimura, J., Gotoh, Y., and Nishida, E. (1996) Eur. J. Biochem. 241, 315-321 [Abstract]
  37. Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982 [Abstract]
  38. Cox, J. A., Felder, C. C., and Henneberry, R. C. (1990) Neuron 4, 941-947 [Medline] [Order article via Infotrieve]
  39. Bessho, Y., Nawa, H., and Nakanishi, S. (1994) Neuron 12, 87-95 [Medline] [Order article via Infotrieve]
  40. Heisenberg, C. P., Thoenen, H., and Lindholm, D. (1992) Neuroreport 3, 685-688 [Medline] [Order article via Infotrieve]
  41. Manev, H., Favaron, M., Guidotti, A., and Costa, E. (1989) Mol. Pharmacol. 36, 106-112 [Abstract]
  42. Ghosh, A., and Greenberg, M. E. (1995) Science 268, 239-247 [Medline] [Order article via Infotrieve]
  43. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) FEBS Lett. 364, 229-233 [CrossRef][Medline] [Order article via Infotrieve]
  44. Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376, 737-745 [CrossRef][Medline] [Order article via Infotrieve]
  45. Rusanescu, G., Qi, H., Thomas, S. M., Brugge, J. S., and Halegoua, S. (1995) Neuron 15, 1415-1425 [Medline] [Order article via Infotrieve]
  46. Farnsworth, C. L., Freshney, N. W., Rosen, L. B., Ghosh, A., Greenberg, M. E., and Feig, L. A. (1995) Nature 376, 524-527 [CrossRef][Medline] [Order article via Infotrieve]
  47. Finkbeiner, S., and Greenberg, M. E. (1996) Neuron 16, 233-236 [Medline] [Order article via Infotrieve]
  48. Tokiwa, G., Dikic, I., Lev, S., and Schlessinger, J. (1996) Science 273, 792-794 [Abstract]
  49. Dawson, V. L., Dawson, T. M., London, E. D., Bredt, D. S., and Snyder, S. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6368-6371 [Abstract]
  50. Lafon-Cazal, M., Pietri, S., Culcasi, M., and Bockaert, J. (1993) Nature 364, 535-537 [CrossRef][Medline] [Order article via Infotrieve]
  51. Lipton, S. A., Choi, Y. B., Pan, Z. H., Lei, S. Z., Chen, H. S., Sucher, N. J., Loscalzo, J., Singel, D. J., and Stamler, J. S. (1993) Nature 364, 626-632 [CrossRef][Medline] [Order article via Infotrieve]
  52. Gunasekar, P. G., Kanthasamy, A. G., Borowitz, J. L., and Isom, G. E. (1995) J. Neurochem. 65, 2016-2021 [Medline] [Order article via Infotrieve]
  53. Moriguchi, T., Toyoshima, F., Gotoh, Y., Iwamatsu, A., Irie, K., Mori, E., Kuroyanagi, N., Hagiwara, M., Matsumoto, K., and Nishida, E. (1996) J. Biol. Chem. 271, 26981-26988 [Abstract/Free Full Text]
  54. Lander, H. M., Jacovina, A. T., Davis, R. J., and Tauras, J. M. (1996) J. Biol. Chem. 271, 19705-19709 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.