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Immunolocalization of p38 MAP kinase in mouse brain
Brain Research 887 (2000) 350–358 www.elsevier.com / locate / bres
Research report
Immunolocalization of p38 MAP kinase in mouse brain Masumi Maruyama a , Tatsuhiko Sudo a , *, Yoshitoshi Kasuya b , Takashi Shiga c , Bing-Ren Hu d , Hiroyuki Osada a a
Antibiotics Laboratory, RIKEN, 2 -1 Hirosawa Wako, Saitama 351 -0198 Japan b School of Medicine, Chiba University, Chiba, Japan c Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan d Queen’ s Medical Center, Hawaii, USA Accepted 3 October 2000
Abstract p38 has been implicated to play a critical role in regulating apoptosis in PC12 and cerebellar granule cells, and is inactivated in cultured fetal neurons in response to insulin. Though p38 is activated in microglia after ischemia, the physiological functions of p38 in the brain are not well understood. As a first step to elucidate the physiological functions of p38 in the central nervous system, we raised a polyclonal antibody against p38 and performed immunohistochemical examination to demonstrate the localization of p38 in mouse brain. Strong p38 immunoreactivity was apparent in fiber bundles including the olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, stria terminalis, fimbria and alveus hippocampi, fornix, stria medullaris, optic chiasm and optic tract. Although similar regions were stained with both anti-p38 and anti-neurofilament antibodies, intense p38 immunoreactivity was often observed in myelin sheath-like structures but not in axons. This is the first demonstration of the localization of p38 in the central nervous system and provides an anatomical basis for understanding physiological roles of p38. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Signal transduction: gene expression Keywords: p38 MAP kinase; Immunohistochemistry; Oligodendrocyte; Central nervous system
1. Introduction p38 was first identified as either an anti-inflammatory drug (CSAID) binding protein [15], a lipopolysaccharide (LPS) activated kinase [8] or a stress responsive kinase [21]. p38 is a member of mitogen-activated protein kinases (MAPK) and is activated by dual phosphorylation of the conserved TGY motif [8,15,21]. p38 has been implicated to play roles in converting extracellular stresses to cellular responses. Thus far, four members of p38 have been identified in the human. Among them, p38a and b have been shown to be expressed relatively ubiquitously, by
*Corresponding author. Tel.: 181-48-467-9542; fax: 181-48-4624669. E-mail address: [email protected] (T. Sudo).
Northern analysis of human tissues [12], whereas p38g is expressed only in skeletal muscle [16] and d has limited expression in kidney and lung [13]. In vitro studies have suggested that p38 regulates gene expression by phosphorylating transcription factors, CREBP1 /ATF2 [19], MEF2C [9], CHOP [25] and a transcriptional regulator, Max [27]. Meanwhile p38 phosphorylates cellular kinases such as MAPKAPK2 / 3 [17,21], resulting in hyperphosphorylation of HSP27 to modulate actin dynamics [7]. Although the functional relevance and significance in physiological conditions are not well understood, p38 may play a role in stress responses. More recently, p38 has been implicated to play a critical role in regulating apoptosis in PC12 [26] and cerebellar granule cells [14] by its activation. Conversely, p38 is inactivated in cultured fetal neurons in response to insulin [10]. Global forebrain ischemia results in apoptosis in hippocampal CA1 neurons and activation of p38 in
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03063-8
M. Maruyama et al. / Brain Research 887 (2000) 350 – 358
microglias [24]. Moreover analysis performed in ATF2 null mice has indicated that ATF2 plays a critical role in the hippocampal CA3 [20]. These results suggest that p38 may play a critical part in maintenance of central nervous system (CNS) through the determination of survival and / or death in response to extracellular stimuli. Here we report the localization of p38 in the adult mouse brain as a first step to understanding its physiological functions in the CNS.
2. Material and methods
2.1. Antibodies A polyclonal anti-p38 antibody was raised by immunizing rabbits with an affinity purified recombinant glutathione-S-transferase-p38a (GST-p38) [22]. The antiGST-p38 serum was repeatedly applied to GST-c-Jun [11] column to remove antibodies against GST and bacterial proteins. Then, partially purified serum was multi-step affinity purified by binding to GST-p38 column. Monoclonal antibodies against neurofilament 68, 160, 200 and CNPase were purchased from Sigma-Aldrich, Japan K. K. Alexa 488– and Alexa 594–conjugated second antibodies were purchased from Molecular Probes. Biotinylated secondary antibodies and ABC reagents were purchased from Vector Laboratories.
2.2. Immunoblot analysis Dissected brain tissues for immunoblotting were obtained from adult mouse and sonicated in buffer containing 50 mM Hepes–KOH (pH 7.4), 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 20 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 1 mM p-nitrophenylphosphate, 20 mM bglycerophosphate and 1% Triton X-100. Then 100 mg of protein was loaded in each lane after boiling for 3 min in sample buffer containing sodium dodecyl sulfate (SDS). The tissue samples were resolved by 12% SDS–polyacrylamide gel electrophoresis and transferred onto Immobilon (Millipore). The filters were blocked for 1 h in 5% non-fat dried milk in buffer containing 10 mM Tris–HCl (pH 7.8) and 144 mM NaCl (TBS) supplemented with 0.05% Tween-20 (TBST). Then filters were incubated for 2 h in 1% non-fat dried milk in TBST containing anti-p38 antibody or pre-immune serum. After several washes, the blots were incubated with horseradish–peroxidase conjugated anti-rabbit IgG (Santa Cruz) in 1% non-fat dried milk containing TBST for 1 h followed by several washes with TBST, and developed using SuperSignal (Pierce).
2.3. Immunohistochemistry Eight-week-old C57BL / 6J male mice (n56) were pur-
351
chased from CLEA Japan Inc. Under deep anesthesia with an intraperitoneal injection of sodium pentobarbital, mice were perfused through the left cardiac ventricle with saline followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4. The brain was then removed and immersion fixed in the same fixative for 12 h at 48C. For paraffin sections, the brain was dehydrated in graded ethanol, embedded in paraffin, and sectioned at 4 mm thickness. For cryostat sections, the brain was immersed in the series of 5, 15 and 30% sucrose solutions in PBS for cryoprotection, frozen in Tissue Tek O.C.T. compound (Miles Inc.), and sectioned at 10 mm thickness. For immunoperoxidase labeling, sections were blocked by incubation with 10% normal horse serum in PBS for 30 min followed by several washes with PBS. They were then incubated with a primary antibody in PBS for 1 h followed by several washes with PBS containing 0.05% Triton X-100 (PBST). Then sections were incubated in biotinylated secondary antibody for 1 h followed by several washes with PBS. They were then incubated in ABC reagent followed by several washes with PBS. Bound peroxidase was visualized by incubating sections with 0.05% diaminobenzidine and 0.01% H 2 O 2 in 50 mM Tris–HCl, pH 7.4. Control sections were incubated with pre-immune serum. All incubations were performed at room temperature. For immunofluorescence double labeling, sections were blocked by incubating with 10% normal goat serum in PBS for 30 min followed by several washes with PBS. Then they were incubated with a primary antibody in PBS with 2% normal goat serum for 1 or 3 h followed by several washes with PBST. Alexa 488– and Alexa 594– conjugated second antibodies were diluted 2000-fold and incubated for 1 h in PBS containing 2% goat serum. After extensive washing with PBS, sections were examined by using fluorescent microscopy (Olympus). Control sections were incubated either with pre-immune serum or antibody preabsorbed with GST-p38. The terminology of brain areas was mainly after Paxinos and Watson [18] and Franklin and Paxinos [6]
3. Results
3.1. Specificity of the anti-p38 antibody To study the localization of p38 MAP kinase at the protein level, we used a rabbit polyclonal antibody against recombinant GST-p38. Immunoblotting experiments showed that the anti-p38 antibody but not preimmune serum specifically recognized a single band of about 42 kD in mouse brain extracts and extracts of murine embryonic fibroblasts prepared from E10.5 of progeny of p38 1 / 2 intercrosses [23] (Fig. 1). Moreover, the immunoreactivity recognized by the anti-p38 antibody disappeared when
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Fig. 1. Specificity of anti-p38 antibody against brain extracts and extracts of murine embryonic fibroblasts (MEF) prepared from E10.5 progeny of p38 1 / 2 intercrosses. 100 mg of brain homogenate (lanes 1, 2) and 10 mg of MEF extracts (lanes 3–8) were applied for immunoblotting experiments. Lanes: 1, 3–5 pre-immune serum was used as 1st antibody (negative control): Lanes: 2, 6–8 anti-p38 antibody recognizes a single band of 42 kD except for p38 2 / 2 MEF (lane 8). The positions of molecular mass markers are indicated on the left.
coronal sections were stained either with preimmune serum or with antibody pre-absorbed with GST-p38 (Fig. 2). These results demonstrated the specificity of the anti-p38 antibody.
3.2. Localization of p38 in mouse brain To determine the localization of p38 in adult mouse brain, the anti-p38 antibody was applied to paraffin-embedded sections and cryostat sections, which resulted in the similar patterns (Figs. 3 and 4). Strong p38 immunoreactivity was observed in fiber bundles, but not in cell bodies or nuclei. In the telencephalon, strong p38 immunoreactivity was observed in the lateral olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, external capsule, stria terminalis, fimbria and alveus hippocampi. In the deeper layers of the neocortex, many fibers running in various directions were also immunoreactive for p38 (Fig. 3B). In the diencephalon, strong immunoreactivity was observed in the fornix, mammilothalamic tract, stria medullaris, fasciculus retroflexus, optic chiasm, and optic tract. In the brainstem, many fiber bundles were strongly immunoreactive for p38, including cerebral peduncles, longitudinal fasciculus and transverse fibers of the pons. In the cerebellum, the white matter (corpus medullare) was strongly immunoreactive for p38 (Fig. 3D). It is noteworthy that no fibers in CA1 through dentate gyrus in the hippocampus were immunoreacitve for p38, whereas many fibers including mossy fibers in CA3 region
Fig. 2. Specific recognition by anti-p38 antibody in brain sections. Coronal sections were incubated with either anti-p38 antibody (A), pre-immune serum (B) or preabsorption of anti-p38 antibody with GSTp38 (C). dlo, dorsolateral olfactory tract; lo, lateral olfactory tract. Scale bar5100 mm.
were immunoreactive for neurofilaments (Fig. 5A, B). Dendrites of Purkinje cells in the molecular layer were not immunoreactive for p38 in the cerebellum (Fig. 5C, D).
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Fig. 3. Distribution of p38 immunoreactivity in the forebrain and the cerebellum in sagittal sections. Intense p38 immunoreactivity is observed in ac, anterior commissure; f, fornix; cc, corpus callosum; cg, cingulum; sm, stria medullaris; fr, fasciculus retroflexus; ox, optic chiasm; lfp, longitudinal fasciculus; tfp, transverse fibers of the pons; wm, white matter in the cerebellum. gcc, genu corpus callosum; MHb, medial habenular nucleus; fr, fasciculus retroflexus; ml, molecular layer; gl, granular layer; Pl, Purkinje layer. (A) to (F) were paraffin-embedded sections and (G) was a cryostat section. Scale bars530 mm for (A,E,G), 60 mm for (B,C,D) and 150 mm for (F).
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Fig. 4. Distribution of p38 immunoreactivity in horizontal and coronal sections. Dense p38 immunoreactivity is observed in dlo, dorsolateral olfactory tract; lo, lateral olfactory tract (A); sm, stria medullaris; cc, corpus callosum (B); alv, alveus hippocampi; bsc, brachium superior colliculus; ec, external capsule; fi, fimbria hippocampus; st, stria terminalis (C) in horizontal sections and cg, cingulum; fr, fasciculus retroflexus (D); fr; ic, internal capsule; opt, optic tract; mt, mammillothalamic tract; cp, cerebral peduncles (E) in coronal section. CPu, caudate putamen; Ld, lambdoid septal zone; MHb, medial habenular nucleus; Rt, reticular thalamic nucleus. Scale bar5150 mm.
3.3. Cellular localization of p38 immunoreactivity Since strong p38 immunoreactivity was observed only in
fiber bundles, it is possible that axons and / or myelin sheaths express p38. To identify p38-immunoreactive structures at the cellular level, sections were first double-
M. Maruyama et al. / Brain Research 887 (2000) 350 – 358
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Fig. 5. Absence of p38 immunoreactivity in dentate gyrus and parallel fibers. Horizontal (A,B) and sagittal sections (C,D) were labeled with either anti-p38 (A,C) or anti-neurofilament antibodies (B,D). CA3, area CA3; DG, dentate gyrus; ml, molecular layer; gl, granular layer; Pl, Purkinje layer; wm, white matter. Scale bar5150 mm.
labeled with anti-p38 and anti-neurofilament 68, 160 and 180 antibodies. At higher resolution, p38 immunoreactivity was often observed in myelin sheath-like structures surrounding axons (Fig. 6A, C, D, F), but not in axons, which were immunoreactive for neurofilaments (Fig. 6B, C, E, F). Localization of p38 in myelin sheath structures was confirmed by double labeling with anti-p38 and antiCNPase antibodies (Fig. 7C, F). These results suggest that in some thick fibers, p38 is predominantly expressed in the myelin sheath but not axons.
4. Discussions We raised a specific anti-p38 MAP kinase antibody and analyzed the localization of p38 immunohistochemically in
adult mouse brain. Previous studies concerning p38 expression in the brain are very limited. In the hippocampus CA1, phosphorylated p38 is increased in microglia but not neurons after ischemia [24]. Phosphorylated p38 is also increased in cultured cerebellar granule cells under glutamate-induced apoptosis [14]. This is the first report regarding the expression of p38 in the central nervous system under physiological conditions. Our results demonstrated that p38 MAP kinase is highly expressed in fiber bundles including the olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, stria terminalis, fimbria and alveus hippocampi, fornix, stria medullaris, optic chiasm, and optic tract. Although similar regions were stained with both anti-p38 and anti-neurofilament antibodies, strong immunoreactivity was often observed in myelin sheath-like structures, but not in axons.
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Fig. 6. Cellular localization of p38 immunoreactivity. Sections were double-labeled with anti-p38 and anti-neurofilament 68, 160 and 180 antibodies. At higher resolution, in the caudate putamen, p38 immunoreactivity (green fluorescence) was often observed in myelin sheath-like structures surrounding axons (A, C, D, F), but not in axons, which were immunoreactive for neurofilaments (red fluorescence, B, C, E, F). Arrowheads mark equivalent positions in A to C or D to F respectively. Scale bar52.5 mm.
Although p38 has been shown to be expressed in various tissues and cultured cells [10,12–14,16,24,26], the present results clearly show the preferential distribution, or rather cell type-specific expression of p38 in the CNS, indicating that additional, yet unreported, physiological functions can be attributed to p38. The proinflammatory cytokine, tumor necrosis factor-a (TNF-a) and nitric oxide (NO) have been reported to be responsible for several CNS disorders, including inflammatory, infectious, traumatic and degenerative diseases [3]. In inflammatory conditions, inducible nitric oxide synthase (iNOS) which produces NO, is expressed predominantly in activated astrocytes and microglia [4]. By using a specific p38 inhibitor, SB203580, p38 has been demonstrated to play an important role in endotoxin-induced cellular response [5]. Key roles of extracellular signal-regulated kinase (ERK) and p38 in iNOS gene regulation in primary cultured glial cells in response to endotoxin have recently been reported [1]. Also, p38 is reported to be highly phosphorylated after ischemia in microglia but not neurons in the hippocampus CA1 region [24]. It is not possible to
rule out the possible existence of a relatively low but functionally sufficient amount of p38 in those cells under physiological conditions. However, the predominant expression of p38 in myelin sheath-like structures may help to understand its physiological roles in cytokine-induced expression of iNOS gene in oligodendrocytes [2]. Further understanding of the physiological functions and regulation of p38 in the CNS will provide insights into the molecular mechanisms of signal transduction pathways. This subject is currently being studied using transgenic mice produced by gene targeting.
Acknowledgements We thank H. Hama, H. Mizuno and M. Yamamoto for helpful discussions, and R. Sato for technical advice. This work was supported by President’s Special Grant from RIKEN and a grant from the Bioarchitect Research Project of RIKEN to T. Sudo.
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Fig. 7. Colocalization of p38 and CNPase immunoreactivities in myelin sheath-like structures. Sections were double-labeled with anti-p38 (green fluorescence) and anti-CNPase (red fluorescence) antibodies. In the caudate putamen, p38 immunoreactivity was often colocalized in myelin sheath-like structures with CNPase. Colocalization can be seen by the yellow-orange color in C and F. Arrowheads mark equivalent positions in A to C or D to F respectively. Scale bar52.5 mm.
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[15] J.C. Lee, J.T. Laydon, P.C. McDonnell, T.F. Gallagher, S. Kumar, D. Green, D. McNulty, M.J. Blumenthal, J.R. Heys, S.W. Landvatter, J.E. Strickler, M.M. McLaughlin, I.R. Siemens, S.M. Fisher, G.P. Livi, J.R. White, J.L. Adams, P. Young, A protein kinase involved in the regulation of inflammatory cytokine biosynthesis, Nature 372 (1994) 739–746. [16] Z. Li, J. Yong, R.J. Ulevitch, J. Han, The primary structure of p38g: A new member of p38 group of MAP kinase, Biochem. Biophys. Res. Commun. 228 (1996) 334–340. [17] M.M. McLaughlin, S. Kumar, P.C. McDonnell, S.V. Horn, J.C. Lee, G.P. Livi, P.R. Young, Identification of mitogen-activated protein kinase-activated protein kinase3, a novel substrate of CSBP p38 MAP kinase, J. Biol. Chem. 271 (1996) 8488–8492. [18] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinate, Vol. 2nd ed., Academic Press, San Diego, 1986. [19] J. Raingeaud, S. Gupta, J.S. Rogers, M. Dickens, J. Han, R.J. Ulevitch, R.J. Davis, Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine, J. Biol. Chem. 270 (1995) 7420–7426. [20] A.M. Reimold, M.J. Grusby, B. Kosaras, J.W.U. Fries, R. Mori, S. Moniwa, I.M. Clauss, T. Collins, R.L. Sidman, M.J. Glimcher, L.H. Glimcher, Chondrodysplasia and neurological abnormalities in ATF2-deficient mice, Nature 376 (1996) 262–265.
[21] J. Rouse, P. Cohen, S. Trigon, M. Morange, A.A. Llamazares, D. Zamanillo, T. Hunt, A.R. Nebreda, A novel kinase cascade triggered by stress and heat shock that stimulate MAPKAP kinase-2 and phosphorylation of small heat shock proteins, Cell 78 (1994) 1027– 1037. [22] T. Sudo, M. Karin, Assays for JNK and p38, in: J.C. Reed (Ed.), Methods in Enzymology, Vol. 322, Academic Press, San Diego, 2000, pp. 388–392. [23] K. Tamura, T. Sudo, U. Senftleben, A.M. Dadak, R. Johnson, M. Karin, Requirement for p38a in erythropoietin expression: a role for stress kinases in erythropoiesis, Cell 102 (2000) 221–231. [24] K.M. Walton, R. DiRocco, B.A. Bartlett, E. Kourny, V.R. Marcy, B. Jarvis, E.M. Schafer, R.V. Bhat, Activation of p38MAPK in microglia after ischemia, J. Neurochem. 70 (1998) 1764–1767. [25] X.Z. Wang, D. Ron, Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP kinase, Science 272 (1996) 1347–1349. [26] Z. Xia, M. Dickens, J. Raingeaud, R.J. Davis, M.E. Greenberg, Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis, Science 270 (1995) 1326–1331. [27] A.S. Zervos, L. Faccio, J.P. Gatto, J.M. Kyriakis, R. Brent, Mxi2, a mitogen-activated protein kinase that recognizes and phosphorylates Max protein, Proc. Natl. Acad. Sci. USA 92 (1995) 10531–10534.
Research report
Immunolocalization of p38 MAP kinase in mouse brain Masumi Maruyama a , Tatsuhiko Sudo a , *, Yoshitoshi Kasuya b , Takashi Shiga c , Bing-Ren Hu d , Hiroyuki Osada a a
Antibiotics Laboratory, RIKEN, 2 -1 Hirosawa Wako, Saitama 351 -0198 Japan b School of Medicine, Chiba University, Chiba, Japan c Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan d Queen’ s Medical Center, Hawaii, USA Accepted 3 October 2000
Abstract p38 has been implicated to play a critical role in regulating apoptosis in PC12 and cerebellar granule cells, and is inactivated in cultured fetal neurons in response to insulin. Though p38 is activated in microglia after ischemia, the physiological functions of p38 in the brain are not well understood. As a first step to elucidate the physiological functions of p38 in the central nervous system, we raised a polyclonal antibody against p38 and performed immunohistochemical examination to demonstrate the localization of p38 in mouse brain. Strong p38 immunoreactivity was apparent in fiber bundles including the olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, stria terminalis, fimbria and alveus hippocampi, fornix, stria medullaris, optic chiasm and optic tract. Although similar regions were stained with both anti-p38 and anti-neurofilament antibodies, intense p38 immunoreactivity was often observed in myelin sheath-like structures but not in axons. This is the first demonstration of the localization of p38 in the central nervous system and provides an anatomical basis for understanding physiological roles of p38. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Signal transduction: gene expression Keywords: p38 MAP kinase; Immunohistochemistry; Oligodendrocyte; Central nervous system
1. Introduction p38 was first identified as either an anti-inflammatory drug (CSAID) binding protein [15], a lipopolysaccharide (LPS) activated kinase [8] or a stress responsive kinase [21]. p38 is a member of mitogen-activated protein kinases (MAPK) and is activated by dual phosphorylation of the conserved TGY motif [8,15,21]. p38 has been implicated to play roles in converting extracellular stresses to cellular responses. Thus far, four members of p38 have been identified in the human. Among them, p38a and b have been shown to be expressed relatively ubiquitously, by
*Corresponding author. Tel.: 181-48-467-9542; fax: 181-48-4624669. E-mail address: [email protected] (T. Sudo).
Northern analysis of human tissues [12], whereas p38g is expressed only in skeletal muscle [16] and d has limited expression in kidney and lung [13]. In vitro studies have suggested that p38 regulates gene expression by phosphorylating transcription factors, CREBP1 /ATF2 [19], MEF2C [9], CHOP [25] and a transcriptional regulator, Max [27]. Meanwhile p38 phosphorylates cellular kinases such as MAPKAPK2 / 3 [17,21], resulting in hyperphosphorylation of HSP27 to modulate actin dynamics [7]. Although the functional relevance and significance in physiological conditions are not well understood, p38 may play a role in stress responses. More recently, p38 has been implicated to play a critical role in regulating apoptosis in PC12 [26] and cerebellar granule cells [14] by its activation. Conversely, p38 is inactivated in cultured fetal neurons in response to insulin [10]. Global forebrain ischemia results in apoptosis in hippocampal CA1 neurons and activation of p38 in
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03063-8
M. Maruyama et al. / Brain Research 887 (2000) 350 – 358
microglias [24]. Moreover analysis performed in ATF2 null mice has indicated that ATF2 plays a critical role in the hippocampal CA3 [20]. These results suggest that p38 may play a critical part in maintenance of central nervous system (CNS) through the determination of survival and / or death in response to extracellular stimuli. Here we report the localization of p38 in the adult mouse brain as a first step to understanding its physiological functions in the CNS.
2. Material and methods
2.1. Antibodies A polyclonal anti-p38 antibody was raised by immunizing rabbits with an affinity purified recombinant glutathione-S-transferase-p38a (GST-p38) [22]. The antiGST-p38 serum was repeatedly applied to GST-c-Jun [11] column to remove antibodies against GST and bacterial proteins. Then, partially purified serum was multi-step affinity purified by binding to GST-p38 column. Monoclonal antibodies against neurofilament 68, 160, 200 and CNPase were purchased from Sigma-Aldrich, Japan K. K. Alexa 488– and Alexa 594–conjugated second antibodies were purchased from Molecular Probes. Biotinylated secondary antibodies and ABC reagents were purchased from Vector Laboratories.
2.2. Immunoblot analysis Dissected brain tissues for immunoblotting were obtained from adult mouse and sonicated in buffer containing 50 mM Hepes–KOH (pH 7.4), 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 20 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 1 mM p-nitrophenylphosphate, 20 mM bglycerophosphate and 1% Triton X-100. Then 100 mg of protein was loaded in each lane after boiling for 3 min in sample buffer containing sodium dodecyl sulfate (SDS). The tissue samples were resolved by 12% SDS–polyacrylamide gel electrophoresis and transferred onto Immobilon (Millipore). The filters were blocked for 1 h in 5% non-fat dried milk in buffer containing 10 mM Tris–HCl (pH 7.8) and 144 mM NaCl (TBS) supplemented with 0.05% Tween-20 (TBST). Then filters were incubated for 2 h in 1% non-fat dried milk in TBST containing anti-p38 antibody or pre-immune serum. After several washes, the blots were incubated with horseradish–peroxidase conjugated anti-rabbit IgG (Santa Cruz) in 1% non-fat dried milk containing TBST for 1 h followed by several washes with TBST, and developed using SuperSignal (Pierce).
2.3. Immunohistochemistry Eight-week-old C57BL / 6J male mice (n56) were pur-
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chased from CLEA Japan Inc. Under deep anesthesia with an intraperitoneal injection of sodium pentobarbital, mice were perfused through the left cardiac ventricle with saline followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4. The brain was then removed and immersion fixed in the same fixative for 12 h at 48C. For paraffin sections, the brain was dehydrated in graded ethanol, embedded in paraffin, and sectioned at 4 mm thickness. For cryostat sections, the brain was immersed in the series of 5, 15 and 30% sucrose solutions in PBS for cryoprotection, frozen in Tissue Tek O.C.T. compound (Miles Inc.), and sectioned at 10 mm thickness. For immunoperoxidase labeling, sections were blocked by incubation with 10% normal horse serum in PBS for 30 min followed by several washes with PBS. They were then incubated with a primary antibody in PBS for 1 h followed by several washes with PBS containing 0.05% Triton X-100 (PBST). Then sections were incubated in biotinylated secondary antibody for 1 h followed by several washes with PBS. They were then incubated in ABC reagent followed by several washes with PBS. Bound peroxidase was visualized by incubating sections with 0.05% diaminobenzidine and 0.01% H 2 O 2 in 50 mM Tris–HCl, pH 7.4. Control sections were incubated with pre-immune serum. All incubations were performed at room temperature. For immunofluorescence double labeling, sections were blocked by incubating with 10% normal goat serum in PBS for 30 min followed by several washes with PBS. Then they were incubated with a primary antibody in PBS with 2% normal goat serum for 1 or 3 h followed by several washes with PBST. Alexa 488– and Alexa 594– conjugated second antibodies were diluted 2000-fold and incubated for 1 h in PBS containing 2% goat serum. After extensive washing with PBS, sections were examined by using fluorescent microscopy (Olympus). Control sections were incubated either with pre-immune serum or antibody preabsorbed with GST-p38. The terminology of brain areas was mainly after Paxinos and Watson [18] and Franklin and Paxinos [6]
3. Results
3.1. Specificity of the anti-p38 antibody To study the localization of p38 MAP kinase at the protein level, we used a rabbit polyclonal antibody against recombinant GST-p38. Immunoblotting experiments showed that the anti-p38 antibody but not preimmune serum specifically recognized a single band of about 42 kD in mouse brain extracts and extracts of murine embryonic fibroblasts prepared from E10.5 of progeny of p38 1 / 2 intercrosses [23] (Fig. 1). Moreover, the immunoreactivity recognized by the anti-p38 antibody disappeared when
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Fig. 1. Specificity of anti-p38 antibody against brain extracts and extracts of murine embryonic fibroblasts (MEF) prepared from E10.5 progeny of p38 1 / 2 intercrosses. 100 mg of brain homogenate (lanes 1, 2) and 10 mg of MEF extracts (lanes 3–8) were applied for immunoblotting experiments. Lanes: 1, 3–5 pre-immune serum was used as 1st antibody (negative control): Lanes: 2, 6–8 anti-p38 antibody recognizes a single band of 42 kD except for p38 2 / 2 MEF (lane 8). The positions of molecular mass markers are indicated on the left.
coronal sections were stained either with preimmune serum or with antibody pre-absorbed with GST-p38 (Fig. 2). These results demonstrated the specificity of the anti-p38 antibody.
3.2. Localization of p38 in mouse brain To determine the localization of p38 in adult mouse brain, the anti-p38 antibody was applied to paraffin-embedded sections and cryostat sections, which resulted in the similar patterns (Figs. 3 and 4). Strong p38 immunoreactivity was observed in fiber bundles, but not in cell bodies or nuclei. In the telencephalon, strong p38 immunoreactivity was observed in the lateral olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, external capsule, stria terminalis, fimbria and alveus hippocampi. In the deeper layers of the neocortex, many fibers running in various directions were also immunoreactive for p38 (Fig. 3B). In the diencephalon, strong immunoreactivity was observed in the fornix, mammilothalamic tract, stria medullaris, fasciculus retroflexus, optic chiasm, and optic tract. In the brainstem, many fiber bundles were strongly immunoreactive for p38, including cerebral peduncles, longitudinal fasciculus and transverse fibers of the pons. In the cerebellum, the white matter (corpus medullare) was strongly immunoreactive for p38 (Fig. 3D). It is noteworthy that no fibers in CA1 through dentate gyrus in the hippocampus were immunoreacitve for p38, whereas many fibers including mossy fibers in CA3 region
Fig. 2. Specific recognition by anti-p38 antibody in brain sections. Coronal sections were incubated with either anti-p38 antibody (A), pre-immune serum (B) or preabsorption of anti-p38 antibody with GSTp38 (C). dlo, dorsolateral olfactory tract; lo, lateral olfactory tract. Scale bar5100 mm.
were immunoreactive for neurofilaments (Fig. 5A, B). Dendrites of Purkinje cells in the molecular layer were not immunoreactive for p38 in the cerebellum (Fig. 5C, D).
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Fig. 3. Distribution of p38 immunoreactivity in the forebrain and the cerebellum in sagittal sections. Intense p38 immunoreactivity is observed in ac, anterior commissure; f, fornix; cc, corpus callosum; cg, cingulum; sm, stria medullaris; fr, fasciculus retroflexus; ox, optic chiasm; lfp, longitudinal fasciculus; tfp, transverse fibers of the pons; wm, white matter in the cerebellum. gcc, genu corpus callosum; MHb, medial habenular nucleus; fr, fasciculus retroflexus; ml, molecular layer; gl, granular layer; Pl, Purkinje layer. (A) to (F) were paraffin-embedded sections and (G) was a cryostat section. Scale bars530 mm for (A,E,G), 60 mm for (B,C,D) and 150 mm for (F).
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Fig. 4. Distribution of p38 immunoreactivity in horizontal and coronal sections. Dense p38 immunoreactivity is observed in dlo, dorsolateral olfactory tract; lo, lateral olfactory tract (A); sm, stria medullaris; cc, corpus callosum (B); alv, alveus hippocampi; bsc, brachium superior colliculus; ec, external capsule; fi, fimbria hippocampus; st, stria terminalis (C) in horizontal sections and cg, cingulum; fr, fasciculus retroflexus (D); fr; ic, internal capsule; opt, optic tract; mt, mammillothalamic tract; cp, cerebral peduncles (E) in coronal section. CPu, caudate putamen; Ld, lambdoid septal zone; MHb, medial habenular nucleus; Rt, reticular thalamic nucleus. Scale bar5150 mm.
3.3. Cellular localization of p38 immunoreactivity Since strong p38 immunoreactivity was observed only in
fiber bundles, it is possible that axons and / or myelin sheaths express p38. To identify p38-immunoreactive structures at the cellular level, sections were first double-
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Fig. 5. Absence of p38 immunoreactivity in dentate gyrus and parallel fibers. Horizontal (A,B) and sagittal sections (C,D) were labeled with either anti-p38 (A,C) or anti-neurofilament antibodies (B,D). CA3, area CA3; DG, dentate gyrus; ml, molecular layer; gl, granular layer; Pl, Purkinje layer; wm, white matter. Scale bar5150 mm.
labeled with anti-p38 and anti-neurofilament 68, 160 and 180 antibodies. At higher resolution, p38 immunoreactivity was often observed in myelin sheath-like structures surrounding axons (Fig. 6A, C, D, F), but not in axons, which were immunoreactive for neurofilaments (Fig. 6B, C, E, F). Localization of p38 in myelin sheath structures was confirmed by double labeling with anti-p38 and antiCNPase antibodies (Fig. 7C, F). These results suggest that in some thick fibers, p38 is predominantly expressed in the myelin sheath but not axons.
4. Discussions We raised a specific anti-p38 MAP kinase antibody and analyzed the localization of p38 immunohistochemically in
adult mouse brain. Previous studies concerning p38 expression in the brain are very limited. In the hippocampus CA1, phosphorylated p38 is increased in microglia but not neurons after ischemia [24]. Phosphorylated p38 is also increased in cultured cerebellar granule cells under glutamate-induced apoptosis [14]. This is the first report regarding the expression of p38 in the central nervous system under physiological conditions. Our results demonstrated that p38 MAP kinase is highly expressed in fiber bundles including the olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, stria terminalis, fimbria and alveus hippocampi, fornix, stria medullaris, optic chiasm, and optic tract. Although similar regions were stained with both anti-p38 and anti-neurofilament antibodies, strong immunoreactivity was often observed in myelin sheath-like structures, but not in axons.
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Fig. 6. Cellular localization of p38 immunoreactivity. Sections were double-labeled with anti-p38 and anti-neurofilament 68, 160 and 180 antibodies. At higher resolution, in the caudate putamen, p38 immunoreactivity (green fluorescence) was often observed in myelin sheath-like structures surrounding axons (A, C, D, F), but not in axons, which were immunoreactive for neurofilaments (red fluorescence, B, C, E, F). Arrowheads mark equivalent positions in A to C or D to F respectively. Scale bar52.5 mm.
Although p38 has been shown to be expressed in various tissues and cultured cells [10,12–14,16,24,26], the present results clearly show the preferential distribution, or rather cell type-specific expression of p38 in the CNS, indicating that additional, yet unreported, physiological functions can be attributed to p38. The proinflammatory cytokine, tumor necrosis factor-a (TNF-a) and nitric oxide (NO) have been reported to be responsible for several CNS disorders, including inflammatory, infectious, traumatic and degenerative diseases [3]. In inflammatory conditions, inducible nitric oxide synthase (iNOS) which produces NO, is expressed predominantly in activated astrocytes and microglia [4]. By using a specific p38 inhibitor, SB203580, p38 has been demonstrated to play an important role in endotoxin-induced cellular response [5]. Key roles of extracellular signal-regulated kinase (ERK) and p38 in iNOS gene regulation in primary cultured glial cells in response to endotoxin have recently been reported [1]. Also, p38 is reported to be highly phosphorylated after ischemia in microglia but not neurons in the hippocampus CA1 region [24]. It is not possible to
rule out the possible existence of a relatively low but functionally sufficient amount of p38 in those cells under physiological conditions. However, the predominant expression of p38 in myelin sheath-like structures may help to understand its physiological roles in cytokine-induced expression of iNOS gene in oligodendrocytes [2]. Further understanding of the physiological functions and regulation of p38 in the CNS will provide insights into the molecular mechanisms of signal transduction pathways. This subject is currently being studied using transgenic mice produced by gene targeting.
Acknowledgements We thank H. Hama, H. Mizuno and M. Yamamoto for helpful discussions, and R. Sato for technical advice. This work was supported by President’s Special Grant from RIKEN and a grant from the Bioarchitect Research Project of RIKEN to T. Sudo.
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Fig. 7. Colocalization of p38 and CNPase immunoreactivities in myelin sheath-like structures. Sections were double-labeled with anti-p38 (green fluorescence) and anti-CNPase (red fluorescence) antibodies. In the caudate putamen, p38 immunoreactivity was often colocalized in myelin sheath-like structures with CNPase. Colocalization can be seen by the yellow-orange color in C and F. Arrowheads mark equivalent positions in A to C or D to F respectively. Scale bar52.5 mm.
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