Calbrain, a Novel Two EF-hand Calcium-binding Protein That Suppresses Ca2+/Calmodulin-dependent Protein Kinase II Activity in the Brain*

A cDNA clone that encodes a novel Ca2+-binding protein was isolated from a human brain cDNA library. The gene for this clone, termed calbrain, encodes a 70-amino acid polypeptide with a predicted molecular mass of 8.06 kDa. The analysis of deduced amino acid sequence revealed that calbrain contains two putative EF-hand motifs that show significantly high homology to those of the calmodulin (CaM) family rather than two EF-hand protein families. By Northern hybridization analysis, an approximate 1.5-kilobase pair transcript of calbrain was detected exclusively in the brain, and in situ hybridization study revealed its abundant expression in the hippocampus, habenular area in the epithalamus, and in the cerebellum. A recombinant calbrain protein showed a Ca2+ binding capacity, suggesting the functional potency as a regulator of Ca2+-mediated cellular processes. Ca2+/calmodulin-dependent kinase II, the most abundant protein kinase in the hippocampus and strongly implicated in the basic neuronal functions, was used to evaluate the physiological roles of calbrain. Studies in vitro revealed that calbrain competitively inhibited CaM binding to Ca2+/calmodulin-dependent kinase II (K i = 129 nm) and reduced its kinase activity and autophosphorylation.

Calcium ion (Ca 2ϩ ) is a universally employed cytosolic messenger in eukaryotic cells. It is involved in many cellular processes such as signal transduction, contraction, secretion, and cell proliferation (1,2). In the central nervous system, Ca 2ϩ plays a major role in the activities and functions of neuronal cells (3)(4)(5). One of the most widely recognized roles of Ca 2ϩ in synaptic function is its action in neurotransmission. Studies on the effects of Ca 2ϩ on neurotransmitter release, synaptic protein phosphorylation, synaptic vesicles, and synaptic membrane interactions have provided experimental evidence that Ca 2ϩ regulates several biochemical and morphological events in synaptic preparations (6,7).
In many cases, the effects of Ca 2ϩ are mediated by the Ca 2ϩ -binding proteins (8). One superfamily of these proteins is the EF-hand protein family. The EF-hand proteins are charac-terized by single or multiple pairs of a common helix-loop-helix motif that coordinates Ca 2ϩ (9,10). For instance, CaM, 1 troponin C, and myosin light chain have four EF-hand motifs/molecule, whereas S100 proteins have only two motifs per molecule. In addition to the role as a Ca 2ϩ -buffering system, the binding of Ca 2ϩ causes a conformational change of EF-hand proteins and enables them to interact with their target proteins. Most of the EF-hand proteins except CaM show specific tissue distribution, suggesting their particular functions in each tissue. CaM has broad distribution within the cell and throughout different tissues and is a multifunctional regulatory protein that, in a Ca 2ϩ -dependent manner, activates a number of enzymes that are involved in a variety of physiological processes (11). Among these enzymes, CaM-kinase II is one of the most abundant Ca 2ϩ -activated protein kinases in the brain, and it plays important roles in a variety of neural functions including receptor function, neurotransmitter release, and synaptic plasticity (12).
CaM-kinase II is activated by binding to the Ca 2ϩ -bound form of CaM, which dramatically increases the affinity of the enzyme for Mg 2ϩ /ATP, thus leading to substrate phosphorylation and autophosphorylation (13)(14)(15). This self-regulation system coupled to Ca 2ϩ /CaM-dependent autophosphorylation may be involved in important physiological roles responding to transient elevation of intracellular Ca 2ϩ (16). During autophosphorylation (of Thr 286 ), trapping of CaM in CaM-kinase II occurs, resulting in prolongation of the activation period of CaM-kinase II. This process is understood as a good model of memory formation (15,17).
In the present study, we cloned and characterized a novel two EF-hand Ca 2ϩ -binding protein, termed calbrain, that is brain-specific and highly expressed in the hippocampus. To characterize the physiological function of this protein in the hippocampus, the effects of calbrain on CaM-kinase II were examined. The results revealed that calbrain competitively inhibited the activity of CaM-kinase II, suggesting that calbrain may be involved in neuronal signal transduction and memory.

EXPERIMENTAL PROCEDURES
Polymerase Chain Reaction and Human cDNA Library Screening-Degenerate primers were originally designed from transmembrane regions of several mammalian G-protein-coupled receptors. Polymerase chain reaction was performed with these primers using 100 ng of human brain cDNA library (CLONTECH) as a template. The cycling condition was 1 min at 95°C, 1 min at 55°C, and 0.5 min at 72°C for 37 cycles. The polymerase chain reaction products were end-repaired * This research was financially supported by Grant-in-aid for Scientific Research (A) of the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) X94700.
ʈ To whom correspondence should be addressed. with T4 DNA polymerase and ligated into pCR-Script SK(ϩ) vector (Stratagene). Samples were transformed, and colonies were picked for subsequent sequencing. The sequence data was obtained using ABI 373 sequencer (Perkin-Elmer) with dye-terminators by the method of Sanger et al. (18). The data was searched against SWISSPROT (Ver.30.0) data base by BLAST algorithm (18). The fragment that showed a similarity to Ca 2ϩ -binding proteins was chosen for further analysis.
About one million recombinants of human brain gt11 cDNA library (CLONTECH) were grown and transferred onto nylon membranes. The polymerase chain reaction fragment was labeled with [␣-32 P]dCTP and used as a probe for the screening. Hybridization-positive plaques were picked and grown to purify the DNA. Inserts from those positive clones were subcloned into EcoRI cloning site of pBluescript KS (ϩ) vector (Stratagene). The sequence was determined, and data was searched as above.
Northern Hybridization Analysis-The full-length coding region of calbrain was labeled with [␣-32 P]dCTP and used as a probe for Northern hybridization analysis. A human multiple tissue Northern blot filter (CLONTECH) containing 2 g of poly(A)ϩRNA in each lane was prehybridized in a solution containing 5ϫ saline/sodium phosphate/EDTA, 10ϫ Denhardt's, 100 g/ml salmon sperm DNA, 50% formamide, and 2% SDS for 3 h at 42°C. The blot was then hybridized in the same solution with the labeled probe at 42°C for 20 h. The blot was washed several times with a mixture of 2ϫ SSC (1ϫ SSC ϭ 0.15 M NaCl and 0.015 M sodium citrate) and 0.05% SDS for 30 min at room temperature and then once with a mixture of 0.1ϫ SSC and 0.1% SDS for 40 min at 50°C. The filter was exposed to Fuji BASIII imaging plates (Fuji Biomedical, Japan), and the image was analyzed by the BAS 1000 phosphor-imaging system (Fuji Biomedical, Japan).
In Situ Hybridization Study-Adult Sprague-Dawley rats (all male, 8 -9 weeks old) were anesthetized, and brains were perfused with phosphate-buffered saline. Brains were then quickly removed and frozen at-80°C. Cryostat sections (10 m-thickness) were mounted onto slides. The sections were fixed in 4% paraformaldehyde in phosphatebuffered saline, incubated with 0.01% proteinase K in Tris-HCl for 30 min, and then acetylated for 15 min with 0.2 M triethanolamine containing 0.2% acetic acid. After sequential dehydration through a graded alcohol series, each section was hybridized either with a digoxigeninlabeled antisense RNA probe or with a labeled sense probe as a control. The RNA probe was made from a rat homologue of calbrain gene that is 99.0% identical in nucleotide level to the human one. After hybridization at 50°C overnight, the slides were incubated with RNase A (10 g/ml) and washed with 2ϫ SSC and then with 0.2ϫ SSC. Slides were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer Mannheim) for 30 min, washed, and developed by incubating at 4°C overnight with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium. Expression and Purification of Recombinant Protein-Recombinant calbrain protein was expressed using the QIA expressionist system (QIAGEN). The gene construct encoding calbrain was subcloned into pQE expression plasmid vector and transformed into Escherichia coli host strain M15. Transformed M15 cells were cultured in 500 ml of LB medium containing 100 g/ml ampicillin and 25 g/ml kanamycin until the A 600 of 0.7 at 37°C. After adding isopropyl-1-thio-␤-D-galactropyanoside to a final concentration of 1 mM, the cells were incubated for another 5 h. As a negative control, a plasmid vector with no insert was transformed and expressed in the same way. The cells were harvested and lysed with 10 ml of lysis buffer containing 1 mg/ml lysozyme before sonication. After removal of cellular debris, the supernatant was collected, and histidine-tagged calbrain protein was purified on nickel nitrilotriacetic acid resin in a batch procedure. The protein-resin com-FIG. 1. Nucleotide sequence and deduced amino acid sequence of human calbrain. The sequence was derived from human brain cDNA clones. Two EF-hand domains are indicated by lines; the inframe methionine and stop codon are shown in bold face type.

FIG. 2. Alignment of the amino acid sequences of EF-hand domains.
The first and second EF-hand domains of calbrain (CALBR1 and CALBR2) and S100␤ (S100B1 and S100B2) and the first, second, third, and fourth domain of CaM and troponin C (CALM1 to CALM4, TROPO1 to TROPO4, respectively) were aligned. The conserved amino acid residues are shown in shaded boxes. The lines with arrows indicate the helix and loop region of EF-hand. The numbers on the right indicate the percentages of similarity between either CALBR1 or CALBR2 and EF-domains in each protein.
plex was set into a column and washed with washing buffer containing 50 mM sodium phosphate, 300 mM NaCl, 10% glycerol, pH 5.7. After washing, the protein was eluted with 0.1-0.5 M imidazole gradient in washing buffer.
Tricine-SDS-PAGE and Electrophoretic Transfer-Tricine-SDS-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) was carried out as described previously (19). Protein samples, CaM protein as a control, and molecular weight markers were denatured and applied to 10% Tricine-SDS-PAGE. Separated protein bands were either stained with Coomassie Brilliant Blue or transferred to a nitrocellulose membrane by the method of Kyhse-Andersen (20).
Detection of Calcium Binding by 45 Ca 2ϩ -The calcium binding study using 45 Ca 2ϩ was performed according to the method of Maruyama et al. (21). Briefly, after the protein transfer, the membrane was soaked in a solution containing 60 mM KCl, 5 mM MgCl 2 , and 10 mM imidazole-HCl (pH 6.8). The membrane was then incubated in the same buffer containing 1 mCi/liter 45 CaCl 2 for 10 min. Nonspecifically bound 45 Ca 2ϩ was removed by washing with 50% ethanol for 5 min, and dried membrane was exposed to imaging plates. The images were analyzed by Fuji phosphor-imaging system as above, under the Northern hybridization analysis.
Ca 2ϩ binding affinity of recombinant calbrain was determined by equilibrium dialysis. Calbrain was first dialyzed overnight against 1000 volumes of a solution containing 150 mM KCl, 10 mM MOPS, pH 7.1, 3 mM MgCl 2 , 1 mM dithiothreitol, and 0.1 mM EGTA to remove bound Ca 2ϩ from the protein. The dialyzed protein was then used for equilibrium dialysis as follows. A 0.5-ml portion of protein at the concentration of 1 mg/ml was dialyzed with shaking for 48 h at 4°C against 100 ml of the same solution as described above for Ca 2ϩ binding affinity but containing various amounts of CaCl 2 and 45 Ca 2ϩ (5 Ci) to achieve the desired free-Ca 2ϩ concentration. The solutions outside and inside the dialysis tubing were removed, the absorbance at 278 nm was determined, and the protein concentration calculated (A 278 1% ϭ 0.958). Samples of these solutions were subjected to liquid scintillation spectrometry. The association constants for metal and H ϩ binding to EGTA were based on values measured by Fabiato (23).
Kinase Activity and Autophosphorylation Assay-The activity of CaM-kinase II was assayed by measuring the Ca 2ϩ -dependent phosphorylation ( 32 P incorporation) of syntide-2 substrate as described previously by Ochiishi et al. (24). The standard reaction mixture contained 50 M[␥-32 P]ATP, 8 mM Mg(CH 3 COO) 2 , 20 M syntide-2, 0.25 mM CaCl 2 , 0.1 mM EGTA, 50 mM HEPES buffer, pH 8.0, and 0.3 g/ml CaM-kinase II (purified from bovine brain, kindly provided by Dr. Yamauchi (25)). A range of CaM or recombinant calbrain (0 -200 nM) was employed in the assay mixture at the total volume of 20 l. The reaction was carried out at 30°C for 1 min and stopped by spotting onto P81 filter paper. The filter papers were washed several times with 75 mM phosphoric acid, and radioactivity was measured by a liquid scintillation counter. An inhibition assay of Ca 2ϩ -dependent activity of CaM-kinase II was performed in the standard mixture as described above in the presence of  4. Localization of calbrain mRNA in rat brain. In situ hybridization was performed. A rat homologue of calbrain probe (99.0% identical to human one) was labeled and hybridized to a coronal section (A) or a sagittal section (B) of adult rat brain. The hippocampal area (C) and the cerebellar cortex (D) are shown in higher magnification. The strong signals were detected in the CA1 to CA3 of the hippocampal gyrus and granular layer of the dentate gyrus (DG) in the hippocampus (C). In the area of cerebellum, the Purkinje cell layer was stained intensively (D). various concentrations of CaM. Autophosphorylation of CaM-kinase II was assayed in the standard mixture at the total volume of 60 l. The reactions were carried out at 0°C for 10 min as described previously (26), then samples were boiled for 3 min and were subjected to SDS-PAGE. After electrophoresis, the gel was stained with Coomassie Brilliant Blue, and the band of CaM-kinase II protein was excised from the gel. Radioactivity of the gel was determined by a liquid scintillation counter.
Inhibition of CaM Binding to CaM-kinase II by Calbrain-The effect of calbrain on CaM binding to CaM-kinase II was examined as described previously (27) with some modifications. The reaction mixture for the binding studies was the same as that used in the kinase activity assay without ATP. Various concentrations (0 -200 M) of 125 I-CaM and calbrain were incubated in the reaction mixture at the total volume of 200 l at 4°C for 1 h, and they were reacted with 3 g of polyclonal anti-CaM-kinase II antibody (Transduction Laboratories) at 4°C for 16 h. The amount of antibody was proved to be adequate for binding to entire CaM-kinase II in the reaction mixture. Then, 10 l of protein G-Sepharose (Amersham Pharmacia Biotech) was added to each reaction mixture and incubated at 4°C for 1 h on a rotating wheel. After 3 washes with the reaction mixture without CaM-kinase II, these samples were centrifuged (5000 rpm, 5 min), and the radioactivity of each pellet was measured by a gamma counter. Double-reciprocal plots and determination of K i values were performed as described by Segel (28).

Calbrain Has Two EF-hand Motifs-
The isolated clone was found to have a 210-nucleotide open reading frame that encodes a 70-amino acid polypeptide with a predicted molecular mass of 8.06 kDa (Fig. 1). We named the clone as calbrain. A motif search suggested the presence of two EF-hands, a motif known to be involved in calcium binding (29). By a data base search, calbrain showed significant high homology to CaM and troponin C proteins. The four EF-hand domains of CaM, troponin C, and two EF-hand domains of S100␤ protein and calbrain were aligned, and the amino acid sequences were compared as shown in Fig. 2. The first domain of calbrain is highly (50.0% each) homologous to the first and the third domain of CaM. The second domain of calbrain is homologous to the second and the fourth domains of CaM (48.1 and 51.9%, respectively) and those of troponin C (51.9% each). The first and the second domains of calbrain showed only 29.6% homology. Although calbrain has only two EF-hand motifs, the similarity between calbrain and two EF-hand protein (S100␤) is very low (Fig. 2).
Brain-specific Expression of Calbrain mRNA-From the results of Northern hybridization analysis under high stringency conditions, an approximate 1.5-kilobase single calbrain transcript was detected exclusively in the brain (Fig. 3). No band was detected in other tissues examined including heart, placenta, lung, liver, muscle, kidney, and pancreas.
Localization of Calbrain mRNA in the Brain-Results of in situ hybridization study for localization of calbrain mRNA in rat brain was conducted on rat brain sections showed strong signals in the pyramidal layers CA1 to CA3 of the hippocampal gyrus and the granular layer of the dentate gyrus in the hippocampus (Fig. 4). The habenular nucleus in the epithalamus was also stained strongly. In the cerebellum, the Purkinje cells were strongly stained. There were also weak and scattering signals in the cerebral cortex and other areas of the brain. No staining was observed when a sense probe was used as a control.
Ca 2ϩ Binding Property of Calbrain-The purified calbrain and CaM as a control were run on a 10% Tricine-SDS-PAGE gel with molecular mass markers (Fig. 5A). The molecular mass based on the amino acid sequence of mammalian CaM is 16.7 kDa, which is approximately double the size of calbrain. On Tricine-SDS-PAGE gel, calbrain protein was observed at approximately 8 kDa, and CaM is observed as the same size of its molecular mass. The control fraction (sample using the nonrecombinant vector) on the same Tricine-SDS-PAGE gel did not show any protein bands. The 45 Ca 2ϩ binding study of calbrain are shown in Fig. 5A. Both CaM and calbrain showed strong radioactive bands at the expected positions, indicating the binding of 45 Ca 2ϩ to these proteins. Fig. 5B shows a saturation curve for the Ca 2ϩ binding to calbrain. Scatchard analysis (30) of the data (Fig. 5B, inset and legend) reveals that, in the The Inhibitory Effect of Calbrain on CaM-kinase II Activity, Autophosphorylation, and CaM Binding-The effect of calbrain on CaM-kinase II activity is shown in Fig. 6, A and B. The activity of CaM-kinase II incubated in the solution containing 0 -200 M calbrain without CaM (Fig. 6A, open circles) revealed that calbrain was not able to activate CaM-kinase II. On the other hand, Fig. 6B showed that calbrain competitively inhibited activation of CaM-kinase II by CaM with a K i value of 143.5 Ϯ 16.4 nM (mean ϮS.E.). The effect of calbrain on the autophosphorylation of CaM-kinase II was examined, and the results were shown in Fig. 7. Calbrain also competitively inhibited CaM-dependent autophosphorylation of CaM-kinase II with a K i value of 189.7 Ϯ 12.4 nM (mean ϮS.E.), whereas calbrain itself did not induce autophosphorylation of CaMkinase II (data not shown). The binding study of CaM and calbrain to CaM-kinase II revealed that calbrain competitively inhibited CaM binding to CaM-kinase II, and the K i value of this inhibition was 128.6 Ϯ 19.7 nM (mean ϮS.E.) (Fig. 8). DISCUSSION In the present study, we have shown that two EF-hand motifs of calbrain, a novel Ca 2ϩ -binding protein, have a significant homology to those of CaM and other related four EF-hand Ca 2ϩ -binding proteins. The first EF-hand motif of calbrain is very similar to the first and the third motifs, and the second EF-hand motif of calbrain very similar to the second and the fourth motifs of CaM and troponin C. Although calbrain is a two EF-hand protein, the homology between calbrain and two EF-hand proteins such as S100␤ was found to be very low. From the high sequence homology between EF-hand motifs, it has been proposed that CaM, troponin C, and myosin light chain gene evolved from a common four-domain molecule (29, 31,32). In each of these proteins, the first and the third and the second and fourth EF-hand domains show high homology, supporting the hypothesis that these proteins evolved from a twodomain precursor by gene duplication (33,34). The significant homology between the EF-hand motifs of calbrain and CaM family suggests that although calbrain is a two EF-hand protein, evolutionally it is related closely to the CaM family rather than two EF-hand protein families. As calbrain is the first two EF-hand protein whose domains appear to be very similar to those of four EF-hand proteins, our findings may be interesting from the aspect of the evolution of EF-hand proteins.
The biological functions of EF-hand proteins are strongly related to the conformational changes of EF-hand domains in response to Ca 2ϩ binding. EF-hand domains that show large conformational changes by binding Ca 2ϩ are known to have a trigger function in the activation of target proteins. The domains that have regulatory roles are termed regulatory domains (35,36), and proteins that have such domain(s) are called Ca 2ϩ sensor proteins. For example, CaM and troponin C, which are classified in this category, enable the cell to detect a stimulatory influx of Ca 2ϩ and thereby transduce this signal into a variety of cellular processes (37). On the other hand, EF-hand domains that exhibit small conformational changes are termed structural or buffer domains (36,38). These domains are responsible for the structural stability, local Ca 2ϩ transport, and function in buffering intracellular Ca 2ϩ . Proteins possessing these domains, such as calbindin and parvalbumin, are called Ca 2ϩ buffer proteins (39,40). From the amino acid sequence in the present study, it is difficult to predict whether calbrain has regulatory or structural domains, and therefore is a Ca 2ϩ sensor or Ca 2ϩ buffer protein. It has been suggested that the interhelical angle changes of EF-hand proteins upon Ca 2ϩ binding become a good index for their classification. Another useful method is to check whether or not the protein can bind to a hydrophobic column in a Ca 2ϩ -dependent manner (36). Ca 2ϩ sensor proteins that cause large conformational changes bind to the column by Ca 2ϩ -induced exposure of the hydrophobic surface in the protein formed by a pair of EF-hands. After that, these proteins can be eluted by removing Ca 2ϩ with EGTA. This method was successfully applied for the purification of CaM (41). We have purified the recombinant calbrain protein by this method, also (data not shown), and the results indicate that calbrain possesses a regulatory domain that shows a large conformational change with Ca 2ϩ binding and, therefore, can be classified as a Ca 2ϩ sensor protein.
The distribution study of calbrain mRNA revealed that calbrain is a brain-specific Ca 2ϩ -binding protein that is expressed abundantly in the hippocampus, in the habenular nucleus of the epithalamus and in the Purkinje cell layer of the cerebellum. The specific tissue distribution of EF-hand proteins has suggested their particular functions in each tissue (42)(43)(44). The localization of calbrain mRNA in the hippocampus and cerebellum, together with its functional potency as a Ca 2ϩ sensor protein being involved in the Ca 2ϩ signal transduction suggest an important role for this protein in the central nervous system. A number of studies on hippocampus have shown that this part of the brain is particularly involved in acquisition and storage of spatial information (45)(46)(47).
CaM-kinase II is a multifunctional serine/threonine protein kinase capable of phosphorylating several endogenous proteins in the brain (48,49) and is highly expressed in the mammalian central nervous system (50 -52). This enzyme is a major component of postsynaptic density (24,53,54) and plays important roles in the regulation of the neurotransmitter synthesis, receptor function, axonal transport, gene expression, and especially in the long-term potentiation, which is an established model of neural plasticity (14,55,56). Many biochemical studies have indicated that phosphorylation induced by CaM-kinase II can act as a molecular switch, conferring properties that are advantageous for long-lasting storage of changes initiated by brief Ca 2ϩ signals (57)(58)(59). The binding study of CaM and calbrain to CaM-kinase II indicated that under nonautophosphorylated conditions, calbrain inhibited CaM binding to CaM-kinase II competitively (Fig. 8). Although calbrain could not activate CaM-kinase II, Fig. 6B showed that calbrain competitively inhibited the activation of CaM-kinase II by CaM. Because K i values of binding and activity inhibition were similar, it was supposed that inhibition of CaM binding by calbrain may caused the reduction of kinase activity. CaM-kinase II activity is regulated by Ca 2ϩ /CaM and autophosphorylation (60 FIG. 8. Competitive inhibition of CaM binding to CaM-kinase II by calbrain. The binding of CaM to CaM-kinase II in the presence of indicated concentrations of calbrain was studied. 125 I-CaM and calbrain were mixed with CaM-kinase II and Ca 2ϩ under nonautophosphorylated conditions (in ATP-free reaction mixture). CaM-kinase II was immunoprecipitated by anti-CaM-kinase II antibody after the reaction, and the radioactivity of each sample was measured. The radioactivity of the bound 125 I-CaM in the presence of 500 nM CaM and 0 nM calbrain was measured and designated as 100%. The relative ratio (%) of radioactivities of the samples were then calculated. 1/v represents 1/CaM binding rate to CaM-kinase II (%). The results are the mean (ϮS.E.) for two successive experiments performed in duplicate. mational change is induced in the regulatory region, and the interaction of inhibitory domain with the active site is disrupted. It allows the active site to become accessible to exogenous substrate. When Ca 2ϩ /CaM is bound, CaM-kinase II is rapidly autophosphorylated on Thr 286 , and autophosphorylation increases CaM binding affinity to the kinase dramatically by decreasing of CaM-releasing time (61). Under both experimental conditions used for the kinase activity assay (incubation at 30°C for 1 min) and autophosphorylation assay (at 0°C for 10 min), autophosphorylation on Thr 286 of CaM-bound kinase occurs (24 -26). Therefore, the activity detected in Fig. 6B may be influenced by a change of CaM affinity caused by autophosphorylation. K i values of kinase activity (143.5 Ϯ 16.4 nM) and autophosphorylation (189.7 Ϯ 12.4 nM) did not significantly differ from that of CaM binding (128.6 Ϯ 19.7 nM) under nonautophosphorylated condition. This is the first report of a novel Ca 2ϩ -binding protein that can inhibit CaM-dependent CaM-kinase II activity. Although calbrain can reduce autophosphorylation, it is supposed that this protein is involved in the physiological regulation of CaMkinase II. As CaM-kinase II has multiple functions and essential roles in the brain, calbrain may also play important roles in the central nerve system.