p38 Mitogen-activated Protein Kinase Regulates Low Potassium-induced c-Jun Phosphorylation and Apoptosis in Cultured Cerebellar Granule Neurons
2001; Elsevier BV; Volume: 276; Issue: 7 Linguagem: Inglês
10.1074/jbc.m007258200
ISSN1083-351X
AutoresSatoru Yamagishi, Masashi Yamada, Yasuyuki Ishikawa, Tomoya Matsumoto, Toshihiko Ikeuchi, Hiroshi Hatanaka,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoCultured rat cerebellar granule neurons are widely used as a model system for studying neuronal apoptosis. After maturation by culturing in medium containing 26 mmpotassium (high K+), changing to medium containing 5 mm potassium (low K+; LK) rapidly induces neuronal apoptosis. Then over 50% of granule cells die within 24 h. However, the molecular mechanisms by which the LK-induced apoptosis occurs in cultured cerebellar granule cells remain unclear. In the present study, we found that p38 MAP kinase (p38) was an important factor for LK-induced apoptosis. Three hours after changing to LK medium, p38 was markedly activated. In addition, SB203580, a specific inhibitor of p38, strongly inhibited the phosphorylation and expression of c-Jun in LK-induced apoptosis of cultured cerebellar granule cells.In vitro kinase assay using glutathioneS-transferase-c-Jun as a substrate showed that p38 directly phosphorylated c-Jun. Furthermore, in the presence of SB203580, about 80% of neurons survived. These results indicate that p38 regulates LK-induced apoptosis of cerebellar granule neurons. Cultured rat cerebellar granule neurons are widely used as a model system for studying neuronal apoptosis. After maturation by culturing in medium containing 26 mmpotassium (high K+), changing to medium containing 5 mm potassium (low K+; LK) rapidly induces neuronal apoptosis. Then over 50% of granule cells die within 24 h. However, the molecular mechanisms by which the LK-induced apoptosis occurs in cultured cerebellar granule cells remain unclear. In the present study, we found that p38 MAP kinase (p38) was an important factor for LK-induced apoptosis. Three hours after changing to LK medium, p38 was markedly activated. In addition, SB203580, a specific inhibitor of p38, strongly inhibited the phosphorylation and expression of c-Jun in LK-induced apoptosis of cultured cerebellar granule cells.In vitro kinase assay using glutathioneS-transferase-c-Jun as a substrate showed that p38 directly phosphorylated c-Jun. Furthermore, in the presence of SB203580, about 80% of neurons survived. These results indicate that p38 regulates LK-induced apoptosis of cerebellar granule neurons. low potassium mitogen-activated protein high potassium c-Jun N-terminal kinase activating transcription factor 2 brain-derived neurotrophic factor insulin-like growth factor-1 glutathione S-transferase minimum essential medium 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide Apoptosis is a fundamental biological process used to eliminate unwanted, superfluous, or potentially harmful cells. In the developing nervous system, about half of all neurons that are produced die by apoptosis around the time of birth (1Oppenheim R.W. Annu. Rev. Neurosci. 1991; 14: 453-501Crossref PubMed Scopus (2754) Google Scholar). Cultured rat cerebellar granule cells are widely used as a model system for studying neuronal apoptosis. These neurons are usually cultured and matured in medium containing 26 mm potassium (high K+; HK).1 After maturation, changing to medium containing 5 mm potassium (low K+; LK) induces neuronal cell death in which the cells show apoptotic features. In addition, this culture system provides a large homogeneous neuronal population. Therefore, these neurons are widely used as a primary cell culture system to investigate the biochemical and molecular mechanisms underlying neuronal apoptosis in the central nervous system. Mitogen-activated protein (MAP) kinases are serine/threonine kinases that play an important role in signal transduction from the cell surface to the nucleus. The mammalian MAP kinases can be subdivided into extracellular signal-regulated kinases, c-Jun N-terminal kinases (JNK), and p38 MAP kinases (p38). p38 is activated by phosphorylation on Thr-180 and Tyr-182 in response to environmental stress (2Treisman R. Curr. Opin. Cell. Biol. 1996; 8: 205-215Crossref PubMed Scopus (1160) Google Scholar, 3Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2278) Google Scholar). Recently, p38 activation has been suggested to be involved in mediating apoptosis in various cell types (4Kummer J.L. Rao P.K. Heidenreich K.A. J. Biol. Chem. 1997; 272: 20490-20494Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 5Horstmann S. Kahle P.J. Borasio G.D. J. Neurosci. Res. 1998; 52: 483-490Crossref PubMed Scopus (124) Google Scholar, 6Behrens M.M. Strasser U. Koh J.Y. Gwag B.J. Choi D.W. Neuroscience. 1999; 94: 917-927Crossref PubMed Scopus (67) Google Scholar). SB203580, a specific inhibitor of p38, prevents several types of cell death, including glutamate-induced apoptosis of cultured cerebellar granule neurons (7Kawasaki H. Morooka T. Shimohama S. Kimura J. Hirano T. Gotoh Y. Nishida E. J. Biol. Chem. 1997; 272: 18518-18521Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar,8Kikuchi M. Tenneti L. Lipton S.A. J. Neurosci. 2000; 20: 5037-5044Crossref PubMed Google Scholar). c-Jun is a member of the activator protein-1 family of transcription factors possessing leucine zippers. c-Jun is phosphorylated on Ser-63 and Ser-73 within its N-terminal region by JNK (9Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1708) Google Scholar). Phosphorylation of c-Jun is necessary for apoptosis of superior cervical ganglion and cerebellar granule neurons (10Ham J. Babij C. Whitfield J. Pfarr C.M. Lallemand D. Yaniv M. Rubin L.L. Neuron. 1995; 14: 927-939Abstract Full Text PDF PubMed Scopus (757) Google Scholar, 11Watson A. Eilers A. Lallemand D. Kyriakis J. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 751-762Crossref PubMed Google Scholar). However, the mechanism of c-Jun phosphorylation remains unclear. In the present study, we investigated whether p38 is necessary for LK-induced apoptosis and whether p38 is involved in the phosphorylation of c-Jun in cerebellar granule neurons. Our results suggest that p38 was activated after lowering potassium concentration, and active p38 phosphorylates c-Jun directly. We found the p38-c-Jun pathway is very important for LK-induced apoptosis in cerebellar granule neurons. Primary cultures of dissociated cerebellar granule neurons were prepared from the cerebella of postnatal day 9 rats (Wistar ST, both sexes) as described previously (12Levi G. Aloisi F. Ciotti M.T. Gallo V. Brain Res. 1984; 290: 77-86Crossref PubMed Scopus (389) Google Scholar, 13Hatanaka H. Tsukui H. Nihonmatsu I. Brain Res. 1988; 467: 85-95Crossref PubMed Scopus (189) Google Scholar, 14Kubo T. Nonomura T. Enokido Y. Hatanaka H. Dev. Brain Res. 1995; 85: 249-258Crossref PubMed Scopus (165) Google Scholar, 15Shimoke K. Kubo T. Numakawa T. Abiru Y. Enokido Y. Takei N. Ikeuchi T. Hatanaka H. Dev. Brain Res. 1997; 101: 197-206Crossref PubMed Scopus (59) Google Scholar, 16Shimoke K. Yamada M. Ikeuchi T. Hatanaka H. FEBS Lett. 1998; 437: 221-224Crossref PubMed Scopus (18) Google Scholar). Briefly, cells were gently dissociated with a plastic pipette after digestion with papain (90 units/ml, Worthington) at 37 °C. The cells were then cultured in medium consisting of 5% precolostrum newborn calf serum (Mitsubishi Kasei), 5% heat-inactivated horse serum (55 °C, 30 min; Life Technologies, Inc.), and 90% 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium containing 15 mmHEPES buffer (pH 7.4), 30 nm selenium, and 1.9 mg/ml sodium bicarbonate, at a final cell density of 5 × 105cells/cm2 on a polyethyleneimine-coated surface in 6-cm-diameter dishes (21 cm2 of culture surface area, Sumitomo Bakelite). After culture for 1 day in a humidified CO2 (5%) incubator, the medium was changed to 26 mm potassium-containing (HK) minimum essential medium (MEM) supplemented with 5% heat-inactivated horse serum and 1 μm cytosine arabinoside. MEM was supplemented with 2.2 mg/ml glucose and 2.2 mg/ml sodium bicarbonate. HK-MEM was prepared by increasing the KHCO3 concentration from the normal low value of 5.4 to 26 mm, with the omission of the corresponding concentration NaHCO3. After 4 days in culture in a 10% CO2 incubator, the medium was changed to serum-free 5.4 mm potassium-containing (LK) MEM or HK-MEM. The assays described below were then performed. Neuronal survival was determined by MTT assay, according to the original procedure (17Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (45846) Google Scholar) with some modifications (18Hansen M.B. Nielsen S.E. Berg K. J. Immunol. Methods. 1989; 119: 203-210Crossref PubMed Scopus (3329) Google Scholar). Briefly, the tetrazolium salt MTT (3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide) was added to cultures to a final concentration of 1 mg/ml. After incubation for 2 h at 37 °C, the assay was stopped by adding 80% volume of lysis buffer (10% SDS in 50%N,N-dimethyl formamide, pH 4.7). The absorbance was measured spectrophotometrically at 570 nm, following overnight incubation at 37 °C. The percent survival was defined as (A(experimental − blank)/A(control − blank)) × 100; the blank value was taken from wells without cells. To stain neurons, the cultured cells were fixed with 4% paraformaldehyde for 20 min and incubated with anti-MAP2 antiserum (1:1000; a gift from Dr. H. Murofushi) overnight at 4 °C. Cells were visualized with a Vectastain ABC kit (Vector Laboratories), followed by exposure to 0.02% 3–3′-diaminobenzidine 4-HCl, 0.3% H2O2 and 0.1% (NH4)2Ni(SO4)2. Cells were lysed in SDS lysis buffer containing 1% SDS, 20 mm Tris-HCl (pH 7.4), 5 mm EDTA (pH 8.0), 10 mm NaF, 2 mmNa3VO4, 0.5 mm phenylarsine oxide, 10 mm Na4P2O4, and 1 mm phenylmethylsulfonyl fluoride. The lysates were boiled for 3 min and then clarified by ultracentrifugation at 60,000 ×g at 8 °C for 30 min. The protein concentration was determined using a BCA protein assay kit (Pierce), and then aliquots of 10 μg of protein were resolved by electrophoresis on 10% SDS-polyacrylamide gels. Proteins were transferred onto polyvinylidene fluoride membranes (Millipore Corp.) in 0.1 m Tris base, 0.192 m glycine, and 20% methanol using a semi-dry electrophoretic transfer system. The membranes were blocked with 0.1% Tween 20, Tris-buffered saline (T-TBS) containing 5% nonfat dried milk at room temperature for 1 h. Membranes were probed with 1:200 anti-phospho-JNK antibody, 1:200 anti-JNK antibody, 1:200 anti-phospho-p38 antibody, 1:1000 anti-p38 antibody, 1:200 anti-phospho-c-Jun antibody, or 1:1000 anti-c-Jun antibody in T-TBS containing 1 or 5% nonfat dried milk at room temperature for 1 h. After washing three times with T-TBS, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or donkey anti-mouse IgG secondary antibody (Zymed Laboratories Inc. or Jackson ImmunoResearch Laboratories, Inc.) diluted 1:1000 with T-TBS at room temperature for 1 h. The membranes were then washed at least 4 times with T-TBS and were visualized using the ECL chemiluminescence system (Amersham Pharmacia Biotech) or Immunostar (Wako). The cell lysates were prepared at 0, 3, 6, and 9 h after changing to LK medium using Triton lysis buffer containing 1% Triton X-100, 20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 5 mm EDTA (pH 8.0), 10 mm NaF, 2 mm Na3VO4, 0.5 mm phenylarsine oxide, 10 mmNa4P2O4, 2 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride. Then 1 μg of anti-p38 antibody was added to the lysates followed by incubation at 4 °C for at least 3 h. Protein G-Sepharose (10-μl gel) was then added and rotated at 4 °C for 1 h. The immune complexes were pelleted by centrifugation at 10,000 × g at 4 °C for 1 min and then washed twice with Triton lysis buffer and twice with kinase buffer containing 40 mm HEPES (pH 7.4), 10 mmMgCl2, 3 mm MnCl2. p38 kinase reaction was carried out for 20 min at 25 °C with 5 μg of GST-ATF2 (rat, residues 1–109) or GST-c-Jun (human, residues 1–125) as a substrate and the immunoprecipitate in kinase buffer containing 10 μCi of [γ-32P]ATP, 20 μm ATP. After SDS-polyacrylamide gel electrophoresis, the incorporation of32P into GST-ATF2-(1–109) or GST-c-Jun-(1–125) was visualized using a Fuji BAS2000 image analyzer. Anti-MAP2 antibody was kindly provided by Dr. H. Murofushi. Anti-phospho-p38, anti-phospho-JNK, and anti-JNK antibodies were purchased from New England Biolabs Inc., and anti-p38, anti-phospho-c-Jun and anti-c-Jun antibodies were from Santa Cruz Biotechnology Inc. SB203580 was obtained from Calbiochem and dissolved at 10 mm in dimethyl sulfoxide (Me2SO) as a stock solution. We utilized primary cultures of cerebellar granule cells from neonatal rats as a model system to investigate the intracellular signaling in LK-induced cell death. This cell death shows characteristic features of apoptosis (14Kubo T. Nonomura T. Enokido Y. Hatanaka H. Dev. Brain Res. 1995; 85: 249-258Crossref PubMed Scopus (165) Google Scholar, 15Shimoke K. Kubo T. Numakawa T. Abiru Y. Enokido Y. Takei N. Ikeuchi T. Hatanaka H. Dev. Brain Res. 1997; 101: 197-206Crossref PubMed Scopus (59) Google Scholar). After maturation for 4 days in HK medium, the medium was switched to serum-free LK medium. At 24 h after reduction of potassium concentration, cells were stained with anti-MAP2 antibody (Fig. 1 A), and cell survival was determined by MTT assay (Fig. 1 B). As a result, 51.7% of granule neurons died in LK medium. Previous studies showed that the level of c-Jun expression was increased, and the N-terminal phosphorylation of c-Jun was necessary for the process of apoptosis (11Watson A. Eilers A. Lallemand D. Kyriakis J. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 751-762Crossref PubMed Google Scholar, 19Miller T.M. Johnson E.M.J. J. Neurosci. 1996; 16: 7487-7495Crossref PubMed Google Scholar). Therefore, we performed Western blotting analysis with monoclonal anti-phosphorylated c-Jun (Ser-63) antibody to detect the phosphorylation of c-Jun (Fig.2 A). Three hours after changing to LK medium, the level of c-Jun phosphorylation was increased, and this phosphorylation was maintained for 12 h. This phosphorylated and activated c-Jun may form homo- or heterodimers with c-Fos, and the dimerized c-Jun may up-regulate the expression of c-Jun itself. The c-Jun up-regulation is known to be important in DNA damage-induced apoptosis (20Araki T. Enokido Y. Inamura N. Aizawa S. Reed J.C. Hatanaka H. Brain Res. 1998; 794: 239-247Crossref PubMed Scopus (39) Google Scholar) and apoptosis induced by the PI3-K inhibitor, LY294002, in cerebellar granule cells (21Shimoke K. Yamagishi S. Yamada M. Ikeuchi T. Hatanaka H. Dev. Brain Res. 1999; 112: 245-253Crossref PubMed Scopus (66) Google Scholar). Therefore, we examined the level of c-Jun protein during LK-induced cell death using a polyclonal anti-c-Jun antibody (Fig. 2 B). Our results indicated that the expression of c-Jun was markedly increased from 6 to 12 h after potassium deprivation. The delay in the changes in the protein level compared with the phosphorylation of c-Jun was considered to be due to the period required for transcription and translation of c-Jun. In contrast, the phosphorylation and protein levels of c-Jun in serum-free HK-MEM were not changed from basal level. Next we investigated what kind of kinase phosphorylates c-Jun in cerebellar granule neurons. The first candidate was c-Jun N-terminal kinase (JNK) that belongs to the MAP kinase superfamily and is activated by phosphorylation on Thr-183 and Tyr-185. JNK activation is known to participate in apoptosis of brain neurons and PC12 cells (22Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5028) Google Scholar, 23Yang D.D. Kuan C.Y. Whitmarsh A.J. Rincon M. Zheng T.S. Davis R.J. Rakic P. Flavell R.A. Nature. 1997; 389: 865-870Crossref PubMed Scopus (1114) Google Scholar, 24Herdegen T. Skene P. Bahr M. Trends Neurosci. 1997; 20: 227-231Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). In cerebellar granule neurons, glutamate-induced exitotoxicity and DNA damage activated JNK (7Kawasaki H. Morooka T. Shimohama S. Kimura J. Hirano T. Gotoh Y. Nishida E. J. Biol. Chem. 1997; 272: 18518-18521Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 25Inamura N. Araki T. Enokido Y. Nishio C. Aizawa S. Hatanaka H. J. Neurosci. Res. 2000; 60: 450-457Crossref PubMed Scopus (28) Google Scholar). We examined whether JNK was activated to phosphorylate c-Jun during LK-induced apoptosis. To examine JNK activation, the lysates from granule cells cultured for 0, 3, 6, 9, and 12 h in serum-free LK or HK medium after maturation were immunoblotted with anti-phospho-JNK (Fig.3 A). We could not detect any increase in the JNK phosphorylation after lowering potassium concentration. This result suggested that JNK is not involved in c-Jun phosphorylation during LK-induced apoptosis of granule cells, consistent with the observations of Watson et al. (11Watson A. Eilers A. Lallemand D. Kyriakis J. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 751-762Crossref PubMed Google Scholar). The next candidate was p38, which also belongs to the MAP kinase superfamily. To examine whether p38 was activated during potassium deprivation-induced apoptosis, lysates from granule cells cultured for 0, 3, 6, 9, and 12 h in serum-free LK or HK medium were immunoblotted with anti-phospho-p38 antibody (Fig. 3 B). p38 was markedly phosphorylated within 3 h, and the increased level of phosphorylation was prolonged to 9 h after potassium deprivation. To confirm this observation, we further performed in vitro kinase assay of p38 (Fig. 3 C). p38 was immunoprecipitated from the lysates with anti-p38 antibody, and the p38 kinase assay was carried out with [γ-32P]ATP using GST-ATF2-(1–109) as a substrate. ATF2 was markedly phosphorylated by p38 3 h after potassium deprivation. These results indicated that p38 is indeed activated during LK-induced cell death. It is an intriguing question whether p38 phosphorylates c-Jun. As shown in Figs.2 A and 3 B, the time courses of phosphorylation of p38 and c-Jun were very similar. To examine whether p38 is involved in the regulation of c-Jun, we investigated the effects of SB203580, a specific inhibitor of p38, on the phosphorylation and expression of c-Jun. The addition of SB203580 to LK medium markedly suppressed the phosphorylation and up-regulation of c-Jun in a dose-dependent manner (Fig.4 A). Next, we examined whether p38 directly phosphorylated c-Jun using in vitro kinase assay. After immunoprecipitation with anti-p38 antibody, the p38 kinase assay was carried out with [γ-32P]ATP, using GST-c-Jun-(1–125) as the substrate. At 3 h after potassium deprivation, c-Jun was phosphorylated by p38 (Fig. 4 B). These results suggested that p38 directly phosphorylates c-Jun during LK-induced apoptosis of granule cells. To examine whether the activation of p38 is involved in the process of LK-induced apoptosis, we utilized SB203580, an inhibitor of p38. When apoptosis was induced by lowering the potassium concentration, the inhibitor was added to LK medium (Fig. 5). After 24 h, cell survival was quantified by counting the number of MAP2-positive cells. Resultingly SB203580 inhibited neuronal apoptosis. In the presence of 3, 10, and 30 μm SB203580, 59, 74, and 81% of cells survived, respectively. This result indicated that p38 is involved in the signaling pathway of LK-induced apoptosis. Several recent studies have suggested that p38 is required for some apoptotic processes (5Horstmann S. Kahle P.J. Borasio G.D. J. Neurosci. Res. 1998; 52: 483-490Crossref PubMed Scopus (124) Google Scholar, 6Behrens M.M. Strasser U. Koh J.Y. Gwag B.J. Choi D.W. Neuroscience. 1999; 94: 917-927Crossref PubMed Scopus (67) Google Scholar). For example, during glutamate-evoked apoptosis of cerebellar granule neurons, p38 was transiently activated, and this apoptosis was prevented by SB203580 (7Kawasaki H. Morooka T. Shimohama S. Kimura J. Hirano T. Gotoh Y. Nishida E. J. Biol. Chem. 1997; 272: 18518-18521Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). However, function of p38 in apoptotic process remains unclear. In this study, we investigated the role of p38 in LK-induced apoptosis of cultured cerebellar granule neurons. Our results indicated that p38 was phosphorylated and activated during LK-induced apoptosis (Fig. 3,B and C). In Fig. 3 B, control neurons in HK medium also showed a transient and slight activation of p38. The transient activation of p38 was considered to be due to changing from serum-containing to serum-free HK-MEM. Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) are known to rescue granule neurons from LK-induced apoptosis (14Kubo T. Nonomura T. Enokido Y. Hatanaka H. Dev. Brain Res. 1995; 85: 249-258Crossref PubMed Scopus (165) Google Scholar, 26D'Mello S.R. Galli C. Ciotti T. Calissano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993Crossref PubMed Scopus (850) Google Scholar, 27Yamagishi S. Fujikawa N. Kohara K. Tominaga-Yoshino K. Ogura A. Neuroscience. 2000; 95: 473-479Crossref PubMed Scopus (8) Google Scholar). We observed that BDNF and IGF-1 rescued about 60 and 80%, respectively, of neurons from cell death (data not shown). Furthermore, the addition of BDNF and IGF-1 to LK medium suppressed the activation of p38 similarly to control neurons in HK medium (data not shown). The mRNA and protein levels of c-Jun were selectively increased after NGF withdrawal in sympathetic neurons (10Ham J. Babij C. Whitfield J. Pfarr C.M. Lallemand D. Yaniv M. Rubin L.L. Neuron. 1995; 14: 927-939Abstract Full Text PDF PubMed Scopus (757) Google Scholar, 28Estus S. Zaks W.J. Freeman R.S. Gruda M. Bravo R. Johnson E.M.J. J. Cell Biol. 1994; 127: 1717-1727Crossref PubMed Scopus (786) Google Scholar). In cerebellar granule neurons, Watson et al. (11Watson A. Eilers A. Lallemand D. Kyriakis J. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 751-762Crossref PubMed Google Scholar) showed that dominant negative c-JunAla, which cannot be phosphorylated, inhibited LK-induced apoptosis. We observed marked changes in the levels of phosphorylation and expression of c-Jun during this apoptotic process. Although the importance of c-Jun is well known, its kinase has remained unclear. The first candidate was JNK, because the phosphorylation of c-Jun by JNK was well known in sympathetic neurons (29Eilers A. Whitfield J. Babij C. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 1713-1724Crossref PubMed Google Scholar). However, in cerebellar granule neurons, the JNK activity was high under basal conditions and was not activated further during apoptosis. The next candidate was p38, because the time courses of the phosphorylation of p38 and c-Jun during apoptosis were very similar. To investigate the role of p38 in the phosphorylation of c-Jun, we utilized SB203580. The addition of this inhibitor completely prevented both phosphorylation and up-regulation of c-Jun (Fig. 4 A). In addition, in vitro kinase assay of p38 showed that p38 could directly phosphorylate c-Jun (Fig. 4 B). Furthermore, we found that SB203580 prevented LK-induced apoptosis in a dose-dependent manner (Fig. 5). This prevention of apoptosis seemed to be due to the inhibition of phosphorylation of c-Jun. It has been reported that SB203580 can block some JNKs at high doses (30Clerk A. Sugden P.H. FEBS Lett. 1998; 426: 93-96Crossref PubMed Scopus (205) Google Scholar, 31Whitmarsh A.J. Yang S.-H. Su M.S.-S. Sharrocks A.D. Davis R.J. Mol. Cell. Biol. 1997; 17: 2360-2371Crossref PubMed Scopus (438) Google Scholar, 32Harada J. Sugimoto M. Jpn. J. Pharmacol. 1999; 79: 369-378Crossref PubMed Scopus (113) Google Scholar). Since we could not detect low potassium-induced activation of JNKs, its contribution to the present findings is likely to be limited but warrants further investigation. SB202190 is also utilized as a specific inhibitor of p38 (33Lee 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. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3128) Google Scholar). We observed a weak but significant inhibitory effect of SB202190 on the apoptosis (data not shown). ATF2 is a member of the ATF/CREB family of basic region leucine zipper (bZIP) DNA-binding proteins. The N-terminal transactivation domain of ATF2 is phosphorylated by both p38 and JNK (34Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1336) Google Scholar, 35van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (569) Google Scholar, 36Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (472) Google Scholar). Therefore, we investigated whether ATF2 was also phosphorylated by p38 during LK-induced apoptosis. The phosphorylation of ATF2 was detected by Western blotting analysis using anti-phospho-ATF2 antibody. ATF2 was markedly phosphorylated at 3 h (data not shown). The time course of ATF2 phosphorylation corresponded to those of phosphorylation of p38 and c-Jun. These observations suggested that p38 is involved in phosphorylation of not only c-Jun but also of ATF2. We observed that p38 showed greater affinity for ATF2 than c-Jun as a substrate in the kinase assay utilizing the same amounts of GST-ATF2 and GST-c-Jun (data not shown). In addition, we investigated whether SB203580 inhibited phosphorylation of ATF2 as well as that of c-Jun. SB203580 indeed inhibited ATF2 phosphorylation (data not shown). This result confirmed that SB203580 suppresses the p38 pathway. The results presented here demonstrate that p38 can directly phosphorylate c-Jun during LK-induced apoptosis in cultured cerebellar granule neurons. However, the mechanisms of c-Jun-mediated apoptosis remain unclear. Therefore, further studies should be performed to identify c-jun target genes and to determine how c-Jun regulates apoptosis. We thank Regeneron Pharmaceutical Co. for the kind gifts of BDNF; Dr. H. Murofushi (The University of Tokyo) for anti-MAP2 antibody; and Dr. N. Nomura (Kazusa DNA Research Institute) for c-Jun DNA.
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