calmodulin-dependent protein kinase α in the rat central nervous system: an immunohistochemical study

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Neuroseience Letters 204 (1996) 61-64

NEUROSCIENC[ LETTERS

Distribution of Ca2+/calmodulin-dependent protein kinase kinase a in the rat central nervous system: an immunohistochemical study Y a s u h i s a N a k a m u r a a,*, S a c h i k o O k u n o b, T a k a k o K i t a n i b, K a z u y o s h i Otakea, F u m i S a t o a, Hitoshi Fujisawa b aDepartment of Anatomy, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan bDepartment of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan Rece:ived 24 November 1995; revised version received 19 December 1995; accepted 20 December 1995

Abstract

Ca2÷/caimodulin-dependent protein kinase IV (CaM-kinase IV) is activated by CaM-kinase IV kinase. We provided a rabbit antiserum against 20 amino acid residues at the carboxyl-terminal end of CaM-kinase IV kinase, and examined regional and intracellular distribution of CaM-kinase IV kinase immunohistochemically in the central nervous system of the rat by light and electron microscopy. The immunoreactivity was found in cellular nuclei of virtually all neurons. However, the immunoreactivity was weak in the nuclei of the granule cells in the cerebellar cortex, although the nuclei of the granule cells were reported to contain high CaM-kinase IV activity. Thus, it was suggested that other types of CaM-kinase IV kinase might exist in the cerebellum, and the present CaM-kinase IV kinase was named as CaM-kinase ldnase a.

Keywords: Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV); CaM-kinase kinase a; Immunohistochemistry; Brain; Spinal cord; Cellular nuclei; Rat

Ca2+/calmodulin-dependent protein kinase IV (CAMkinase IV), a Ca2÷-responsive multifunctional protein kinase [6], is markedly activated upon phosphorylation by CaM-kinase IV kinase [9-11,16,17]. CaM-kinase IV occurred predominantly in cellular nuclei of many neurons in the central nervous system (CNS) [2,7,8,13,14]. In the present study, the distribution pattern of CaM-kinase IV kinase which we named as CaM-kinase kinase a (to be discussed later) was examined immunohistochemically to compare with that of CaM-kinase IV. Approximately 1.15 mg of a synthetic peptide, CGEGGKSPELPGVQEDEAAS, corresponding to the carboxyl-terminal 20 amino acids of CaM-kinase kinase a was conjugated to 1.6 mg of keyhole limpet hemocyanin (Sigma) by using m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce) as the coupling reagent [5] in the presence of dithiothreitol after reduction of the peptide with sodium borohydride [1]. Two Japanese white rabbits were immunized with the conjugate; approximately 275/zg of the conjugate in Freund's complete ad* Corresponding author. Tel.: +81 3 58035148; fax: +81 3 58035151.

juvant were intradermally injected. Four weeks later the rabbits were again injected with 275/zg of the conjugate in Freund's incomplete adjuvant. Ten days later the rabbits were three times boosted intravenously with 140#g each of the conjugates in buffered saline every 10 days. The antiserum was harvested 1 week after the final injection. For immunoblot analysis, the cerebral cortex of the rat was homogenized with a Teflon/glass homogenizer in 3 vol. of 20 mM HEPES (pH 7.5) containing 1 mM dithiothreitol, 0.1% Triton X-100, and 20#g/ml each of leupeptin, pepstatin, antipain A, and chymostatin (Peptide Institute, Osaka), and the residue was removed by centrifugation at 105 000 × g for 60 min to generate the crude extract. The crude extract and the purified CaMkinase kinase a [10] were subjected to SDS-polyacrylamide gel electrophoresis on 7.5% gel [3], and the protein bands separated were transferred onto a polyvinylidene difluoride membrane (Pall). The membrane was blocked with phosphate-buffered saline containing 5% non-fat dried milk, and then incubated with the antiserum at 1:100 dilution in the blocking buffer, followed by incu-

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1 Fig. 1. Western blot analysis of the rat brain crude extract oy mm~lu, . . . . . . . . . . . . . . . . . . , , , , ~ ~,. r~vp~oxlmamJy z o l t g protein (lane 1) and 10/~g protein (lane 2) of the crude extract of rat cerebral cortex and 20 ng of CaM-kinase kinase a (lane 3) were subjected. Positions of the molecular weight markers are indicated on the left in kDa. Fig. 2. A sagittal section through a rat brain, showing CaM-kinase kinase a-like immunoreactivity. CblCx, cerebellar cortex; CbrCx, cerebral cortex; CP, caudate-putamen; dTh, dorsal thalamus; Hip, hippocampal formation; Hyp, hypothalamus; IC, inferior colliculus; OB, olfactory bulb; PN, pontine nuclei; SC, superior colliculus. Bar = 5 mm. bation with g o a t antirabbit i m m u n o g l o b u l i n conjugated with peroxidase. T h e positive bands w e r e detected with 0.05% 3 , 3 ' - d i a m i n o b e n z i d i n e tetrahydrochloride ( D A B ) and 0.03% H202 in the p r e s e n c e o f 0.02% cobalt chloride.

For i m m u n o h i s t o c h e m i c a l observations, four y o u n g male rats (Wistar, 7 w e e k s old) w e r e used. T h e y were perfused with phosphate buffered 4 . 0 % p a r a f o r m a l d e h y d e solution through the ascending aorta under d e e p pento-

Fig. 3. CaM-kinase kinase a-like immunoreactivity in the parietal cortex (a) and cerebellar cortex (b). In the cerebral cortex, the immunoreactivity is seen in cellular nuclei of all neurons from layer I to layer VI. In the cerebellar cortex, the immunoreactivity is seen in the stellate cells (arrow heads) in the molecular layer (Mo), Purkinje cells (P-arrows), and possible Golgi cells (Go-arrows). The nuclei of granule cells (Gr-arrows) in the granular layer are also weakly immunoreactive. Bars = 100/.tin.

1". Nakamura et al. / Neuroscience Letters 204 (1996) 61-64

barbital anesthesia (50 mg/kg, i.p.). For light microscopy, three brains were cut ~,~erially at 30/zm into frontal or sagittal sections on a freezing microtome; for electron microscopy, a brain was cut at 150/zm with a microslicer. Procedures for immunohistochemistry for light and electron microscopy were as reported previously [7]. Briefly, free-floating sections in 0.1 M phosphate buffer containing 0.1 or 0.05% Triton X-100 were incubated for 2 h at room temperature with the antiserum (1:5000), then with biotinylated antirabbit immunoglobulin, reacted with Vectastain ABC kit. To visualize bound peroxidase, 0.05% DAB was used with 0.03% H20 2, 0.005% nickel chloride and cobalt acetate in the phosphate buffer. For light microscopy, the sections were mounted on gelatinized glass slides; some of them were counterstained with 0.5% Cresyl violet. For electron microscopy, the immunostained sections were trimmed and embedded in Epon after osmification,, dehydration and block-stain with 2% uranium acetate. Ultrathin sections, which were not stained with uranium or lead, were observed under a JEM 100C electron microscope. Fig. 1 shows the results of immunoblot analysis of the crude extract of the cerebral cortex of the rat and purified brain CaM-kinase kinase a with the antiserum raised against the carboxyl-terminal segment of CaM-kinase kinase a. The extract gave a single band in the same position as the purified enzyme. The estimated molecular weight was 66 000. This finding indicated that the antiserum reacted specifically with CaM-kinase kinase a. CaM-kinase kinase .~z-like immunoreactivity was observed throughout the CNS (Fig. 2); it was found in cellular nuclei of virtually all neurons (Figs. 2 and 3). However, the immunoreactivity in the nuclei of the granule cells in the cerebellar cortex was obviously weaker than in the nuclei of the other neurons (Fig. 3b). Cytoplasm of large neurons in the rubral, Deiters' or motor nuclei also showed weak immunostaining. Intranuclear localization of the reaction products was confirmed by electron microscopy (Fig. 4). The present results indicated that CaM-kinase kinase a was localized in cellular nuclei of neurons. This pattern of intracellular distribution of CaM-kinase kinase a was the same as that of CaM-kinase IV [7], although CaMkinase kinase a was distributed more widely than CaMkinase IV in the rat CNS. In the granule cells of the cerebellar cortex, CaM-kinase kinase a-like immunoreactivity was very weak, whereets CaM-kinase IV-like immunoreactivity was highest in the rat CNS [7,8]. It was thus assumed that CaM-kinase IV kinase other than the present CaM-kinase kinase a might exist in the granule cells of the cerebellar cortex. In fact, immunotitration of a crude extract of rat cerebellum with the antiserum used in the present study removed only less than 20% of the activity of CaM-kinase IV kin,tse (unpublished data). Therefore, the present CaM-kinase IV kinase was named as CaMkinase kinase a.

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Fig. 4. An electron micrographof a neuronal cell body in the parietal cortex, showing immunoreactionproducts (arrows) in the cellular nucleus (Nu). Small dots indicate the contour of the neuronal cell body. Bar = 5/zm. CaM-kinase I, another Ca2÷/calmodulin-dependent multifunctional protein kinase, has recently been reported to be activated upon phosphorylation by CaM-kinase I kinase [4]. Both CaM-kinase I kinase [15] and CaMkinase IV kinase [17] activate both CaM-kinase I and IV. However, CaM-kinase I is present in cytoplasm, but not in cellular nuclei [12]. In contrast, CaM-kinase IV is located almost exclusively in cellular nuclei [7]. Thus, CaM-kinase kinase a may be responsible for the activation of CaM-kinase IV. CaM-kinase kinase a-like immunoreactivity was observed in cellular nuclei in the globus pallidus, substantia nigra, thalamic reticular nucleus and motor nuclei where no CaM-kinase IV-like immunoreactivity was detected [7]. Neurons in these CNS regions might contain only a small amount of CaM-kinase IV which was not detectable immunohistochemically. The authors are grateful to Ms. Mie Taguchi for technical help. This work was supported in part by a Grant-inAid from the Ministry of Education, Science and Culture of Japan. [1] Gailit, J., Restoring free sulfhydryl groups in synthetic peptides, Anal. Biochem.,214 (1993) 334-335. [2] Jensen, K.F., Ohmstede, C.-A., Fisher, R.S. and Sahyoun, N., Nuclear and axonal localization of Ca2+/calmodulin-dependent protein kinase type Gr in rat cerebellar cortex, Proc. Natl. Acad. Sci. USA, 88 (1991) 2850-2853.

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