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Differential activation of c-Jun N-terminal protein kinase and p38 in rat hippocampus and cerebellum after electroconvulsive shock
Neuroscience Letters 271 (1999) 101±104
Differential activation of c-Jun N-terminal protein kinase and p38 in rat hippocampus and cerebellum after electroconvulsive shock Seung Wook Oh a, Yong Min Ahn b, Ung Gu Kang b, Yong Sik Kim b, Joo-Bae Park c,* a
Department of Biochemistry, Seoul National University College of Medicine, Seoul 110±799, South Korea b Department of Psychiatry, Seoul National University College of Medicine, Seoul 110±799, South Korea c Department of Biochemistry and Molecular Biology, Sungkyunkwan University School of Medicine, 300 Chunchundong, Jangangu, Suwon 440±746, South Korea Received 13 April 1999; received in revised form 15 June 1999; accepted 25 June 1999
Abstract Electroconvulsive shock (ECS), an effective treatment for psychiatric diseases, has been reported to induce immediateearly genes (IEGs) and to activate p42 and p44 MAPKs (ERK-1 and ERK-2) in rat brain. In this study, we examined the activation of the other members of MAPK family, c-Jun N-terminal protein kinase (JNK/SAPK) and p38. Following ECS, the phosphorylation of p38 was substantially increased in both hippocampus and cerebellum, but the increase of JNK phosphorylation was observed only in hippocampus. We also investigated the phosphorylation of their upstream kinases, SEK-1, MKK6 and MKK3. In both hippocampus and cerebellum, the phosphorylation of MKK6 showed closer correlation with p38 phosphorylation than that of MKK3. However, SEK-1, known as upstream kinase of JNK and p38 in vitro, corresponded with none of MAPKs. These results, with previous reports on the activation of ERK, indicate that ECS activates three MAPKs differentially in rat hippocampus and cerebellum, and suggest the possibility that unknown MAPKK may be involved in the activation of JNK in rat brain after ECS. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Electroconvulsive shock; Hippocampus; Cerebellum; c-Jun N-terminal protein kinase; p38; MAPK kinase
Electroconvulsive shock (ECS) is an effective treatment for major affective disorders such as depression and schizophrenia. However, the mechanism of its therapeutic effectiveness has not been elucidated yet. Recent molecular approaches to the effects of ECS on the brain have revealed that ECS induces the expressions of various immediateearly genes (IEGs) such as c-fos and jun-B [4], and activates signaling molecules [11,12] in various brain regions. The expressions of IEGs are regulated by transcription factors, such as Elk-1, c-Jun, CREB, ATF-2 etc, and the activities of these molecules are regulated by the complicated signaling cascades of protein kinases. The most well-known kinases for the activation of these transcription factors are MAPK family, which consists of three different protein kinase groups, 42 and 44 kDa MAPKs (ERK-1 and ERK-2), c-Jun N-terminal protein kinase (JNK/SAPK, stress-activated protein kinase) and p38. Three groups of mammalian MAPK are activated by * Corresponding author. Tel.: 182-331-299-6130; fax: 182-331299-6149. E-mail address: [email protected] (J.-B. Park)
different extracellular stimuli and mediate distinct cellular responses including IEG expression. The ERKs are activated by growth factors and phorbol ester, and induce cellular differentiation or proliferation [16]. Another class of MAPK family members, JNK, is strongly activated by stressful environments and apoptotic signals [15]. The p38 is a more recently described member of MAPK family and is responsive to osmotic shock and apoptotic signals as well as many stressful stimuli [9]. The members of MAPK family are activated by distinct protein kinases in the upstream, which are collectively called as MAPK kinases (MKKs) and MAPK kinase kinases (MKKKs). Until now, about 10 different MKKs have been cloned in mammals, of which diversity is believed to enable cells to properly respond to various stimuli [5]. Among these, SEK-1 (MKK4/JNKK1), JNKK2, and MKK7 are known to activate JNK [5,14,18] and SEK-1, MKK3, MKK6 are known to activate p38 [5,7]. Interestingly, SEK-1 was reported to activate both JNK and p38 in vitro [13]. Previously, we and others demonstrated that ERKs were
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 53 5- 2
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activated a few minutes after ECS in rat hippocampus [11,17]. ERK-2 was also activated in rat cerebellum after ECS but in a much lower degree [17]. Besides ERKs, no further advances have been made on JNK and p38 activation after ECS. In this paper, we studied the activation of other two members of MAPK family, JNK and p38, in rat hippocampus and cerebellum after ECS. Electroconvulsive shock was administered to male Sprague±Dawley rats (150±200 g) via ear-clip electrode (130 V, 0.5 s, Medicraft B-24). Control animals were identically handled except for electric current (sham treatment). Rats were decapitated at the given time points (0, 1, 2, 5, 10, 30 and 60 min after ECS). The hippocampus and cerebellum were homogenized in 10 vols. (v/w) of pre-chilled buffer containing 25 mM HEPES (pH 7.9), 150 mM NaCl, 0.2% NP-40, 1 mM DTT, 2 mM EDTA, 1 mM PMSF, 20 mM b glycerophosphate, 1 mM NaF, 1 mM Na3VO4, and 2 mg/ml leupeptin. The protein samples were prepared by boiling with the Laemmli's sampling buffer. The immunoblotting was performed with anti-phospho-JNK (Thr183/Tyr185), phospho-SEK-1 (Thr223), phospho-p38 (Thr180/Tyr182), phospho-MKK3/6 (Ser189/Ser207) antibodies (New England Biolab.). After visualization by ECL system (Pierce), the membranes were reprobed with anti-JNK, SEK-1, p38, MKK3, and MKK6 antibodies (Santa Cruz). We ®rst examined whether ECS increased the phosphorylation of JNK as well as p42 and p44 MAPKs. The slight increase in phosphorylation of p46 JNK was observed in hippocampus (Fig. 1; top left), where the phosphorylation of p54 JNK was rarely detected in rat brain. The phosphorylation of JNK increased right after ECS treatment, reached peak at 5 min, but did not completely return to its basal level
even 60 min after ECS. We observed a 50% average increase in phosphorylation at peak time through several independent experiments. In cerebellum, however, there was no signi®cant increase in phosphorylation of JNK after ECS (Fig. 1; top right). In both tissues, high basal levels of phosphorylation were observed. In order to con®rm whether the phosphorylation re¯ected the actual activity in vivo, we measured the activity of JNK at sham and 5 min after ECS. The activity of JNK was measured with bacterially expressed GST-c-Jun (1±79) fusion protein as substrate. Fifteen micrograms of supernatants were added to reaction mixture (20 mM Tris (pH 7.5), 20 mM MgCl2, 2 mM DTT, 20 mM cold-ATP, 0.17 mCi/ml g - 32P-ATP, 0.2 mg/ml GST-c-Jun (1±79) fusion protein) and incubated at 308C for 3 min. In hippocampus, the activity of JNK 5 min after ECS was 142 ^ 13:8% (P , 0:01, n 5) of that of sham animal, but in cerebellum there was no meaningful increase in the activity after ECS (103:6 ^ 10:8%, 5 min after ECS). We next examined the phosphorylation of SEK-1, which was known as the upstream kinase of JNK. In hippocampus, the phosphorylation of SEK-1 started to increase 2 min after ECS, reached peak at 5 min, but did not return to its basal level even 60 min after ECS (Fig. 1; bottom left), which was similar to that of JNK phosphorylation. At the peak, however, we observed more than 150% increase in phosphorylation of SEK-1 in hippocampus, which was much higher than that of JNK. It discords with the general thought that the signal is ampli®ed traveling downstream in the MAPK signal transduction pathway [16]. Moreover, the increase of SEK-1 phosphorylation was also observed in cerebellum, where JNK was not shown to be activated. In
Fig. 1. Phosphorylation of JNK and SEK-1 in rat hippocampus and cerebellum after ECS. After ECS, rats were decapitated at the indicated times. Hippocampus and cerebellum were homogenized and subjected to SDS-PAGE. The proteins were transferred to membranes, and the membranes were immunoblotted with anti-phospho-JNK (top) and anti-phospho-SEK-1 (bottom) antibodies. After visualization with ECL, deprobed membranes were immunoblotted again with anti-JNK (top) and anti-SEK-1 (bottom) antibodies to analyze protein amounts. Each protein band is indicated by the arrows. The asterisk (*) indicates, phosphorylated ERK, which crossreacts with anti-phospho-JNK antibody (top left).
S.W. Oh et al. / Neuroscience Letters 271 (1999) 101±104
addition, SEK-1 was phosphorylated more heavily in hippocampus than in cerebellum of sham rats, although the phosphorylations of JNK at basal levels were similar in both tissues (Fig. 1; top). These results suggest the possibility that SEK-1 may not be the JNK kinase in rat brain and that MKK other than SEK-1 may activate JNK in rat hippocampus. The phosphorylation of p38, another member of MAPK family, rapidly increased 1±2 min after ECS and decreased after that in rat hippocampus, but did not return to its basal level even at 60 min (Fig. 2; top left). The phosphorylation of p38 in cerebellum increased in the same pattern. However, in contrast to hippocampus, it did not prolong; rather immediately decreased after 5 min, and completely returned to its basal level 10 min after ECS (Fig. 2; top right). ECS also increased the phosphorylation of MKK6 and MKK3, known as the upstream kinases of p38. The phosphorylation of MKK3 slightly increased between 5 min and 30 min after ECS in hippocampus, when the phosphorylation of p38 decreased (Fig. 2; bottom left). In cerebellum, the basal level of phosphorylation of MKK3 was considerably higher than that in hippocampus although basal levels of p38 phosphorylation were almost the same in both tissues. In addition, there was no signi®cant increase in the phosphorylation of MKK3 in cerebellum (Fig. 2; bottom right). Our results clearly indicate that MKK3 is not responsible for the phosphorylation of p38 in both regions after ECS. On the contrary, there is more intimate relevancy between the phosphorylation of p38 and that of MKK6. In hippocampus, the increase in phosphorylation of MKK6 was observed 1 min, reached peak between 5 and 10 min after ECS, and then decreased. However, it did not completely return to its basal level 60 min after ECS (Fig. 2; bottom left). In cerebellum, the phosphorylation of MKK6 was also observed 1 min after ECS, reached peak between 2 and 5
103
min, and then rapidly returned to its basal level (Fig. 2; bottom right). In both hippocampus and cerebellum, there was a discrepancy in the temporal pattern of the phosphorylation of p38 and MKK6. The peak time of p38 was laid between 1 and 2 min after ECS, but that of MKK6 came a few min later in both tissues. Nonetheless, the temporal patterns of p38 and MKK6 phosphorylation coincide in duration as well as the time point when the phosphorylation begins to increase considerably. These results suggest the possibility that MKK6 functions as p38 kinase in rat brain after ECS. However, we should not also rule out the possibility that the other kinases mediate the phosphorylation of p38. Recent studies have demonstrated that p38 kinases, MKK3 and MKK6, are differentially activated by apoptotic signal [8], and activated MKK3 or MKK6 brings about selective activation of p38 isoforms [6]. Our results also show that MKK3 and MKK6 are differentially activated by ECS and suggest that they would play distinct roles in rat brain. It has been generally accepted that each group of MAPK family members has separate signaling pathways, and that JNK and p38 mediate different, even opposite, events from those by ERK. In neuronal cells, JNK and p38 have been reported to be important in neuronal apoptosis, whereas ERK to induce differentiation or cell growth [19]. However, there are evidences that each of MAPK family members cross-talks and converges on some points of cascade to balance the separate pathways [3]. In previous study, we observed that ERK was activated in hippocampus after ECS [11]. Here, we showed that both JNK and p38 were activated in rat hippocampus after ECS. These results indicate that ECS activates all three members of MAPK family in rat hippocampus. However, the consequences of the activation of MAPK family are not clear yet. ECS induces IEGs in rat hippocampus and MAPKs are known as kinases for transcription factors such as TCF and ATF-2 [10]. There-
Fig. 2. Phosphorylation of p38 and MKK3/6 in rat hippocampus and cerebellum after ECS. After ECS, rats were decapitated at the indicated times. The membranes, prepared as describe in Fig. 1, were immunoblotted with anti-phospho-p38 (top) and anti-phosphoMKK3/6 (bottom) antibodies. After visualization with ECL, deprobed membranes were immunoblotted again with anti-p38 (top) and anti-MKK6 (bottom) antibodies to analyze the protein amounts. Each protein band is indicated by the arrows.
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fore, it is quite possible that the activated MAPKs activate transcription factors, which, in turn, induce transcription of IEGs. However, it was reported that activated ERKs in rat hippocampus after ECS were not translocated to nucleus and thus were not able to activate Elk-1 [1]. Therefore, it remains to be elucidated what kinds of transcription factors are activated by each of MAPK family members. Furthermore, ECS does not induce neuronal death in rat brain. Thus, the activated JNK in rat hippocampus after ECS may not be an apoptotic signal and is different from that of glutamate receptor stimulation. We also found that MAPK family members were differentially activated in hippocampus and cerebellum. In cerebellum, the activation of JNK was not observed. Previously, we reported that ECS activated ERKs in rat hippocampus and cerebellum, but the activity of ERKs in cerebellum was less than one forth of that in hippocampus. These results indicate that there are some differences in the signaling system for the activation of each MAPK family in hippocampus and cerebellum, and suggest that three members of MAPK family are activated by distinct upstream signaling pathways after ECS. Furthermore, the phosphorylation of p38 more rapidly returned to its basal level in cerebellum, whereas it was prolonged more than one hour in hippocampus. Chen et al. [2] reported that this type of differences in duration of MAPK activation could determine cell fate upon apoptotic signal in human Jurkat T cells. Therefore, the differences in the duration of p38 in different regions suggest the possibility that MAPKs may play a different role according to the types of tissue. Consequently, this study shows that three MAPKs are differentially activated in rat hippocampus and cerebellum. In addition, our results suggest that neuronal cell response to ECS is determined by a dynamic balance of ERK, JNK, and p38 activities. Finally, we suggest that MKK6, not MKK3, may be the candidate for p38 kinase in both hippocampus and cerebellum after ECS, and that the activation of JNK after ECS may be mediated by a kinase other than SEK-1 in rat brain. Further studies are needed to elucidate the possible JNK kinase(s) in rat hippocampus for the activation of JNK after ECS, and the target molecules for each family of MAPKs.
[4] [5]
[6]
[7]
[8]
[9]
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[13]
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[15]
[16]
This study was supported by Seoul National University Hospital Research Fund (03-1996-090-0). [1] Bhat, R.V., Engber, T.M., Finn, J.P., Koury, E.J., Contreras, P.C., Miller, M.S., Dionne, C.A. and Walton, K.M., Region speci®c targets of p42/p44 MAPK signaling in rat brain. J. Neurochem., 70 (1998) 558±571. [2] Chen, Y., Wang, X., Templeton, D., Davis, R.J. and Tan, T., The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and n radiaton. Duration of JNK activation may determine cell death and proliferation. J. Biol. Chem., 271 (1996) 31929±31936. [3] Cheng, H.L. and Feldman, E.L., Bidirectional regulation of
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p38 kinase and c-Jun N-terminal protein kinase by insulinlike growth factor-I. J. Biol. Chem., 273 (1998) 14560±14565. Cole, A.J., Abu±Shakra, S. and Saffen, D.W., Rapid rise in transcription factor mRNAs in rat brain after electroshockinduced seizures. J. Neurochem., 55 (1990) 1920±1927. Derijard, B., Raingeaud, J., Barrett, T., Wu, I.H., Han, J., Ulevitch, R.J. and Davis, R.J., Independent human MAP kinase signal transduction pathways de®ned by MEK and MKK isoforms. Science, 267 (1995) 682±685. Enslen, H., Raingeaud, J. and Davis, R.J., Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. J. Biol. Chem., 273 (1998) 1741±1748. Han, J., Lee, J.D., Jiang, Y., Li, Z., Feng, L. and Ulevitch, R.J., Characterization of the structure and function of a novel MAP kinase kinase (MKK6). J. Biol. Chem., 271 (1996) 2886±2891. Huang, S., Jiang, Y., Li, Z., Nishida, E., Mathias, P., Lin, S., Ulevitch, R.J., Nemerow, G.R. and Han, J., Apoptosis signaling pathway in T cells is composed of ICE/Ced-3 family proteases and MAP kinase kinase 6b. Immunity, 6 (1997) 739±749. Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K. and Gotoh, Y., Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signalling pathways. Science, 275 (1997) 90±94. Janknecht, R., Ernst, W.H., Pingoud, V. and Nordheim, A., Activation of ternary complex factor Elk-1 by MAP kinases. EMBO J., 12 (1993) 5097±5104. Kang, U.G., Hong, K.S., Jung, H.Y., Kim, Y.S., Soeng, Y.S., Yang, Y.C. and Park, J.B., Activation and tyrosine phosphorylation of 44-kDa mitogen-activated protein kinase (MAPK) induced by electroconvulsive shock in rat hippocampus. J. Neurochem., 63 (1994) 1979±1982. Lee, Y.H., Ryu, S.H., Suh, P.G., Park, J.B., Ahn, Y.M. and Kim, Y.S., Tyrosine phosphorylation of PCL-g1 induced by electroconvulsive shock in rat hippocampus. Biochem. Biophys. Res. Commun., 194 (1993) 665±670. Lin, A., Minden, A., Martinetto, H., Claret, F.X., Lange± Carter, C., Mercurio, F., Johnson, G.L. and Karin, M., Identi®cation of a dual speci®city kinase that activates the Jun kinases and p38-Mpk2. Science, 268 (1995) 286±290. Lu, X., Nemoto, S. and Lin, A., Identi®cation of c-Jun NH2terminal protein kinase (JNK)-activation kinase 2 as an activator of JNK but not p38. J. Biol. Chem., 272 (1997) 2475± 24754. Rosette, C. and Karin, M., Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science, 274 (1996) 1194±1197. Seger, R. and Krebs, E.G., The MAPK signalling cascade. FASEB J., 9 (1995) 726±735. Stratton, K.R., Worley, P.F., Litz, J.S., Parsons, S.J., Huganir, R.L. and Baraban, J.M., Electroconvulsive treatment induces a rapid and transient increase in tyrosine phosphorylation of a 40-kilodalton protein associated with microtubule-associated protein 2 kinase activity. J. Neurochem., 56 (1991) 147±152. Tournier, C., Whitmarsh, A.J., Cavanagh, J., Barrett, T. and Davis, R.J., Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase. Proc. Natl. Acad. Sci. USA, 94 (1997) 7337±7342. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J. and Greenberg, M.E., Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science, 270 (1995) 1326±1331.
Differential activation of c-Jun N-terminal protein kinase and p38 in rat hippocampus and cerebellum after electroconvulsive shock Seung Wook Oh a, Yong Min Ahn b, Ung Gu Kang b, Yong Sik Kim b, Joo-Bae Park c,* a
Department of Biochemistry, Seoul National University College of Medicine, Seoul 110±799, South Korea b Department of Psychiatry, Seoul National University College of Medicine, Seoul 110±799, South Korea c Department of Biochemistry and Molecular Biology, Sungkyunkwan University School of Medicine, 300 Chunchundong, Jangangu, Suwon 440±746, South Korea Received 13 April 1999; received in revised form 15 June 1999; accepted 25 June 1999
Abstract Electroconvulsive shock (ECS), an effective treatment for psychiatric diseases, has been reported to induce immediateearly genes (IEGs) and to activate p42 and p44 MAPKs (ERK-1 and ERK-2) in rat brain. In this study, we examined the activation of the other members of MAPK family, c-Jun N-terminal protein kinase (JNK/SAPK) and p38. Following ECS, the phosphorylation of p38 was substantially increased in both hippocampus and cerebellum, but the increase of JNK phosphorylation was observed only in hippocampus. We also investigated the phosphorylation of their upstream kinases, SEK-1, MKK6 and MKK3. In both hippocampus and cerebellum, the phosphorylation of MKK6 showed closer correlation with p38 phosphorylation than that of MKK3. However, SEK-1, known as upstream kinase of JNK and p38 in vitro, corresponded with none of MAPKs. These results, with previous reports on the activation of ERK, indicate that ECS activates three MAPKs differentially in rat hippocampus and cerebellum, and suggest the possibility that unknown MAPKK may be involved in the activation of JNK in rat brain after ECS. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Electroconvulsive shock; Hippocampus; Cerebellum; c-Jun N-terminal protein kinase; p38; MAPK kinase
Electroconvulsive shock (ECS) is an effective treatment for major affective disorders such as depression and schizophrenia. However, the mechanism of its therapeutic effectiveness has not been elucidated yet. Recent molecular approaches to the effects of ECS on the brain have revealed that ECS induces the expressions of various immediateearly genes (IEGs) such as c-fos and jun-B [4], and activates signaling molecules [11,12] in various brain regions. The expressions of IEGs are regulated by transcription factors, such as Elk-1, c-Jun, CREB, ATF-2 etc, and the activities of these molecules are regulated by the complicated signaling cascades of protein kinases. The most well-known kinases for the activation of these transcription factors are MAPK family, which consists of three different protein kinase groups, 42 and 44 kDa MAPKs (ERK-1 and ERK-2), c-Jun N-terminal protein kinase (JNK/SAPK, stress-activated protein kinase) and p38. Three groups of mammalian MAPK are activated by * Corresponding author. Tel.: 182-331-299-6130; fax: 182-331299-6149. E-mail address: [email protected] (J.-B. Park)
different extracellular stimuli and mediate distinct cellular responses including IEG expression. The ERKs are activated by growth factors and phorbol ester, and induce cellular differentiation or proliferation [16]. Another class of MAPK family members, JNK, is strongly activated by stressful environments and apoptotic signals [15]. The p38 is a more recently described member of MAPK family and is responsive to osmotic shock and apoptotic signals as well as many stressful stimuli [9]. The members of MAPK family are activated by distinct protein kinases in the upstream, which are collectively called as MAPK kinases (MKKs) and MAPK kinase kinases (MKKKs). Until now, about 10 different MKKs have been cloned in mammals, of which diversity is believed to enable cells to properly respond to various stimuli [5]. Among these, SEK-1 (MKK4/JNKK1), JNKK2, and MKK7 are known to activate JNK [5,14,18] and SEK-1, MKK3, MKK6 are known to activate p38 [5,7]. Interestingly, SEK-1 was reported to activate both JNK and p38 in vitro [13]. Previously, we and others demonstrated that ERKs were
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 53 5- 2
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activated a few minutes after ECS in rat hippocampus [11,17]. ERK-2 was also activated in rat cerebellum after ECS but in a much lower degree [17]. Besides ERKs, no further advances have been made on JNK and p38 activation after ECS. In this paper, we studied the activation of other two members of MAPK family, JNK and p38, in rat hippocampus and cerebellum after ECS. Electroconvulsive shock was administered to male Sprague±Dawley rats (150±200 g) via ear-clip electrode (130 V, 0.5 s, Medicraft B-24). Control animals were identically handled except for electric current (sham treatment). Rats were decapitated at the given time points (0, 1, 2, 5, 10, 30 and 60 min after ECS). The hippocampus and cerebellum were homogenized in 10 vols. (v/w) of pre-chilled buffer containing 25 mM HEPES (pH 7.9), 150 mM NaCl, 0.2% NP-40, 1 mM DTT, 2 mM EDTA, 1 mM PMSF, 20 mM b glycerophosphate, 1 mM NaF, 1 mM Na3VO4, and 2 mg/ml leupeptin. The protein samples were prepared by boiling with the Laemmli's sampling buffer. The immunoblotting was performed with anti-phospho-JNK (Thr183/Tyr185), phospho-SEK-1 (Thr223), phospho-p38 (Thr180/Tyr182), phospho-MKK3/6 (Ser189/Ser207) antibodies (New England Biolab.). After visualization by ECL system (Pierce), the membranes were reprobed with anti-JNK, SEK-1, p38, MKK3, and MKK6 antibodies (Santa Cruz). We ®rst examined whether ECS increased the phosphorylation of JNK as well as p42 and p44 MAPKs. The slight increase in phosphorylation of p46 JNK was observed in hippocampus (Fig. 1; top left), where the phosphorylation of p54 JNK was rarely detected in rat brain. The phosphorylation of JNK increased right after ECS treatment, reached peak at 5 min, but did not completely return to its basal level
even 60 min after ECS. We observed a 50% average increase in phosphorylation at peak time through several independent experiments. In cerebellum, however, there was no signi®cant increase in phosphorylation of JNK after ECS (Fig. 1; top right). In both tissues, high basal levels of phosphorylation were observed. In order to con®rm whether the phosphorylation re¯ected the actual activity in vivo, we measured the activity of JNK at sham and 5 min after ECS. The activity of JNK was measured with bacterially expressed GST-c-Jun (1±79) fusion protein as substrate. Fifteen micrograms of supernatants were added to reaction mixture (20 mM Tris (pH 7.5), 20 mM MgCl2, 2 mM DTT, 20 mM cold-ATP, 0.17 mCi/ml g - 32P-ATP, 0.2 mg/ml GST-c-Jun (1±79) fusion protein) and incubated at 308C for 3 min. In hippocampus, the activity of JNK 5 min after ECS was 142 ^ 13:8% (P , 0:01, n 5) of that of sham animal, but in cerebellum there was no meaningful increase in the activity after ECS (103:6 ^ 10:8%, 5 min after ECS). We next examined the phosphorylation of SEK-1, which was known as the upstream kinase of JNK. In hippocampus, the phosphorylation of SEK-1 started to increase 2 min after ECS, reached peak at 5 min, but did not return to its basal level even 60 min after ECS (Fig. 1; bottom left), which was similar to that of JNK phosphorylation. At the peak, however, we observed more than 150% increase in phosphorylation of SEK-1 in hippocampus, which was much higher than that of JNK. It discords with the general thought that the signal is ampli®ed traveling downstream in the MAPK signal transduction pathway [16]. Moreover, the increase of SEK-1 phosphorylation was also observed in cerebellum, where JNK was not shown to be activated. In
Fig. 1. Phosphorylation of JNK and SEK-1 in rat hippocampus and cerebellum after ECS. After ECS, rats were decapitated at the indicated times. Hippocampus and cerebellum were homogenized and subjected to SDS-PAGE. The proteins were transferred to membranes, and the membranes were immunoblotted with anti-phospho-JNK (top) and anti-phospho-SEK-1 (bottom) antibodies. After visualization with ECL, deprobed membranes were immunoblotted again with anti-JNK (top) and anti-SEK-1 (bottom) antibodies to analyze protein amounts. Each protein band is indicated by the arrows. The asterisk (*) indicates, phosphorylated ERK, which crossreacts with anti-phospho-JNK antibody (top left).
S.W. Oh et al. / Neuroscience Letters 271 (1999) 101±104
addition, SEK-1 was phosphorylated more heavily in hippocampus than in cerebellum of sham rats, although the phosphorylations of JNK at basal levels were similar in both tissues (Fig. 1; top). These results suggest the possibility that SEK-1 may not be the JNK kinase in rat brain and that MKK other than SEK-1 may activate JNK in rat hippocampus. The phosphorylation of p38, another member of MAPK family, rapidly increased 1±2 min after ECS and decreased after that in rat hippocampus, but did not return to its basal level even at 60 min (Fig. 2; top left). The phosphorylation of p38 in cerebellum increased in the same pattern. However, in contrast to hippocampus, it did not prolong; rather immediately decreased after 5 min, and completely returned to its basal level 10 min after ECS (Fig. 2; top right). ECS also increased the phosphorylation of MKK6 and MKK3, known as the upstream kinases of p38. The phosphorylation of MKK3 slightly increased between 5 min and 30 min after ECS in hippocampus, when the phosphorylation of p38 decreased (Fig. 2; bottom left). In cerebellum, the basal level of phosphorylation of MKK3 was considerably higher than that in hippocampus although basal levels of p38 phosphorylation were almost the same in both tissues. In addition, there was no signi®cant increase in the phosphorylation of MKK3 in cerebellum (Fig. 2; bottom right). Our results clearly indicate that MKK3 is not responsible for the phosphorylation of p38 in both regions after ECS. On the contrary, there is more intimate relevancy between the phosphorylation of p38 and that of MKK6. In hippocampus, the increase in phosphorylation of MKK6 was observed 1 min, reached peak between 5 and 10 min after ECS, and then decreased. However, it did not completely return to its basal level 60 min after ECS (Fig. 2; bottom left). In cerebellum, the phosphorylation of MKK6 was also observed 1 min after ECS, reached peak between 2 and 5
103
min, and then rapidly returned to its basal level (Fig. 2; bottom right). In both hippocampus and cerebellum, there was a discrepancy in the temporal pattern of the phosphorylation of p38 and MKK6. The peak time of p38 was laid between 1 and 2 min after ECS, but that of MKK6 came a few min later in both tissues. Nonetheless, the temporal patterns of p38 and MKK6 phosphorylation coincide in duration as well as the time point when the phosphorylation begins to increase considerably. These results suggest the possibility that MKK6 functions as p38 kinase in rat brain after ECS. However, we should not also rule out the possibility that the other kinases mediate the phosphorylation of p38. Recent studies have demonstrated that p38 kinases, MKK3 and MKK6, are differentially activated by apoptotic signal [8], and activated MKK3 or MKK6 brings about selective activation of p38 isoforms [6]. Our results also show that MKK3 and MKK6 are differentially activated by ECS and suggest that they would play distinct roles in rat brain. It has been generally accepted that each group of MAPK family members has separate signaling pathways, and that JNK and p38 mediate different, even opposite, events from those by ERK. In neuronal cells, JNK and p38 have been reported to be important in neuronal apoptosis, whereas ERK to induce differentiation or cell growth [19]. However, there are evidences that each of MAPK family members cross-talks and converges on some points of cascade to balance the separate pathways [3]. In previous study, we observed that ERK was activated in hippocampus after ECS [11]. Here, we showed that both JNK and p38 were activated in rat hippocampus after ECS. These results indicate that ECS activates all three members of MAPK family in rat hippocampus. However, the consequences of the activation of MAPK family are not clear yet. ECS induces IEGs in rat hippocampus and MAPKs are known as kinases for transcription factors such as TCF and ATF-2 [10]. There-
Fig. 2. Phosphorylation of p38 and MKK3/6 in rat hippocampus and cerebellum after ECS. After ECS, rats were decapitated at the indicated times. The membranes, prepared as describe in Fig. 1, were immunoblotted with anti-phospho-p38 (top) and anti-phosphoMKK3/6 (bottom) antibodies. After visualization with ECL, deprobed membranes were immunoblotted again with anti-p38 (top) and anti-MKK6 (bottom) antibodies to analyze the protein amounts. Each protein band is indicated by the arrows.
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fore, it is quite possible that the activated MAPKs activate transcription factors, which, in turn, induce transcription of IEGs. However, it was reported that activated ERKs in rat hippocampus after ECS were not translocated to nucleus and thus were not able to activate Elk-1 [1]. Therefore, it remains to be elucidated what kinds of transcription factors are activated by each of MAPK family members. Furthermore, ECS does not induce neuronal death in rat brain. Thus, the activated JNK in rat hippocampus after ECS may not be an apoptotic signal and is different from that of glutamate receptor stimulation. We also found that MAPK family members were differentially activated in hippocampus and cerebellum. In cerebellum, the activation of JNK was not observed. Previously, we reported that ECS activated ERKs in rat hippocampus and cerebellum, but the activity of ERKs in cerebellum was less than one forth of that in hippocampus. These results indicate that there are some differences in the signaling system for the activation of each MAPK family in hippocampus and cerebellum, and suggest that three members of MAPK family are activated by distinct upstream signaling pathways after ECS. Furthermore, the phosphorylation of p38 more rapidly returned to its basal level in cerebellum, whereas it was prolonged more than one hour in hippocampus. Chen et al. [2] reported that this type of differences in duration of MAPK activation could determine cell fate upon apoptotic signal in human Jurkat T cells. Therefore, the differences in the duration of p38 in different regions suggest the possibility that MAPKs may play a different role according to the types of tissue. Consequently, this study shows that three MAPKs are differentially activated in rat hippocampus and cerebellum. In addition, our results suggest that neuronal cell response to ECS is determined by a dynamic balance of ERK, JNK, and p38 activities. Finally, we suggest that MKK6, not MKK3, may be the candidate for p38 kinase in both hippocampus and cerebellum after ECS, and that the activation of JNK after ECS may be mediated by a kinase other than SEK-1 in rat brain. Further studies are needed to elucidate the possible JNK kinase(s) in rat hippocampus for the activation of JNK after ECS, and the target molecules for each family of MAPKs.
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