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J Biol Chem, Vol. 274, Issue 32, 22556-22562, August 6, 1999

Ca²⁺/Calmodulin-dependent Protein Kinase Cascade in Caenorhabditis elegans
IMPLICATION IN TRANSCRIPTIONAL ACTIVATION^*

Koh Eto^§, Naomi Takahashi, Yoshishige Kimura, Yasuhiko Masuho, Ken-ichi Arai^§, Masa-aki Muramatsu^¶, and Hiroshi Tokumitsu

From the  Helix Research Institute, Inc., 1532-3 Yana, Kisarazu-shi, Chiba 292-0812, the ^§ Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo Shirokanedai, Minato-ku, Tokyo 108-0071, and the ^¶ Department of Biological Cybergenetics, Medical Research Institute, Tokyo Medical Dental University, Bunkyo-ku, Tokyo 113-8510, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We have recently demonstrated that Caenorhabditis elegans Ca²⁺/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803-15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CeCaM-KI), which has approximately 60% identity to mammalian CaM-KI. CeCaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by CeCaM-KK through phosphorylation of Thr¹⁷⁹ in a Ca²⁺/CaM-dependent manner, resulting in a 30-fold decrease in the K_m of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-KI, CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH₂-terminal six residues (²PLFKRR⁷) contain a functional nuclear localization signal. We have also demonstrated that CeCaM-KK and CeCaM-KI reconstituted a signaling pathway that mediates Ca²⁺-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of CeCaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved in C. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca²⁺-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Ca²⁺/calmodulin-dependent protein kinases (CaM-K)¹ are implicated in the regulation of a wide variety of biological events mediated by intracellular Ca²⁺, such as muscle contraction, neurotransmitter release, and gene expression (1-4). Recent studies have indicated that so-called multifunctional CaM-kinases, including CaM-KI, -II, and -IV, are regulated by phosphorylation either by itself or by upstream protein kinase (4, 5). In contrast to CaM-KII, which converts the Ca²⁺/CaM-independent kinase by autophosphorylation, both CaM-KI and CaM-KIV are activated by CaM-KK through phosphorylation of a Thr residue (Thr¹⁷⁷ in CaM-KI and Thr¹⁹⁶ in CaM-KIV) in their "activation loops," resulting in an increase in their catalytic efficiency (5-10).

The  isoform of CaM-KK was originally purified and cloned from rat brain as a regulatory protein kinase for CaM-KIV and was later demonstrated to be an activator for CaM-KI (6-8, 11). It has been shown that CaM-KK is regulated by an intrasteric mechanism through its autoinhibitory domain (residue 436-441) and activated by binding of the Ca²⁺·CaM complex to the overlapping CaM-binding region (residue 438-463 in the  isoform) also common to other CaM-kinases (12-14) and conserved in the recently cloned  isoform (15, 16). Therefore, a dual action of Ca²⁺/CaM binding to both CaM-KK and its downstream target CaM-Ks is required to activate the CaM-kinase cascade (10, 12). Recently, we have identified an Arg/Pro-rich insert region (the RP domain, residue 167-189 in rat  CaM-KK) in the catalytic domain of CaM-KK, which is involved in the recognition of target CaM-kinases (17). The RP domain is also conserved in the  isoform and in the Caenorhabditis elegans homologue.

The CaM-kinase cascade has been functionally demonstrated in various mammalian cells such as transfected COS-7 cells (12), Jurkat cells (18), PC-12 cells (19), and cultured hippocampal neurons (20), which are strictly regulated by intracellular Ca²⁺. One of the targets for the CaM-KK/CaM-KIV cascade has been demonstrated to be CREB, which plays a role in long term memories that depend on altered gene expression. Extensive studies have demonstrated that the CaM-KK/CaM-KIV cascade is involved in Ca²⁺-dependent regulation of transcriptional activation through phosphorylation of CREB at Ser¹³³ (20-24), which is consistent with nuclear localization of CaM-KIV (25). On the other hand, CaM-KI, another target for CaM-KK, is predominantly a cytoplasmic enzyme (36), and the physiological function(s) of the CaM-KK/CaM-KI cascade is not well known. A recent study has shown that CaM-KK may mediate the anti-apoptotic effect of modest elevations of Ca²⁺ through phosphorylation and activation of protein kinase B (26). This result also indicates that multiple protein kinases might be phosphorylated and activated by CaM-KK, resulting in the regulation of a wide variety of functions.

Most of the studies of the CaM-kinase cascade have been done in mammalian systems. The C. elegans homologue of CaM-KK (CeCaM-KK) was initially identified in the GenBank data base (27) and proven to activate mammalian CaM-KIV in vitro (17), suggesting that CaM-kinase cascade may exist in C. elegans, although the target(s) for CaM-KK in C. elegans has not been identified.

In this study, we have cloned a novel target for CaM-KK in C. elegans that is highly homologous to mammalian CaM-KI. Our characterization of CeCaM-KI, including its activation by CeCaM-KK, subcellular localization, and function in intact cells, strongly suggests that this organism also shares the CaM-kinase cascade, which has a potential role in transcriptional activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- The C. elegans N2 strain embryonic stage cDNA library in ZAP phage vector was kindly provided from Dr. Yuuji Kohara (National Institute of Genetics, Mishima, Japan). C. elegans cDNAs were generated using mRNA prepared from the C. elegans N2 strain. Recombinant rat CaM was expressed in Escherichia coli BL21(DE3) using pET-CaM, which was kindly provided from Dr. Nobuhiro Hayashi (Fujita Health University, Toyoake, Japan) and purified by phenyl-Sepharose column chromatography (28). C. elegans CaM-KK cDNA (GenBank^TM/EBI accession no. AB016838) was cloned as recently described (17). Rat CaM-KI cDNA was obtained by reverse transcriptase-PCR as described (17).

cDNA Cloning of C. elegans CaM-KI-- Two oligonucleotides used for PCR amplification of CeCaM-KI were designed on the basis of the sequence in a C. elegans cosmid (K07A9) in the data base as follows: 5'-GGTGGAGAGCTTTTCGATAGAATTGTT-3' and 5'-CAAATATAAAACATCGAAAATTACCTTTTTCCA-3'. These two primers were used in PCR for 35 cycles at 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min using C. elegans cDNAs as a template. A 0.6-kilobase pair of amplified DNA fragment was subcloned into a pT7Blue vector (Novagen) and sequenced, which encoded residue 103-307 of CeCaM-KI (see Fig. 1A). To isolate the full-length clone of CeCaM-KI, the 0.6-kilobase pair PCR product was used as a probe to screen a C. elegans N2 strain embryonic stage cDNA library in ZAP vector. Among 2 × 10⁵ plaques screened, one positive clone was identified, and the cDNA insert was isolated from the phage. The nucleotide sequence of full-length cDNA was completely determined in both strands by an Applied Biosystems model 377 automated DNA sequencer.

Construction of Expression Plasmids-- To express a recombinant protein of CeCaM-KI (wild type) in COS-7 cells, the full-length cDNA was cloned into the mammalian expression plasmid pME18s, which was constructed as follows. The coding region of the cDNA in pBluescript SK() was amplified using a sense oligonucleotide, 5'-CCGGAATTCATGCCCCTTTTTAAGCG-3', and an antisense oligonucleotide, 5'-GCGGATTGATTTCTCGAGAGTTCAAATTCTGTGG-3'. The PCR product was digested by EcoRI and XhoI, and the fragment was ligated into pME18s. For GST-fusion constructs (GST·CeCaM-KIs), CeCaM-KI wild type and mutant cDNAs were amplified by PCR to introduce convenient restriction sites using a sense primer, 5'-CCGGAATTCCCCCTTTTTAAGCG-3', and an antisense primer, 5'-GCGGATTGATTTCTCGAGAGTTCAAATTCTGTGG-3'. The sense primer lacked the initial ATG for the first methionine of the cDNA. The PCR products were digested with EcoRI and XhoI and then ligated into pGEX-4T-1 (Amersham Pharmacia Biotech). For the GFP-fusion construct (GFP·CeCaM-KIs), CeCaM-KI wild type and NH₂-terminal deletion mutant cDNA were amplified by PCR using sense primers as follows: wild type, 5'-CCGGAATTCCCCCTTTTTAAGCG-3'; NH₂-terminal deletion (residue 1-7) mutant, 5'-CCGGAATTCGATGGGAGTGGTCCCGCGCCGAACGCC-3'; and an antisense primer, 5'-CTTTGGGGGGTCGGGTACCTCAAAAGCGTATTACTG-3'. The PCR products were digested with EcoRI and KpnI and then ligated into pEGFP-C2 (CLONTECH). A GFP-rat CaM-KI expression construct was generated by subcloning the cDNA from pT7Blue (Novagen) as a SacI-SalI fragment into pEGFP-C2.

Mutagenesis-- Site-directed mutagenesis of the CeCaM-KI cDNA in pBluescript SK() was performed using the GeneEditor^TM in vitro site-directed mutagenesis system (Promega) followed by subcloning into the expression plasmids described above. For construction of the point substitution mutants, mutagenic oligonucleotides are described as follows: T179A, 5'-GTAATGGCTGCAGCGTGTGG-3'; T179D, 5'-CAGGAGTAATGGCTGACGCGTGTGGAACACCGGG-3'; K52A, 5'-CAGATGTATGCAGTCGCATGCATTGACAAAAAAGC-3'. The COOH-terminal truncated mutant of CeCaM-KI cDNA at position 295 was constructed by PCR using a sense oligonucleotide, 5'-CCGGAATTCCCCCTTTTTAAGCG-3', and an antisense-oligonucleotide, 5'-CCGCTCGAGTCAATGTACGGCGACAGTTCCGTGGAATATCGTG-3', and then subcloned into pGEX-4T-1. The nucleotide sequence of each mutant CeCaM-KI cDNA was confirmed.

Cell Culture and Transient Transfection-- COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in an atmosphere of 5% CO₂. For transient transfection, cells grown in 10-cm-diameter dishes were transfected with 5-10 µg of the appropriate expression constructs using 60 µg of LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's protocol. The cells were cultured in serum-free medium (Opti-MEM, Life Technologies, Inc.) for 5 h after transfection, followed by culture in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum for the indicated times. The cells were observed for GFP fluorescence using Axiovert 135 fluorescence microscopy (Carl Zeiss) or lyzed for purification of the recombinant CaM-KKs, CaM overlay assay, Western blotting, and CRE reporter gene assay.

Expression and Purification of Recombinant CeCaM-KI and CeCaM-KK-- E. coli (JM109) carrying the expression plasmid (pGEX-CeCaM-KI) was precultured in LB broth containing 100 µg/ml ampicillin at 37 °C overnight. An overnight culture of E. coli (1 ml) was added into 100 ml of LB broth containing 100 µg/ml ampicillin, the culture was continued to A₆₀₀ at 0.8, and then 0.4 mM isopropyl-1-thio--D-galactopyranoside was added. After 4 h of culture, the E. coli was harvested by centrifugation. All of the purification steps described below were carried out at 4 °C. The bacterial pellet was resuspended in 10 ml of phosphate-buffered saline containing 0.2 mM PMSF and lyzed by sonication. After centrifugation at 15,000 × g for 15 min, the supernatant was loaded onto a 1-ml bed volume of glutathione-Sepharose (Amersham Pharmacia Biotech) affinity column. After washing the column with 20 ml of phosphate-buffered saline containing 0.2 mM PMSF, recombinant CeCaM-KI was eluted with 10 mM glutathione in 50 mM Tris-HCl (pH 8.0) and 0.2 mM PMSF and then dialyzed against 100 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, 2 mM DTT, and 0.2 mM PMSF. The recombinant protein was mixed with equal an volume of 80% glycerol and 20% ethylene glycol and stored at 20 °C. The extracts were prepared from COS-7 cells transfected with either an empty vector or pME-CeCaM-KK by lysis at 4 °C by adding 1 ml/10-cm-diameter dish of lysis buffer (150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, 0.2 mM PMSF, 10 mg/liter leupeptin, 10 mg/liter pepstatin A, 10 mg/liter trypsin inhibitor) containing 0.5 mM EGTA. After centrifugation at 15,000 × g for 15 min, 5 mM CaCal₂ was added into the supernatant. Then the supernatant was applied to a CaM-Sepharose (Amersham Pharmacia Biotech) column (0.5-ml bed volume) equilibrated with buffer A (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.2 mM PMSF, 10 mg/liter leupeptin, 10 mg/liter pepstatin A, 10 mg/liter trypsin inhibitor) containing 0.5 mM CaCl₂. The column was washed with 10 ml of buffer A containing 0.5 mM CaCl₂ followed by washing with buffer A containing 0.5 mM CaCl₂ and 1 M NaCl. After the column was washed with buffer A containing 0.5 mM CaCl₂, recombinant CeCaM-KK was eluted with 2 mM EGTA in buffer A and stored in a final concentration of 40% glycerol and 10% ethylene glycol at 20 °C.

Activation and Phosphorylation of CeCaM-KI by CeCaM-KK-- GST·CeCaM-KI (0.5 µg) was incubated with recombinant CaM-KK (9 ng) expressed in COS-7 cell at 30 °C for 10 min in 10 µl of 50 mM HEPES (pH7.5), 10 mM Mg(Ac)₂, 1 mM DTT, and 400 µM ATP containing either 2 mM CaCl₂, 8 µM CaM or 2 mM EGTA. The reaction was terminated by a 20-fold dilution at 4 °C with 50 mM HEPES (pH 7.5), 2 mg/ml bovine serum albumin, 10% ethylene glycol, and 2 mM EDTA. CaM-KI activity was measured at 30 °C for 10 min in 25 µl of 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)₂, 1 mM DTT, 400 µM [-³²P]ATP (1000-2000 cpm/pmol), 40 µM syntide-2 containing either 2 mM CaCl₂, 8 µM CaM or 2 mM EGTA. The reaction was initiated by the addition of 5 µl of CaM-KI and terminated by spotting aliquots (15 µl) onto phosphocellulose paper (Whatman P-81) followed by washing in 75 mM phosphoric acid. Phosphorylation of CeCaM-KI by CeCaM-KK was essentially the same as in the activation assay except for using 400 µM [-³²P]ATP (1000-2000 cpm/pmol). After a 10-min incubation at 30 °C, the reaction was terminated by adding 5 µl of SDS-PAGE sample buffer. Then the samples were subjected to SDS-15% PAGE followed by autoradiography.

Enrichment of CaM-binding Proteins in C. elegans-- C. elegans (mixed stage) was harvested from four plates (6-cm-diameter dish) and extracted in 3 ml of lysis buffer (50 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 10% glycerol, 0.2 mM PMSF, 10 mg/l leupeptin, 10 mg/l pepstatin A, 10 mg/l trypsin inhibitor) with sonication. After centrifugation at 15,000 × g for 20 min, CaCl₂ was added to the extract at a final concentration of 5 mM, and then the extract (1.2 mg of protein) was applied to a CaM-Sepharose column (60 µl bed volume) that was pre-equilibrated with 2 mM CaCl₂-containing buffer. After washing the column extensively, CaM-binding proteins were eluted with 2 mM EGTA-containing buffer (0.5 M NaCl. 20 mM Tris-HCl pH7.5, 0.05% Tween 20). Eluate was collected and concentrated to 50 µl with a microconcentrator (Microcon 30, Amicon) followed by Western blot analysis using 10 µl of the sample.

Detection of CREB Phosphorylation-- COS-7 cells were transfected with a combination of expression constructs (pME18s) encoding CeCaM-KI wild type or mutants (4 µg) and/or CeCaM-KK (2 µg). After incubation for 40 h, the cells were deprived of serum for 6 h and then stimulated with 1 µM calcium ionophore and 10 mM CaCl₂ for 10 min. The cells were immediately lyzed with 1 ml of lysis buffer (150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, 0.2 mM PMSF, 10 mg/liter leupeptin, 10 mg/liter pepstatin A, 10 mg/liter trypsin inhibitor, 50 mM sodium fluoride, 0.1 mM Na₃VO₄, 100 nM okadaic acid, and 1 µM microcystin-LR). Each cell extract (18 µg) was subjected to SDS-10% PAGE followed by Western blotting for detection of either CREB, using anti-CREB antibody (New England Biolab), or phosphorylated CREB, using anti-phospho-CREB antibody specific for CREB phosphorylated at Ser¹³³ (New England Biolab). Detection of the immunoreactive band was carried out using an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).

Luciferase Assay-- COS-7 cells were transfected with 4 µg of a plasmid pFR-4xCRE-luciferase (Stratagene) and a combination of expression plasmids (pME18s) carrying either CeCaM-KI wild type or mutants (4 µg) and/or CeCaM-KK (2 µg). A cDNA (2 µg) of the catalytic subunit of PKA was also used as a positive control. After incubation for 40 h, the cells were deprived of serum for 6 h and then stimulated with 1 µM calcium ionophore and 10 mM CaCl₂ for 6 h. Then the cells were lyzed with 1 ml of lysis buffer (25 mM glycyl glycine (pH 7.8), 8 mM MgSO₄, 1 mM EDTA, 1% Triton X-100, 5% glycerol, and 1 mM DTT), and the luciferase activity of each cell extract (10 µl) was measured by the luciferase assay kit (PicaGene, Toyo Ink).

Anti-CeCaM-KI Antiserum-- After the recombinant GST·CeCaM-KI (wild type) was subjected to SDS-PAGE, electroeluted GST·CeCaM-KI from the excised gels was used to immunize a Japanese White rabbit (approximately 500 µg/injection). The rabbit received booster injections at 14-day intervals. The presence of anti-CeCaM-KI antibody was assayed by Western blotting using the extract of mock- and CeCaM-KI-transfected COS-7 cells. The antiserum was applied to a GST-bound glutathione-Sepharose column twice to remove anti-GST antibodies, and the flow-through fraction was collected and used for Western blotting and immunoprecipitation.

Other Methods-- General techniques for the culture and handling of worms have been described (43). The C. elegans Bristol (N2) stock was used as the wild type strain. CaM overlay was performed as described previously (8). Anti-GFP antibody (CLONTECH) was used for detection of the GFP-fusion protein expressing in COS-7 cells. Protein concentration was estimated by Coomassie dye binding (Bio-Rad) using bovine serum albumin as a standard.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cloning of CaM-KI Homologue from C. elegans-- Recent studies identified C. elegans CaM-KK (CeCaM-KK) in the data base (GenBank^TM accession no. U11029) (27) and demonstrated that recombinant CeCaM-KK (GenBank^TM accession no. AB016838) was able to activate mammalian CaM-KIV in a Ca²⁺/CaM-dependent manner (17). To identify the target(s) for CeCaM-KK, we searched the C. elegans genome data base and found a C. elegans cosmid (K07A9) containing a protein kinase catalytic domain that is highly homologous to mammalian CaM-KI. Because the cosmid does not contain a full-length sequence, we used a combination of reverse transcriptase-PCR, to amplify the portion of the protein kinase cDNA (residues 129-308), and screening of C. elegans ZAP phage cDNA library, using the reverse transcriptase-PCR product as a probe. A cDNA of 1546 base pairs encoding 348 amino acid residues in the open reading frame was isolated, which was approximately 60% identical with rat (29, 30) and human CaM-KI (10) (Fig. 1A). We transfected the cDNA into COS-7 cells and detected an approximately 40-kDa CaM-binding protein on SDS-PAGE by the CaM overlay method; this is consistent with the calculated M_r value of 39,066 (Fig. 1B). Therefore, we have termed this gene product C. elegans CaM-KI (CeCaM-KI). Anti-CeCaM-KI antiserum recognized the endogenous CeCaM-KI in the partially purified fraction from C. elegans extract by CaM-Sepharose column chromatography (Fig. 1C, right lane), which had the same mobility on SDS-PAGE as the overexpressed enzyme in COS-7 cells (Fig. 1C, left lane). We also detected 40-kDa CaM-binding protein by the CaM overlay method in the immunoprecipitated fraction from C. elegans extract with the anti-CeCaM-KI antiserum (data not shown). These results suggest that the isolated cDNA encodes full-length CeCaM-KI and the methionine at position 1 in CeCaM-KI is likely the translation initiation. CeCaM-KI contains a Thr residue (Thr¹⁷⁹) in the catalytic domain equivalent to the phosphorylation-activation Thr¹⁷⁷ in mammalian CaM-KI. It is noteworthy that a basic cluster (KRR⁷) of a potential NLS is inserted at the NH₂-terminal region in CeCaM-KI but not in the mammalian CaM-KI, as described in detail below.

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Cloning and expression of C. elegans CaM-kinase I. A, amino acid sequence comparison of C. elegans CaM-KI with mammalian CaM-KIs. CaM-KI cDNA (GenBankTM accession no. AB021864) was obtained from C. elegans N2 strain embryonic stage cDNA library in ZAP vector as described under "Experimental Procedures." The putative initiation methionine is encoded by ATG at nucleotide 57. The termination codon TGA is denoted at nucleotide 1101. The deduced amino acid sequence of CeCaM-KI was aligned with those of rat (29, 30) and human CaM-KI (10). The respective amino acid numbers are shown at both sides. The positions where at least two of the three sequences are identical are indicated by the lighter shaded boxes. The catalytic domain is indicated by a solid line box. The ATP-binding region is overlined above the amino acid sequences. The phosphorylation site Thr179 in CeCaM-KI for activation by CaM-KK, equivalent to Thr177 in rat and human CaM-KI is indicated by an asterisk. A potential NLS of CeCaM-KI is overlined with dashes. The regulatory region containing CaM-binding and autoinhibitory domains is indicated by black boxes (31). B, expression of recombinant CeCaM-KI and CeCaM-KK. Mock, CeCaM-KI, or CeCaM-KK cDNA in pME18s vector was transfected into COS-7 cells. Each cell extract (approx. 20 µg) was subjected to SDS-10% PAGE followed by CaM overlay as described under "Experimental Procedures." C, expression of endogenous CeCaM-KI. CaM-binding proteins were enriched from the extract of C. elegans (mixed stage) by CaM-Sepharose column chromatography as described under "Experimental Procedures" and then subjected to SDS-10% PAGE followed by Western blotting using anti-CeCaM-KI antiserum (1/1000 dilution, right lane). The extract of COS-7 cells expressing wild type CeCaM-KI (as shown in B) was also analyzed in the left lane.

Activation Mechanism of CeCaM-KI by CeCaM-KK-- We expressed and purified CeCaM-KI as a GST-fusion protein in E. coli to test its activity and activation by recombinant CeCaM-KK. As shown in Fig. 2, recombinant wild type CeCaM-KI has a Ca²⁺/CaM-dependent protein kinase activity toward the peptide substrate (syntide-2), and the activity is enhanced approximately 10-fold by CeCaM-KK treatment in a Ca²⁺/CaM-dependent manner. Furthermore, phosphorylation of CeCaM-KI by CeCaM-KK was strongly induced only in the presence of Ca²⁺/CaM, whereas basal CeCaM-KI underwent weak autophosphorylation in a Ca²⁺/CaM-dependent manner, which did not induce the activity. We also observed that both the  and  isoforms of rat CaM-KK were able to activate CeCaM-KI in a manner similar to CeCaM-KK (data not shown). When we used the T179A mutant of CeCaM-KI, it was no longer activated and phosphorylated by CeCaM-KK, indicating that Thr¹⁷⁹ is a primary phosphorylation-activation site for CeCaM-KK. This finding is also consistent with the observation that the mutation of Thr¹⁷⁹ to Asp resulted in an approximately 5-fold increase in the basal Ca²⁺/CaM-dependent activity without activation. These results clearly demonstrated the activation of CeCaM-KI by CeCaM-KK through Ca²⁺/CaM-dependent phosphorylation of Thr¹⁷⁹ in vitro. Truncation at residue position 295 generated a constitutively active form of CeCaM-KI, which was incapable of binding Ca²⁺/CaM (data not shown), suggesting the existence of an autoinhibitory domain and CaM-binding region in the COOH-terminal from position 295. The regulatory region of CeCaM-KI has 50% identity with that of mammalian CaM-KI (31). Based on the amino acid sequence comparison, Trp³⁰⁵ in CeCaM-KI is conserved in many CaM-binding proteins including mammalian CaM-KI (Fig. 1A) as one of the anchoring residues to the COOH-terminal hydrophobic pocket of CaM (32). According to NMR and x-ray structure determination of the CaM·MLCK peptide (M13) complex (33, 34) and the CaM·CaM-KII peptide complex (35), both skeletal and smooth muscle MLCK peptides have 14 residues between two key hydrophobic residues, and the CaM-KII peptide has 10 residues between them. Therefore Leu³¹⁸ can be predicated as another anchoring residue in CeCaM-KI to the NH₂-terminal hydrophobic pocket of CaM, which appears to be of the MLCK type. The truncation mutant was still activated and phosphorylated by CeCaM-KK in a complete Ca²⁺/CaM-dependent manner, indicating that CeCaM-KK also requires Ca²⁺/CaM for phosphorylation and activation of CeCaM-KI, consistent with previous observation by using a constitutively active mutant of mouse CaM-KIV (17).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Activation of CeCaM-KI by CeCaM-KK. Either wild type or mutants (positions 1-295, T179A, T179D) of GST·CeCaM-KI (0.5 µg), which was expressed and purified from E. coli, was incubated with either buffer () or 9 ng of recombinant CeCaM-KK (+) at 30 °C for 10 min in the absence () or presence (+) of 2 mM CaCl2, 8 µM CaM. After terminating the activation reaction, protein kinase activity of each CeCaM-KI toward syntide-2 was measured at 30 °C for 10 min in the absence (open bar) or presence (solid bar) of 2 mM CaCl2, 8 µM CaM as described under "Experimental Procedures." The results are presented as the mean and S.E. of three experiments. 32P incorporated into each recombinant CeCaM-KI during the activation reaction under the same condition as described above (except [-32P]ATP was used), was analyzed by SDS-15% PAGE followed by autoradiography (inset).

Effect of Activation on Kinetic Parameters of CeCaM-KI-- CeCaM-KI was incubated with activation reaction including Ca²⁺/CaM, Mg-ATP, and either recombinant CeCaM-KK or buffer for 60 min. EDTA/EGTA-containing buffer was added to stop activation, and CeCaM-KK, Mg-ATP, and excess CaM were removed by glutathione-Sepharose column chromatography. Both basal and activated CeCaM-KIs were eluted by the addition of 10 mM glutathione followed by kinetic constants determination of both enzymes for syntide-2 and ATP. From the results shown in Fig. 3, it is clear that the main effect of activation by CeCaM-KK was to lower the K_m for syntide-2. Phosphorylation of Thr¹⁷⁹ by CeCaM-KK decreased the K_m of CeCaM-KI for syntide-2 from 657 to about 20 µM with little effect on either the V_max or K_m for ATP (Fig. 3, A and B), which is similar to the activation mechanism of mammalian CaM-KIV by CaM-KK (7). However, the V_max of recombinant CeCaM-KI (approximately 0.1 µmol/min/mg) for syntide-2 obtained in the present study was about 1-5% that of recombinant mammalian CaM-KI (2-12 µmol/min/mg (10, 44)) but comparable with that of CaM-KIV (0.15-0.5 µmol/min/mg (7, 45, 46)). This may be due to the structural difference of catalytic domain between C. elegans and mammalian CaM-KI, because approximately 25% of the amino acid residues in the catalytic domain are not identical between both CaM-KIs (Fig. 1A).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Kinetic analysis of CeCaM-KI activation by CeCaM-KK. After recombinant wild type CeCaM-KI was incubated with either buffer (basal CeCaM-KI, open circle) or CeCaM-KK (activated CeCaM-KI, closed circle) at 30 °C for 60 min with a standard activation reaction (see "Experimental Procedures"), the reaction was terminated by the addition of EDTA/EGTA-containing buffer and then purified by glutathione-Sepharose column chromatography. The kinetic properties of both enzymes were analyzed and presented as double-reciprocal plots (Lineweaver-Burk). For the titration of syntide-2 (A), 400 µM [-32P]ATP and 20-1000 µM syntide-2 were used. For the titration of ATP (B), 500 µM syntide-2 and 20-1000 µM [-32P]ATP were used. The experiments were performed in triplicate for each point. Km and Vmax values determined from each double-reciprocal plot (Lineweaver-Burk) are indicated in each panel as the mean and S.E. of three experiments.

Nuclear Localization of CeCaM-KI-- It has already been reported that mammalian CaM-KI is localized mainly in the cytoplasm (36). To visualize the subcellular localization of CeCaM-KI, we transfected GFP-fusion constructs of CeCaM-KI into COS-7 cells. Expression of the GFP-fusion protein of each CaM-KI was confirmed by Western blotting using anti-GFP antibody (Fig. 4D) and the CaM overlay method (data not shown). In contrast to rat CaM-KI localized in cytoplasm (Fig. 4C) consistent with a previous report (36), CeCaM-KI (wild type, Pro²-Ala³⁴⁸) is highly concentrated in the nucleus (Fig. 4A). When we used a mutant CeCaM-KI lacking 6 residues (Pro²-Arg⁷) at the NH₂-terminal region, it was no longer staying in the nucleus (Fig. 4B), suggesting that the NH₂-terminal 6 residues contain a potential NLS. This region includes the basic cluster Lys⁵-Arg⁶-Arg⁷, which is similar to the NLS (KKRK) in the delta B isoform of CaM-KII (37), but it is lacking in the mammalian CaM-KI (Fig. 1A). We have detected GFP·CeCaM-KK localized in both the cytoplasm and the nuclei of transfected cells (data not shown).

View larger version (71K): [in this window] [in a new window]   Fig. 4.   Subcellular localization of CeCaM-KI. COS-7 cells were transfected with GFP-fusion constructs carrying CeCaM-KI wild type (residue 2-348, A), the NH2-terminal deletion mutant, which lacks residue 2-7 (B), or rat CaM-KI wild type (C). After 20 h post-transfection, the cells expressing each GFP-fusion protein were observed for GFP fluorescence by a fluorescence microscopy and then lyzed to monitor the expression level of each GFP·CaM-KI by Western blotting using anti-GFP antibody (panel D, lane a, CeCaM-KI wild type; lane b, NH2-terminal deletion mutant of CeCaM-KI; lane c, rat CaM-KI wild type). Results are representative of experiments repeated at least four times.

Transcriptional Activation by C. elegans CaM-kinase Cascade-- Nuclear localization of CeCaM-KI gave us an idea that the CaM-KK/CaM-KI cascade in C. elegans might be involved in the regulation of transcriptional activation analogous to the CaM-KK/CaM-KIV/CREB pathway in mammalian cells. It has been shown that CREB appears to be a good substrate for mammalian CaM-KI in vitro (38) and that overexpressed mammalian CaM-KI can stimulate CREB-dependent transcriptional activation (39). However, mammalian CaM-KI has been shown to be localized in the cytoplasm in intact cells (36), and therefore the involvement of this kinase in the activation of CREB-dependent transcriptional activation is still controversial. To analyze the C. elegans CaM-kinase cascade, we used mammalian cells because there is little information available about CREB and CREB-dependent transcriptional activation in C. elegans, although there is one predicted CREB gene in C. elegans (42). First, we tried to detect the phosphorylation of endogenous CREB at Ser¹³³ upon stimulation with 1 µM calcium ionophore in COS-7 cells, which was transfected with various combinations of plasmids carrying CeCaM-KI and/or CeCaM-KK (Fig. 5A). Detection of CREB phosphorylation was carried out using anti-phospho-CREB antibody. A 10-min stimulation with calcium ionophore induced significant phosphorylation of CREB only in the cells transfected with both CeCaM-KI wild type and CeCaM-KK as well as in PKA-transfected cells (Fig. 5A). We detected an immunoreactive band migrating faster than the phosphorylated CREB, which was also induced by co-transfection of the components of the C. elegans CaM-kinase cascade upon stimulation with calcium ionophore as well as PKA transfection. Because the antibody used for detection of the phosphorylated form of CREB also detects the phosphorylated form of the CREB-related proteins, activating transcription factor-1 (ATF-1) and cAMP response element binding modulator (CREM), the lower band is possibly ATF-1. This is consistent with a previous report that ATF-1 can be activated by increasing cAMP and Ca²⁺ concentrations (40). Next we tested the activation of CRE-dependent transcriptional activation by the C. elegans CaM-kinase cascade using the CRE reporter gene assay in transfected COS-7 cells (Fig. 5B). Again, stimulation with calcium ionophore induced CRE-dependent transcriptional activation at 4-5-fold only in those cells transfected with both CeCaM-KI wild type and CeCaM-KK, which is consistent with the induction of CREB phosphorylation as shown in Fig. 5A. Interestingly, unlike overexpressed mammalian CaM-KI, which alone enhanced CREB-dependent transcription by membrane depolarization (39), CeCaM-KI alone could not significantly induce the phosphorylation of CREB and activate CRE-dependent transcription upon stimulation with calcium ionophore, indicating this cascade to be strictly regulated by upstream CaM-KK. We confirmed that COS-7 cells transfected with both the T179A mutant and the K52A mutant (kinase deficient) of CeCaM-KI with CeCaM-KK did not respond with calcium ionophore stimulation for both phosphorylation of CREB and CRE-dependent transcriptional activation (Fig. 5, A and B). Taken together, these results suggest that CeCaM-KI is activated by CeCaM-KK through phosphorylation of Thr¹⁷⁹ by Ca²⁺ mobilization in intact cells and subsequently phosphorylates CREB at Ser¹³³, resulting in the activation of CRE-dependent transcription.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   CREB-dependent transcriptional activation by C. elegans CaM-kinase cascade. A, enhancement of CREB phosphorylation by C. elegans CaM-kinase cascade. COS-7 cells were transfected with a combination of the expression plasmids carrying either CeCaM-KI wild type or mutants and/or CeCaM-KK as indicated. After depletion of serum for 6 h, the cells were stimulated with (+) or without () 1 µM calcium ionophore (Ca-ionophore) in the presence of 10 mM CaCl2 for 10 min, and then the cell extracts (18 µg) were subjected to SDS-10% PAGE followed by Western blotting using either anti-CREB antibody (upper panel) or anti-phospho-CREB antibody (lower panel) as described under "Experimental Procedures." The position of CREB is indicated by arrows. The catalytic subunit of PKA cDNA (PKA) was transfected as a positive control (right lane). B, transcriptional activation of the CRE-luciferase gene by C. elegans CaM-kinase cascade. COS-7 cells were transfected with the reporter gene plasmid (CRE-TATA-luciferase) with the expression plasmid carrying either CeCaM-KI wild type or mutants in the absence or presence of CeCaM-KK as indicated. After depletion of serum for 6 h, the cells were stimulated with (solid bars) or without (open bars) 1 µM calcium-ionophore in the presence of 10 mM CaCl2 for 6 h and then extracted for the measurement of luciferase activity as described under "Experimental Procedures." The results were presented as the mean and S.E. obtained from three independent transfections.

Conclusion-- The results presented in this paper demonstrate the existence of a CaM-kinase cascade (CaM-KK/CaM-KI) in C. elegans; this cascade operates functionally both in vitro and in intact cells, as do its mammalian counterparts. Ca²⁺-dependent transcriptional regulation through a CaM-kinase cascade seems to be conserved in C. elegans, which is consistent with nuclear localization of CeCaM-KI. Therefore, the CaM-KK/CaM-KIV pathway, which is thought to be involved in Ca²⁺-dependent transcriptional regulation in mammalian cells (20-24, 41), may be replaced by the CaM-KK/CaM-KI cascade in C. elegans. This reasoning is also consistent with the fact that the CaM-KIV homologue cannot be found in the C. elegans genome data base. Identification and characterization of the components in the CaM-kinase cascade in C. elegans described in this paper provide useful tools for evaluating the physiological significance of this protein kinase cascade. The question of the physiological function(s) mediated by the CaM-KK/CaM-KI cascade in C. elegans still remains unanswered and is now under investigation with genetic approaches.

	ACKNOWLEDGEMENT

We thank Dr. Henrik T. Yudate (Helix Research Institute) for critical reading of the manuscript.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The 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/EMBL Data Bank with accession number(s) AB021864.

To whom correspondence should be addressed. Tel.: 81-438-52-3967; Fax: 81-438-52-3952; E-mail: tokumitu@hri.co.jp.

	ABBREVIATIONS

The abbreviations used are: CaM-K, Ca²⁺/calmodulin-dependent protein kinase; CaM, calmodulin; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; CRE, cAMP response element; CREB, cAMP response element-binding protein; NLS, nuclear localization signal; Ce, C. elegans; PKA, cAMP-dependent protein kinase; GST, glutathione S-transferase; GFP, green fluorescent protein; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DTT, dithiothreitol; MLCK, myosin light chain kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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