Isoform-specific Activation and Structural Diversity of Calmodulin Kinase I*

We earlier confirmed that there are isoforms of Ca2+/calmodulin (CaM)-dependent protein kinase I (CaM kinase I) (CaM kinase Iβ1 and Iγ) beside CaM kinase Iα by cDNA cloning (Yokokura, H., Terada, O., Naito, Y., and Hidaka, H. (1997) Biochim. Biophys. Acta 1338, 8–12). Here, we demonstrate the existence of an isoform-specific activation mechanism of CaM kinase I and alternative splicing specifically regulating CaM kinase I (CaM kinase Iβ2) in the central nervous system. To cast light on isoform structure-enzyme activity relationships, CaM kinase Iβ1, Iβ2, and Iα were expressed separately using a baculovirus/Sf9 cell expression system. The novel CaM kinase Iβ2 isoform demonstrated similar catalytic activity to those of CaM kinase Iβ1 and Iα. Interestingly, CaM kinase Iβ1 and Iβ2 both can activate CaM kinase Iα activity via phosphorylation at Thr177. Reverse transcribed-polymerase chain reaction analysis showed that CaM kinase Iβ2 is dominant in the cerebrum and cerebellum, whereas CaM kinase Iβ1 is present in peripheral tissues such as liver, heart, lung, kidney, spleen, and testis. CaM kinase Iβ2 was also detected with an anti-CaM kinase Iβ2 antibody in PC12 cells. The results indicate that alternative splicing is a means for tissue-specific expression of CaM kinase Iβ. Thus the Thr177 residue of CaM kinase Iα is phosphorylated by not only CaM kinase kinase but also CaM kinase Iβ for activation of the enzyme.

The Ca 2ϩ /calmodulin (CaM) 1 -dependent protein kinase (CaM kinase) is known to mediate signals associated with elevation of intracellular Ca 2ϩ . CaM kinases constitute a family of structurally related enzymes that include phosphorylase kinase, myosin light chain kinase, and CaM kinases I-IV. CaM kinase I was first identified in rat brain based on its ability to phosphorylate the synaptic vesicle-associated protein, synapsin I (1). It has since been purified and characterized from bovine (2) and rat brain (3,4). This monomeric enzyme has been revealed to demonstrate multiple 37-43-kDa polypep-tides by SDS-PAGE (2)(3)(4)(5). We have purified and reported partial amino acid sequences of a novel CaM kinase, closely related to CaM kinase I (5,6). Following the cloning of one cDNA for CaM kinase I (CaM kinase I␣) (7,8) we have recently cloned two isoforms of CaM kinase I from a rat fetal brain cDNA library and termed them CaM kinase I␤ and I␥ (9).
It has been recognized that CaM kinase I␣ must be phosphorylated at the Thr 177 residue for maximal activity (10 -14) and that this occurs via autophosphorylation or by the action of CaM kinase kinase. Several groups have identified and purified CaM kinase kinases (10,11,(15)(16)(17), and a cDNA for a 68-kDa rat CaM kinase kinase has been cloned (18). Some kinase kinases are known to be able to phosphorylate both CaM kinase I and IV (14,18,19), suggesting the existence of a CaM kinase cascade, similar to the mitogen-activated protein kinase cascade. Moreover, we have reported that CaM kinases I and IV can phosphorylate each other (14). For further understanding of the relationships between the members of the CaM kinase cascade and identification of the role of CaM kinase I isoforms, more information on their characteristics is necessary.
Here we report cDNA cloning of a novel isoform of CaM kinase I, CaM kinase I␤2, which is expressed in the central nervous system, along with an analysis of the kinetic character of the expressed enzyme. The relationship between CaM kinases I␣ and I␤2 is described, and evidence is presented that CaM kinase I␤2 may have a significant role in the CaM kinase cascade.
[␣-32 P]dCTP and [␥-32 P]ATP were purchased from Amersham Corp., and all other chemicals were purchased from standard suppliers.
cDNA Cloning of CaM Kinase I Isoform-Oligo(dt)-primed cDNA library of embryonic rat brain in ZAP II were kindly provided by Dr. S. Nakanishi (Institute of Immunology, Kyoto University Faculty of Medicine, Kyoto, Japan). About 5.4 ϫ 10 5 plaques were screened using the EcoRI-SacI fragment (430 bp) of clone N2 (9), CaM kinase I␤, labeled with [␣-32 P]dCTP as a probe. A single cDNA clone of insert size 1.6 kb was isolated and sequenced.
Construction of Plasmids-CaM kinase I␤2 and I␤1 cDNA were inserted into the PstI-NotI cut PVL1392 baculovirus transfer vector. * This work was supported in part by Grants 06404019 and 06507001 from the Ministry of Education, Science, Sports 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) AB004267.
CaM kinase I␣ cDNA was inserted into BamHI-EcoRI cut PVL1393. The expression plasmid for CaM kinase kinase was made as follows. Two primers were synthesized: sense 5Ј-ATGAATTCATGGAGCGCAG-TCCAGCCGT-3Ј and antisense 5Ј-ATGCGGCCGCTCAGGATGCAGC-CTCATCTT-3Ј, based on the published cDNA sequence of CaM kinase kinase (18). A PCR reaction was performed using LA PCR kit ver. 2 (Takara Shuzo, Shiga, Japan) with a cDNA library of embryonic rat brain as the template under the following conditions: 30 cycles of 72°C for 3 min, 94°C for 1 min, and 55°C for 2 min. After digestion with EcoRI and NotI, the PCR fragment was subcloned into the bacterial expression vector, pGEX4T-1 (Pharmacia Biotech Inc.). A Thr 177 3 Ala mutant of CaM kinase I␣ was made using the Sculptor in vitro mutagenesis system (Amersham Corp.), a cDNA of CaM kinase I␣ subcloned into the BamHI-EcoRI site of pGEX2T (Pharmacia Biotech Inc.) and a mutagenic oligonucleotide (5Ј-AGTGTGCTCTCCGCAGCCTGTGGGA-CT-3Ј).
Protein Preparation-CaM was purified from bovine brain as described previously (20). CaM kinase I␤2, I␤1, and I␣ were expressed in the baculovirus expression system (Invitrogen). Sf9 cells expressing recombinant enzymes were harvested by centrifugation. Soluble protein extracts were prepared by sonication of the cells in buffer A (10 mM Tris-HCl, pH 8.0, 1 mM EGTA, 5 mM 2-mercaptoethanol, 10 g/ml leupeptin, 1 g/ml pepstatin, and 0.4 mM phenylmethylsulfonyl fluoride). After centrifugation at 8,000 ϫ g for 15 min, the supernatant supplemented with 2 mM CaCl 2 was applied to CaM-Sepharose 4B (Sigma) and washed with 40 mM Tris-HCl, pH 8.0, 1 mM CaCl 2 1 M NaCl. Elution was carried out with buffer B (40 mM Tris-HCl, pH 8.0, 2 mM EGTA, 50 mM NaCl). To evaluate CaM dependence, CaM kinase I␤2 was purified with phenyl Sepharose (Pharmacia Biotech Inc.). Glutathione S-transferase fusion proteins were prepared as follows. JM109 cells containing the expression plasmids were grown overnight at 37°C. The fully grown culture was diluted 100-fold in fresh LB medium and grown for an additional 2.5 h. Isopropyl ␤-D-thiogalactopyranoside was then added to a final concentration of 0.1 mM. After a 12-h further incubation at 25°C, the cells were collected by centrifugation, resuspended in 10 ml of buffer A, sonicated, and centrifuged at 8,000 ϫ g for 15 min. The supernatant was loaded onto glutathione-Sepharose 4B (Pharmacia Biotech Inc.), washed with 40 mM Tris-HCl, pH 8.0, 2 mM EGTA, 1 M NaCl, and eluted in buffer B containing 5 mM glutathione. The CaM kinase I␣-glutathione S-transferase fusion protein was prepared as described previously (21).
Western Blot Analysis-PC12 cells were harvested, sonicated in phosphate-buffered saline containing 10 g/ml leupeptin, 1 g/ml pepstatin, and 0.4 mM phenylmethylsulfonyl fluoride and centrifuged at 10,000 ϫ g for 15 min. The supernatant and CaM kinase Is were separated by 1.0% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Anti-CaM kinase I antiserum was diluted in phosphate-buffered saline and hybridized to the blot for 1 h. Immunoreactive bands were visualized by chemiluminescence (Amersham Corp.).
CaM Kinase Activation-CaM kinase Is were incubated with 21 nM CaM kinase I␣, I␤2, or CaM kinase kinase at 30°C for 5-10 min in 35 mM HEPES, pH 8.0, 10 mM MgCl 2 , 1 mM CaCl 2 , 1 mM dithiothreitol, 0.005% Tween 20, 1.5 M CaM, and 100 M ATP. The activation reactions were terminated by 15-fold dilution with a solution containing 35 mM HEPES, 2 mg/ml bovine serum albumin, 10% glycerol, 1 mM EDTA. The kinase activity was determined at 30°C for 10 min using standard assay conditions as described above with 50 M syntide-2 and 100 M [␥-32 P]ATP as the substrate. For stoichiometrical analysis, 200 nM CaM kinase I␣ was incubated in 300 M [␥-32 P]ATP with 21 nM CaM kinase I␤2 or CaM kinase kinase for 60 min. Reactions were stopped by the addition of loading buffer followed by electrophoresis on SDS-PAGE. Gels were stained with Coomassie Blue and dried. Quantitation of 32 P incorporation into CaM kinase I␣ was achieved by cutting out the appropriate gel pieces and determining their radioactivity by scintillation counting.
Other Methods-DNA sequences were determined for both strands by automatic sequencing using a model 373A (Applied Biosystems Inc.). SDS-PAGE was carried out by the method of Laemmli (23). Protein concentrations were measured by the Bradford (24) method.

Isolation and Analysis of cDNA Clones of CaM Kinase
I␤2-We earlier reported the existence of isoforms of CaM kinase I (CaM kinase I␤1 and I␥) other than CaM kinase I␣ by cDNA cloning. Further screening of a rat brain oligo(dT)primed cDNA library yielded a 1.6-kb clone. Sequencing of this clone indicated that it contained the appropriate nucleotide sequence of CaM kinase I␤ with an additional 137-bp sequence after nucleotide 954. This sequence contains a stop codon and codes for 25 amino acids that do not share significant sequence homology with CaM kinase I␤ (Fig. 1). The CaM kinase I␤ reported previously and this novel isoform are tentatively termed CaM kinase I␤1 and CaM kinase I␤2, respectively.
Tissue Distribution of CaM Kinase I␤1 and I␤2-Northern blot analysis of rat tissues was performed using a cDNA fragment of CaM kinase I␤2 sharing 561 bp with CaM kinase I␤1 as a probe. An approximately 1.8-kb mRNA was detected in the cerebrum and cerebellum, and a 4-kb faint band in heart, lung, kidney, spleen, and testis (Fig. 2).
To evaluate the tissue-specific expression of CaM kinase I␤1 and I␤2, we conducted RT-PCR of rat tissues with overlapping pairs of primers spanning the alternatively spliced segment. In all samples we examined, cDNAs were amplified. The larger fragment amplified from cerebrum and PC12 cells exhibited the same apparent size as that from the CaM kinase I␤2 cDNA (Fig. 2). The fragment was extracted and the sequence was analyzed and found to be identical to CaM kinase I␤2. The smaller fragment, exhibiting the same apparent size as that from CaM kinase I␤1, was dominantly amplified in the liver, heart, kidney, lung, spleen, and testis (Fig. 2).
When a crude extract of PC12 cells was subjected to Western blot analysis with antibody against CaM kinase I, which can recognize CaM kinase I␣, I␤1, and I␤2, a clear band at the position corresponding to CaM kinase I␤2 (M r 38,000) and a weaker band at the position corresponding to CaM kinase I␣ (M r 41,000) were detected (Fig. 3).
Characterization of Recombinant CaM Kinase I␤2-To examine their enzymatic characteristics, CaM kinase I␣, I␤1, and I␤2 were expressed in Sf9 cells and purified with CaM-Sepha-  rose. These enzymes had similar catalytic activity (Table I) and were activated by CaM in a dose-dependent manner (Fig. 4). CaM kinase I␤2, I␤1, and I␣ demonstrated CaM requirements with EC 50 values of approximately 35, 80, and 150 nM, respectively. For analysis of of substrate specificity, we tested four peptides. The phosphorylation capacities of CaM kinase I␣, CaM kinase I␤1, and CaM kinase I␤2 were similar except with CREB (Fig. 5), which was found to be a highly preferred substrate for CaM kinase I␣, but not I␤1 or I␤2. Kinetic analysis data showed the V max of the CaM kinase I␣ for the CREB peptide to be 5 times larger than those for the CaM kinase I␤1 and I␤2 (Table I).
Activation of CaM Kinase I␤2 by CaM Kinase Kinase-It is well established that CaM kinase I␣ and IV are phosphorylated and activated by CaM kinase kinase. To examine the effects of CaM kinase kinase on the activity of CaM kinase I␤2, we expressed a glutathione S transferase-CaM kinase kinase fusion protein in Escherichia coli and purified it on glutathione-Sepharose. CaM kinase I␤2 was incubated with or without CaM kinase kinase for 10 min, and its activity was determined. CaM kinase I␤2 was activated 6.7-fold by autophosphorylation and 30-fold by CaM kinase kinase (Fig. 6). Under the same conditions, CaM kinase I␣ and I␤1 were activated 2.9-and 2.9-fold by autophosphorylation and 40-and 16-fold by CaM kinase kinase, respectively (Fig. 6). Unlike CaM kinase II or CaM kinase IV, CaM kinase I␤1 or I␤2 activated by CaM kinase kinase did not generate Ca 2ϩ /CaM-independent activity.
Phosphorylation of CaM Kinase I␣ by CaM Kinase I␤1 and I␤2-We have suggested the existence of a new CaM kinase cascade like that involving mitogen-activated protein kinases (14). Therefore, we determined whether CaM kinase I␣ and I␤ could associate with each other for their activation. CaM kinase I␣ was phosphorylated by CaM kinase I␤2 that had been activated by CaM kinase kinase, and this resulted in activation. However, CaM kinase I␤2 was not phosphorylated by CaM kinase I␣ that had been activated by CaM kinase kinase (Fig.  7). Similar results were obtained when CaM kinase I␤1 was used instead of CaM kinase I␤2 (data not shown). Stoichiometry analysis showed that 0.99 Ϯ 0.05 molecule of 32 P was incorporated per CaM kinase I␣ by CaM kinase I␤2 and 1.47 Ϯ 0.02 molecules of 32 P by CaM kinase kinase. Since it has been reported that CaM kinase I␣ is phosphorylated by CaM kinase kinase at Thr 177 , we constructed a mutant CaM kinase I␣ with a substitution of Thr 177 by Ala (T177A) and expressed it in E. coli as a glutathione S-transferase fusion protein. The purified mutant CaM kinase I␣ was not phosphorylated by CaM kinase I␤2 (Fig. 8) and was not activated (data not shown).

DISCUSSION
The data presented in this study show the existence of a novel isoform of CaM kinase I, generated by alternative splicing between nucleotide 955 and 956 of the CaM kinase I␤1 cDNA. Northern blot analysis revealed 1.8-and 4-kb mRNAs for the CaM kinase I␤s (Fig. 2). Considering the RT-PCR data, the 1.8-kb mRNA that is highly expressed in the central nervous system but lacking in the liver, heart, lung, kidney, spleen, and testis might be CaM kinase I␤2. The 4-kb mRNA could encode CaM kinase I␤1, but the possibility that it represents an unspliced intermediate or a spurious hybridization could not be excluded. Western blot analysis revealed that CaM kinase I␤ is expressed in PC12 cells and RT-PCR data showed the form to be I␤2. The results indicate that CaM kinase I␤2 might have a significant role as an effector enzyme for brain specific functions.
CaM kinase I␤1 and I␤2 contain the same catalytic and putative CaM binding domains and show similar kinetic activities. CaM kinase I␤2, however, has a lower CaM requirement (Fig. 4). Since the amino acid sequence of the putative CaM binding domain in CaM kinase I␤2 is similar to that in CaM kinase I␣ (91% similarity), the difference in the CaM requirement is interesting. Thus, CaM may interact with as yet un- known domains of CaM kinase I␤2 or I␤1, in addition to the canonical CaM binding domain. In this context, the C-terminal sequence in CaM kinase I␤2 might be a good candidate since it is absent in CaM kinase I␤1. The C-terminal sequences of CaM kinase I␣ and I␤1 contain proline-rich sections, whereas CaM kinase I␤2 does not. It is therefore also possible that this prevents CaM kinase I␣ or I␤1 from forming a secondary structure necessary for additional CaM binding.
Activation of CaM kinase I and IV is dependent upon phosphorylation by CaM kinase kinase for maximal activity (10 -18, 25-27). Our data demonstrated that CaM kinase I␤2 also possesses CaM kinase kinase activity. Recently, another two CaM kinase kinases were purified from rat brain (28) and from rat cerebellum (29). The molecular weight of the former is about 73,000 and that of the latter is about 66,000 as determined by SDS-PAGE. The predicted molecular weight of CaM kinase I␤2 is 38,423, and the lack of any sequence identity indicates that it is a totally different protein. The level of phosphorylation of CaM kinase I␣ catalyzed by CaM kinase kinase appears greater than that catalyzed by CaM kinase I␤2. Stoichiometrical analysis showed that 1 molecule of 32 P was incorporated per CaM kinase I␣ molecule by CaM kinase I␤2, whereas CaM kinase kinase provided additional phosphorylation. The T177A mutant was also phosphorylated by CaM kinase kinase, although the phosphorylation level of the mutant was lower than that of the wild type (Fig. 8). Since we previously reported another site phosphorylated by CaM kinase kinase besides Thr 177 (14), these results indicate that the level of phosphorylation might be different due to multiple phosphorylation by CaM kinase kinase.
Recently, Aletta et al. (30) reported the existence of an intracellular CaM kinase I phosphorylation cascade in PC12 cells, and we showed, here, that they express CaM kinase I␤2 (Fig. 3). These data suggest that CaM kinase I␤2 might be involved in a CaM kinase I cascade in PC12 cells. However, CaM kinase I␤2 phosphorylates peptides other than those targeted by CaM kinase kinase, so it is possible that there might be specific substrates. Further work is required to clarify these points.