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First published online July 9, 2004; 10.1104/pp.104.041970 Plant Physiology 135:1280-1293 (2004) © 2004 American Society of Plant Biologists Characterization of a Novel Calcium/Calmodulin-Dependent Protein Kinase from Tobacco1,[w]Key Lab of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China (L.M., S.L., Y.-T.L.); and Department of Plant and Microbial Biology, University of California, Berkeley, California 94720 (R.L.J.)
A cDNA encoding a calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CaMK) from tobacco (Nicotiana tabacum), NtCaMK1, was isolated by protein-protein interaction-based screening of a cDNA expression library using 35S-labeled CaM as a probe. The genomic sequence is about 24.6 kb, with 21 exons, and the full-length cDNA is 4.8 kb, with an open reading frame for NtCaMK1 consisting of 1,415 amino acid residues. NtCaMK1 has all 11 subdomains of a kinase catalytic domain, lacks EF hands for Ca2+-binding, and is structurally similar to other CaMKs in mammal systems. Biochemical analyses have identified NtCaMK1 as a Ca2+/CaMK since NtCaMK1 phosphorylated itself and histone IIIs as substrate only in the presence of Ca2+/CaM with a Km of 44.5 µM and a Vmax of 416.2 nM min–1 mg–1. Kinetic analysis showed that the kinase not previously autophosphorylated had a Km for the synthetic peptide syntide-2 of 22.1 µM and a Vmax of 644.1 nM min–1 mg–1 when assayed in the presence of Ca2+/CaM. Once the autophosphorylation of NtCaMK1 was initiated, the phosphorylated form displayed Ca2+/CaM-independent behavior, as many other CaMKs do. Analysis of the CaM-binding domain (CaMBD) in NtCaMK1 with truncated and site-directed mutated forms defined a stretch of 20 amino acid residues at positions 913 to 932 as the CaMBD with high CaM affinity (Kd = 5 nM). This CaMBD was classified as a 1-8-14 motif. The activation of NtCaMK1 was differentially regulated by three tobacco CaM isoforms (NtCaM1, NtCaM3, and NtCaM13). While NtCaM1 and NtCaM13 activated NtCaMK1 effectively, NtCaM3 did not activate the kinase.
Calcium as a universal second messenger regulates a range of cellular and physiological processes in plants and animals (Poovaiah and Reddy, 1993  ; Gilroy and Trewavas, 1994; Sanders et al., 2002). It is believed that changes in cytosolic Ca2+ are sensed by a group of Ca2+-binding proteins, including calmodulin (CaM)-like domain protein kinases (CDPKs) and CaM (Chin and Means, 2000; Harmon et al., 2000; Luan et al., 2002). CDPKs, well-characterized kinases, have been shown to be modulated by binding of Ca2+ to a carboxy-terminal CaM-like sequence with EF hands and mediate many of the effects of Ca2+ in plant cells (Poovaiah and Reddy, 1987; Roberts and Harmon, 1992; Harper et al., 1994; Harmon et al., 2000; Cheng et al., 2002).
CaM is a ubiquitous intracellular Ca2+ receptor involved in transducing a variety of extracellular signals (Bush, 1995
Although typical Ca2+/CaMKs with activities similar to CaMK in animals have not been identified in plants (Luan et al., 2002 Here, we report on the isolation and identification of a cDNA from tobacco that encodes a CaMK (NtCaMK1). We show that substrate phosphorylation and autophosphorylation of NtCaMK1 is Ca2+/CaM dependent. Capillary electrophoresis of phosphoamino acids revealed that phosphorylation of NtCaMK1 occurs on Thr residues. Kinetic analysis showed that NtCaMK1 specifically bound CaM with high affinity (Kd = 5 nM) via a CaM-binding domain (CaMBD) having a 1-8-14 motif. Although the kinase was effectively activated by both NtCaM1 and NtCaM13, NtCaM3 failed to stimulate the enzymatic activation of NtCaMK1. These data indicate that NtCaMK1 is a novel Thr kinase modulated by Ca2+/CaM.
Cloning and Characterization of NtCaMK1 cDNA
A cDNA library constructed with the expression vector
The deduced amino acid sequence of NtCaMK1 consists of 1,415 residues with a calculated molecular mass of 156 kD, and contains all 11 subdomains characteristic of a protein kinase catalytic domain (Hanks et al., 1988 ). A database comparison to identify homologies between NtCaMK1 and other genes with known functions suggests that NtCaMK1 is a CaMK homolog (Fig. 1B). NtCaMK1 shares high sequence identity with many CBKs, including rat CaMKII  -subunit (30%; accession no. J02942; Lin et al., 1987), tobacco NtCBK2 (56%; accession no. AF435452; Hua et al., 2003), maize (Zea mays) MCKI (50%; accession no.1839597; Lu et al., 1996), lily CCaMK (31%; accession no. 860675; Patil et al., 1995); and CDPKs such as soybean (Glycine max) CDPK (44%; accession no. P28583; Harper et al., 1991), Arabidopsis AtCDPK3 (29%; accession no. 1220099; Urao et al., 1994), and Arabidopsis CRK5 (62%; accession no. At3g50530; Fig. 1B). Furthermore, the nonkinase part of NtCaMK1 shares 17% homology with a potato calmodulin-binding protein (accession no. AF378084; Reddy et al., 2002), a 5 kb-long sequence that encodes a protein of 1,309 amino acids. The NtCaMK1 sequence was also analyzed using programs found on the ExPASy Proteomics tools Web page (www.expasy.ch/tools). Using MotifScan, we found three strong PEST sequences with PEST scores (452–466 [+8.75], 481–506 [+9.02], 1241–1272 [+8.63]), respectively (Fig. 1A). Unlike CDPKs and CCaMKs, NtCaMK1 does not possess Ca2+-binding EF hands at its C terminus (Fig. 1B). To rule out the possibility that NtCaMK1 resulted from some artifacts during the construction of the cDNA library, a PCR-based strategy was used to isolate the genomic sequence for NtCaMK1, and two PCR fragments, named P1 (13.3 kb) and P2 (13.5 kb), were obtained (data not shown). The sequence data (AY562224) indicated that these two PCR fragments have 2,266 bp of overlapping sequence, and the 24.6-kb genomic sequence has 21 exons interrupted by 20 introns in the full-length cDNA. The intron-exon borders were determined by comparison with the cDNA sequence, and they all conform to the canonical GT/AG 5' and 3' splice sites (Fig. 1C).
Since NtCaMK1 was isolated by screening a tobacco cDNA library with 35S-labeled CaM implying its CaM-binding ability, we characterized its ability to bind tobacco CaM (Fig. 4A). When purified NtCaMK1 was used for CaM binding with either 35S-labeled CaM or biotinylated CaM, CaM bound only in the presence of Ca2+ (Fig. 4, C and E). CaM binding was lost in the presence of EGTA (Fig. 4, B and D), indicating that NtCaMK1 is a CaM-binding protein that binds CaM in a Ca2+-dependent manner. This conclusion was also supported by the observation that, during purification, NtCaMK1 was eluted from CaM-Sepharose-4B only in the absence of Ca2+.
To further define the CaMBD of NtCaMK1, several expression plasmids were constructed to produce different truncated forms of NtCaMK1 that were used for CaM-binding assays (Fig. 5A). All recombinant forms of NtCaMK1 lacked CaM-binding ability in the presence of EGTA (Fig. 5C). However, in the presence of Ca2+, constructs NtCaMK1p932 and NtCaMK1p950 containing the putative CaMBD (amino acid residues 913–932) bound CaM (Fig. 5B, lanes 2 and 3), as did full-length NtCaMK1 (Fig. 5B, lane 1). Constructs NtCaMK1p865 and NtCaMK1p912 lacking the putative CaMBD lacked CaM-binding ability (Fig. 5B, lanes 4 and 5), indicating that the CaMBD is located among the 20 to 30 amino acid residues at the C terminus of NtCaMK1p932.
The prediction of secondary structure of the 20 amino acids from Ala-913 to Lys-932 of NtCaMK1 using the Anthwin 45 package (Microsoft, Redmond, WA) suggests a basic amphiphilic -helix (data not shown). The helical wheel projection for NtCaMK1 CaMBD shows a typical CaMBD feature, with segregation of basic and hydrophobic residues on opposite sides of the helix (O'Neil and DeGrado, 1990; Putnam-Evans et al., 1990; Lu and Harrington, 1994). To experimentally identify this tentative CaMBD, construct pCaMBD encoding these 20 amino acid residues was made with vector pET32a and used for CaM-binding assay. The protein expressed by pCaMBD bound CaM in a Ca2+-dependent manner (Fig. 5B, lane 6, and C, lane 6), demonstrating that it is these 20 amino acid residues at positions 913 to 932 that are likely responsible for CaM binding. To test whether the truncated C terminus has a CaMBD, the C-terminal amino acid sequence from 933 to 1,415 was expressed and assayed for CaM binding. The results indicated that the C terminus of NtCaMk1 did not have CaM-binding ability (Fig. 5D).
Comparative analyses of the CaM-binding regions of the many reported CaM-binding proteins has provided multiple sequence motifs required for CaM complex formation. Two motifs for Ca2+-dependent binding, named 1-8-14 and 1-5-10, were suggested based on the position of conserved hydrophobic residues (Rhoads and Friedberg, 1997
To confirm the role of these amino acid residues in CaM binding in NtCaMK1, amino acid substitutions at several residues that were expected to play a critical role in the interaction between CaM and NtCaMK1 were introduced. The hydrophobic 916Leu at the first position of the 1-8-14 motif was replaced by Arg (L916R), and 919Trp was replaced by Arg (W919R; Fig. 6A). Expression constructs for these mutations were introduced into Escherichia coli and the expressed proteins separated on SDS-PAGE and transferred to polyvinylidine difluoride (PVDF) membranes for CaM-binding assays with biotinylated CaM (Reddy et al., 1993
We also modified the CaMBD of NtCaMK1 by introducing four negatively charged Glu residues instead of the positively charged Arg and Lys residues at positions Arg-924 and Lys927 (913AKALKKWRMMARKKKAVSHK932 to 913AKALKKWRMMAEEEEAV SHK932), resulting in the breakage of basic amphiphilic -helix structure (Fig. 6A). CaM-binding assays with this modified CaMBD showed that activity was lost (Fig. 6B, lane RK4E). However, when positively charged amino acid residues were replaced by other positively charged amino acid residues, such as K918N or K925N (Fig. 6A), the substitutions did not affect complex formation between the CaMBD and CaM (Fig. 6B, lanes K918N and K925N), demonstrating the role of positive charges of amino acid residues at certain positions in the CaMBD of NtCaMK1.
It has been reported that different CaM-binding proteins have different binding affinities for CaM, resulting in subtle regulation by different concentrations of either CaM and/or CaMBPs (Sikela and Hahn, 1987
Autophosphorylation and Substrate Phosphorylation of NtCaMK1 NtCaMK1 was purified and used to analyze autophosphorylation and substrate phosphorylation activity. Both autophosphorylation and histone IIIs phosphorylation activities were Ca2+/CaM-dependent (Fig. 8A, lanes c and f) and no phosphorylation activity was detected in the presence of either EGTA or calcium alone (Fig. 8A, lanes a, b, d, and e). This result was further supported by time course analyses for kinase activity of NtCaMK1. Time course analyses showed that NtCaMK1 rapidly phosphorylated itself and histone IIIs as substrate, but only in the presence of Ca2+/CaM (Fig. 8, B and C). The time to maximal autophosphorylation activity was 5 min and for maximal activity of substrate phosphorylation 30 min. Autophosphorylation and substrate phosphorylation can be catalyzed by autophosphorylated NtCaMK1 in a Ca2+/CaM-independent manner as shown in Figure 8B. These data show that autophosphorylation of NtCaMK1 occurred when it was initiated by Ca2+/CaM and then blocked with excess EGTA. Autophosphorylation of NtCaMK1 occurs when an autophosphorylation reaction was initiated for as little as 30 s in the presence of Ca2+/CaM, followed by incubation in excess EGTA, although maximal autophosphorylation activity was lower in the presence of excess EGTA than in the presence of Ca2+/CaM alone. Autophosphorylated NtCaMK1 showed substrate phosphorylation activity in the presence of EGTA (Fig. 8D).
Kinetic parameters of the kinase activity of NtCaMK1 were determined from double-reciprocal analyses of data for phosphorylation of various concentrations of histone IIIs in the presence of 100 µM ATP. The Km for histone IIIs was calculated to be 44.5 µM and the Vmax 416.2 nM min–1 mg–1 when Ca2+/CaM was added. A synthetic peptide substrate for CaMKII, syntide-2 (Hashimoto and Soderling, 1987 ), was also used as substrate in phosphorylation assays with NtCaMK1. Kinetic analysis showed that unphosphorylated NtCaMK1 had a Km for syntide-2 of 22.1 µM and a Vmax of 644.1 nM min–1 mg–1 when assayed in the presence of Ca2+/CaM.
To explore the effects of the regulatory region, including the CaMBD, on the enzymatic properties of NtCaMK1, two truncated forms of NtCaMK1 were utilized for enzymatic assays. NtCaMK1p932, which includes the CaMBD, carried out autophosphorylation and substrate phosphorylation in the presence of Ca2+/CaM and lost kinase activity in the presence of either calcium or EGTA (Fig. 9A), showing the same Ca2+/CaM dependence as full-length NtCaMK1. Time course analyses showed that purified NtCaMK1p932 rapidly phosphorylated histone IIIs in the presence of Ca2+/CaM, with 5 min to maximal activity of autophosphorylation and 30 min to maximal activity of substrate phosphorylation (Fig. 10A), consistent with enzymatic characteristics of full NtCaMK1. NtCaMK1p865, lacking the CaMBD, carried out autophosphorylation and substrate phosphorylation in the presence of both Ca2+/CaM and EGTA (Fig. 9B). Time course analyses showed that purified NtCaMK1p865 rapidly phosphorylated histone IIIs in the presence of either EGTA or Ca2+ in the absence of CaM (Fig. 10B). Differences in the enzymatic characteristics of NtCaMK1p865 and NtCaMK1p932 suggest that amino acids 865 to 932 may inhibit NtCaMK1 activity in the absence of Ca2+/CaM, and that this inhibition can be abolished by binding CaM to the CaMBD of NtCaMK1.
Activation of NtCaMK1 by NtCaM Isoforms To investigate whether this NtCaMK1 is differentially regulated by various CaM isoforms, three tobacco CaM isoforms (NtCaM1, NtCaM3, and NtCaM13) and bovine brain CaM were used, and the Ka value of NtCaMK1 activity was assayed with the addition of increasing amounts of CaM in the presence of 1 mM Ca2+ (Fig. 11). Our experiments indicated that NtCaM1, NtCaM13, and bovine brain CaM half-maximally activated NtCaMK1 with a Ka of 26 nM, 60 nM, and 40 nM, respectively, using histone IIIs as substrate. However, NtCaM3 produced essentially no activation of NtCaMK1 (Fig. 11A). When syntide-2 was employed as substrate, NtCaMK1 can be activated by NtCaM1, NtCaM13, and bovine brain CaM, but with different Ka values (56 nM, 31 nM, and 45 nM, respectively). NtCaM3 still cannot stimulate the enzymatic activation of NtCaMK1 with syntide-2 (Fig. 11B). Thus, NtCaM13 was a better activator than NtCaM1 when syntide-2 was used as substrate, whereas NtCaM1 was better when histone IIIs was used. However, NtCaM3 failed to activate the kinase with both substrates. These data suggest differential regulation of the kinase by different CaM isoforms. This was further verified when the autophosphorylation and substrate phosphorylation products of NtCaMK1 were analyzed by SDS-PAGE. While both substrate phosphorylation and autophosphorylation activities were detected with NtCaM1 (Fig. 11C, lanes a and e) and NtCaM13 (Fig. 11C, lanes b and f) as activators, no phosphorylation activity was detected in the presence of 1 µM NtCaM3 (Fig. 11C, lanes c and g), even in the presence of extremely high concentrations (10 µM) of NtCaM3 (Fig. 11C, lanes d and h).
Phosphoamino Acid Analysis Capillary electrophoresis was used to identify the amino acids that become phosphorylated during autophosphorylation of NtCaMK1. When the hydrolysis products of purified NtCaMK1 that were not autophosphorylated in vitro were separated by capillary electrophoresis, no phosphoamino acids were detected, demonstrating that the purified NtCaMK1 used in our kinase activity assays was not phosphorylated in vivo (Fig. 12A). Capillary electrophoretic analyses of autophosphorylated NtCaMK1 showed that Thr residues were phosphorylated (Fig. 12B), indicating that NtCaMK1 was a Thr protein kinase, as suggested by our database analysis.
Plant cells generate Ca2+ signals with different amplitudes, frequencies, and durations in response to a variety of internal and external stimuli (Knight et al., 1991 ; Price et al., 1994; Bush, 1995; McAinsh and Hetherington, 1998). As a major calcium sensor and target modulator, CaM plays a vital role in transducing Ca2+ signals by regulating the activity of numerous target proteins, including CaMKs (Zielinski, 1998). CaMKs are key components in many calcium/CaM-mediated pathways in animals and yeast (Saccharomyces cerevisiae). However, typical Ca2+/CaMK similar to CaMKs have not been identified at the molecular and biochemical levels in plants. In contrast, plants contain a unique class of protein kinases, the CDPKs, that bind Ca2+ but not CaM (Poovaiah and Reddy, 1987; Roberts and Harmon, 1992; Harper et al., 1994). In this study, we have identified a novel plant protein kinase, NtCaMK1, that shares the features of animal and yeast CaMKs. The full length of cDNA for NtCaMK1 is 4.8 kb and contains an ORF consisting of 1,415 amino acid residues. This cDNA sequence is unusually long, but it does not result from a cloning artifact because our genomic sequence data demonstrate that this sequence is from one gene. At least one other plant CaM-binding protein has an unusually long cDNA. The cDNA of potato CaM-binding protein is about 5.0 kb long and shares 17% homology with NtCaMK1 (Reddy et al., 2002).
Several lines of evidence support our conclusion that NtCaMK1 is a protein kinase of the CaMK class and not a CDPK. Biochemical analyses indicate that autophosphorylation and phosphorylation of histone IIIs by NtCaMK1 occurs only in the presence of Ca2+/CaM, showing a regulatory role of CaM on NtCaMK1 activity. In contrast, plant CDPKs bind their own intramolecular CaM-like domain, but do not bind free CaM unless the C-terminal CaM-like domain is removed (Yoo and Harmon, 1996
CaM-binding proteins have characteristic CaMBDs that allow protein-protein interactions, and comparative analyses have revealed multiple sequence motifs required for CaM complex formation. Most of the CaMBDs identified in CaM-binding proteins are stretches of 16 to 35 amino acid residues that form
Unlike mammalian systems, plant cells have many different isoforms of CaM. The differential expression and subcellular locations of plant CaMs in response to environmental stimuli and during plant development provide the mechanisms for altering Ca2+ signal transduction and the intrinsic means of selectively activating or inhibiting specific CaM target enzymes in plants (Lee et al., 1995
Several other aspects of the sequence of NtCaMK1 are interesting. A 33 amino acid sequence (IRPRAKSVANVETVKSAGSVDVKRLETSGRSES) of NtCaMK1 at positions 266 to 298 of the N terminus is perfectly repeated at the C terminus at positions 1,383 to 1,415. This repeated sequence of unknown function is unique and so far not found in any other protein. We also find a number of PEST signals in NtCaMK1 that are present in rapidly degraded enzymes, transcription factors, and components of receptor signaling pathways (Rechsteiner, 1988 In summary, this article describes the isolation and characterization of a novel CaMK from tobacco. Its CaMBD has high affinity for CaM and the CaMBD was mapped to a short stretch of amino acid residues between 913Ala and 932Lys, and was identified as a 1-8-14 motif. The protein kinase activity of NtCaMK1 is observed only in the presence of Ca2+/CaM and can be differentially regulated by different isoforms of CaM.
Materials
Tobacco cells (Nicotiana tabacum L. cv Wisconsin-38) were grown in the dark at 23°C as suspension cultures in Gamborg's B-5 medium. Cells from mid-log-phase cultures (7 d old) were used in all experiments (Harrington and Alm, 1988
The construction and screening with 35S-labeled CaM of a cDNA library made with
Based on the sequence of NtCaMK1 cDNA, four gene-specific primers with 5'-ATGTCCTTACAATCTCATCTAGAG-3' and 5'-CTTTAAGTCCAAGAGTACGGC-3' for 5' end, and with 5'-GCGGACAAGATTCTGATGACC-3' and 5'-CTATGACTCTGACCGGCCACTAG-3' for 3' end of NtCaMK1 were designed and used for PCR with tobacco genomic DNA as template. PCR was carried out with 150 ng each of primers, along with 200 µM of dNTPs, 5 units of TaKaRa LA tag, and 1 µg of tobacco genomic DNA as template in a 50-µL reaction volume. The PCR amplification conditions were 94°C for 1 min, 55°C for 1 min, and 72°C for 15 min, for 30 cycles. The amplified PCR products were separated in agarose gel, cloned into pGEM-T Easy Vector, and sequenced.
Tobacco calmodulin (NtCaM1, NtCaM3, and NtCaM13) cDNAs cloned into the pET15 expression vector (a gift from Dr. Y. Ohashi, National Institute of Agrobiological Sciences, Ibaraki, Japan; Yamakawa et al., 2001
Total RNA from tobacco was isolated with TRIZOL as described by the manufacturer (GIBCO, Carlsbad, CA) and northern blotting was carried out as described previously (Lu et al., 1996
Tobacco genomic DNA was extracted using the cetyl-trimethyl-ammonium bromide (CTAB) method (Wagner et al., 1987
For mapping the CaMBD of NtCaMK1, several C-terminal deletion constructs were prepared with a pET32a vector. The cDNAs for the full ORF and four truncated forms (NtCaMK1p865, NtCaMK1p912, NtCaMK1p932, and NtCaMK1p950) of NtCaMK1 (Fig. 5A) were amplified using Platinum Pfx DNA polymerase (GIBCO) with a 5' primer (5'-ATGTCCTTACAATCTCATCTAGAGA-3') and the following five 3' primers: 3' primer (5'-CTATGACTCTGACCGGCCACTAG-3') for the full ORF of NtCaMK1; 3' primer (5'-TGACGTCCTTAACCATGGATGACT-3') for NtCaMK1p865 encoding the N-terminal 865 amino acid residues; 3' primer (5'-CTGCTCCTTCAGATAAAACAGTTC-3') for NtCaMK1p912 encoding the N-terminal 912 amino acid residues; 3' primer (5'-TTTATGAGAAACAGCTTTCTTTTT-3') for NtCaMK1p932 encoding the N-terminal 932 amino acid residues; and 3' primer (5'-TTCTTCTAACAAAGCAAACTGCTC-3') for NtCaMK1p950 encoding the N-terminal 950 amino acid residues. The C-terminal amino acid sequence from 933 to 1,415 was amplified using a 5' primer (5'-TCAACGGTGGTGTCATGTATG-3') and 3' primer (5'-CTATGACTCTGACCGGCCACTAG-3'). All of these C-terminal deletion mutants were prepared with a pET32a vector at the EcoRV site. To precisely determine the putative CaMBD (amino acid residues at positions 913–932) of NtCaMK1, two complementary primers with BamHI and HindIII sites, respectively, were synthesized as follows: 5'-GATCCGCCAAAGCCCTGAAAAAGTGGCGTATGATGGCTCGTAAAAAGAAAGCTGTTTCTCATAAAA-3' and 5'-ACGTTTATGAGAAACAGCTTTCTTTTTACGAGCCATCATACGCCACTTTTTCAGGGCTTTGGCG-3', annealed and cloned into a BamHI- and HindIII-digested pET32a vector (denoted by pCaMBD). To identify the critical amino acid residue(s) within the CaMBD of NtCaMK1 for the interaction between CaM and NtCaMK1, several point mutations were introduced into the putative CaMBD region. Substitutions of single amino acids were performed using the QuikChange XL site-directed mutagenesis kit (Stratagene) with pCaMBD and four pair of forward and reverse primers as follows: forward primer 5'-GCCAAAGCCCGGAAAAAGTGGCGTATG-3' and reverse primer 5'-CATACGCCACTTTTTCCGGGCTTTGGC-3' for L916R in which Arg was substituted for Leu at position 916; forward primer 5'-GCCCTGAAAAAGCGGCGTATGATGG CTCG-3' and reverse primer 5'-CGAGCCATCATACGCCGCTTTTTCAGGGC-3' for W919R in which Arg was substituted for Trp at position 919; forward primer 5'-GCCCTGAAAAATTGGCGTATGATGGC-3' and reverse primer 5'-GCCATCATACGCCAATTTTTCAGGGC-3' for K918N in which Asn was substituted for Lys at position 918; forward primer 5'-GGCGTATGATGGCTCGTAATAAGAAAGCTG-3' and reverse primer 5'-CAGCTTTCTTATTACGAGCCATCATATACGCC-3' for K925N in which Asn was substituted for Lys at position 925; and forward primer 5'-GGCGTATGATGGCTGAGGAGGAGGAGGCTGTTTCTC-3' and reverse primer 5'-GAGAAACAGCCTCCTCCTCCTCAGCCATCATACGCC-3' for RK4E in which 924Arg-Lys-Lys-Lys-927 was substituted for 924Glu-Glu-Glu-Glu-927 at positions of 924 to 927. All constructs mentioned above were confirmed by DNA sequencing.
The cDNAs for the full ORF and two truncated forms of NtCaMK1 (NtCaMK1p865 and NtCaMK1p932) were amplified with Platinum Pfx DNA polymerase and ligated into the EcoRI site of pFastBacHTb that was blunted by Klenow enzyme. After sequence confirmation, recombinant plasmids were transformed into DH10BAC competent cells containing the bacmid with a mini-att Tn7 target site and helper plasmid. The mini Tn7 element on the pFastBacHTb donor plasmid can transpose to the mini-att Tn7 element on the bacmid in the presence of transposition proteins provided by the helper plasmid. Clones containing recombinant bacmid were identified based on the disruption of the lacZ
Insect sf-9 cells were infected with the recombinant virus for 72 h and harvested at room temperature. Cells were washed once with Grace's medium, resuspended in 5 mL of lysis buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol, 1% Nonidet P-40, 0.2 mM phenylmethanesulfonyl fluoride), and sonicated for 30 s, followed by centrifugation at 12,000g for 10 min. The supernatant was applied to Ni-NTA resin column pre-equilibrated with buffer A (50 mM potassium phosphate, pH 6.0, 300 mM KCl, 10% glycerol). After extensive washing with buffer A, followed by buffer A containing 25 mM imidazole, NtCaMK1 was eluted with buffer A containing 200 mM imidazole. The eluted protein was dialyzed against 25 mM Tris-HCl, pH 7.5, containing 2 mM CaCl2 for 6 h and loaded onto a CaM Sepharose-4B (Amersham Pharmacia Biotech, Uppsala) column pre-equilibrated with buffer B (25 mM Tris-HCl, pH 7.5, 2 mM CaCl2). After washing with buffer B plus 200 mM NaCl, NtCaMK1 was eluted with buffer C (25 mM Tris-HCl, pH 7.5, 2 mM EGTA). The purified NtCaMK1 was used immediately for SDS-PAGE and enzymatic analyses. Protein concentration was determined by the method of Bradford (1976)
NtCaMK1 and its truncated forms expressed in Escherichia coli were separated by SDS-PAGE and blotted onto PVDF membrane. For biotinylated CaM-binding assay, the membrane was blocked in 2% BSA/TBS (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 50 mM MgCl2, plus 1 mM CaCl2 or 2 mM EGTA) and washed three times with TBS for 15 min each. After incubation in TBS containing biotinylated CaM for 3 h at room temperature and then washed with TBS, the membrane was treated with avidin-HRP conjugate (Bio-Rad, Hercules, CA) and dissolved in TBS for 1 h. Protein bound to biotinylated CaM was visualized by color development with 4-chloro-1-naphthol and H2O2. For 35S-CaM-binding assay, the proteins were electrophoretically transferred onto nitrocellulose filters and incubated in binding buffer (10 mM Tris-HCl, pH 7.5 ,150 mM NaCl, and 1% [w/v] nonfat dry milk) containing 35S-CaM (0.5 x 106 cpm/µg) plus either 1 mM CaCl2 or 2 mM EGTA as described by Lu and Harrington (1994)
CaM-binding affinity assays were carried out as described (Takezawa et al., 1996
NtCaMK1 autophosphorylation was carried out in 100-µL reaction mixture containing 25 mM Tris-HCl, pH 7.5, 0.5 mM dithiothreitol, 10 mM magnesium acetate, 100 µM ATP, 10 µCi [
Substrate phosphorylation by NtCaMK1 was performed in a 100-µL reaction mixture containing 25 mM Tris-HCl, pH 7.5, 0.5 mM dithiothreitol, 10 mM magnesium acetate, 100 µM ATP, 10 µCi [ For time course assays with histone IIIs as substrate, aliquots for the zero time point were taken immediately after the addition of NtCaMK1 to initiate the reaction and the reaction was terminated by adding one-fifth volume 5x SDS-PAGE sample buffer. Aliquots were separated by SDS-PAGE with a discontinuous 8%/20% separating gel and, after staining with 0.1% Coomassie Brilliant Blue, the gels were vacuum-dried and exposed to x-ray film at –80°C. The amount of phosphates transferred to histone IIIs was determined by counting the radioactivities of the excised histone IIIs bands in a liquid scintillation counter. The experiments were repeated three times in duplicate. For time course assays with syntide-2 as substrate, the experiments with two parallel reactions for different treatments were repeated three times. Aliquots for the zero time point were taken immediately after the addition of NtCaMK1 to initiate the reaction, and the reaction was terminated by adding one-fifth volume 5x SDS-PAGE sample buffer. Aliquots from one reaction were separated by SDS-PAGE with 10% separating gel. The amount of phosphates transferred to the kinase was determined by counting the radioactivities of the excised NtCaMK1 bands in a liquid scintillation counter. Aliquots from another reaction were applied to P81 phosphocellulose filters (2 x 2-cm squares; Whatman, Clifton, NJ) for total 32P incorporation of the kinase and syntide-2. Filters were washed four times for 10 min each in 75 mM phosphoric acid and rinsed in 100% ethanol and air-dried. 32P incorporation was determined by liquid scintillation counting. The amount of phosphates transferred to syntide-2 substrate was determined by subtracting 32P incorporation of NtCaMK1 from total 32P incorporation of NtCaMK1 plus syntide-2.
The reaction for NtCaMK1 substrate phosphorylation was performed as described above. Aliquots (10 µL) were removed and applied to P81 phosphocellulose filters (2 x 2-cm squares; Whatman). Filters were washed four times for 10 min each in 75 mM phosphoric acid, rinsed in 100% ethanol, and air-dried. 32P incorporation was determined by liquid scintillation counting (Beckman LS 6500). The experiments were repeated three times in duplicate.
Unphosphorylated and autophosphorylated NtCaMK1 were hydrolyzed in 6 M HCl for 12 h at 110°C, then dried and dissolved in 20 µL of 10 mM borate buffer (pH 10.0). The hydrolyzed product was mixed with 20 µL of 1 mM fluorescein isothiocyanate (FITC), dissolved in acetone containing 0.05% pyridine (Sigma), and incubated in the dark for 12 h at room temperature. To prepare FITC-tagged standard amino acids and phosphoamino acids, 2 µL standard solution containing each amino acid and phosphoamino acid (0.5 mM each) were mixed with 46 µL of 1 mM FITC, 100 µL of 20 mM borate buffer (pH 10.0), and 52 µL of water. The mixture was incubated in the dark for 12 h at room temperature.
FITC-tagged amino acids were analyzed by capillary electrophoresis as described by Liu et al. (2001) Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers GenBank AF550608 for NtCaMK1; GenBank AY56224 for genomic NtCaMK1.
We thank Dr Y. Ohashi for tobacco calmodulin cDNAs (NtCaM1, NtCaM3, and NtCaM13). Received March 2, 2004; returned for revision March 22, 2004; accepted April 7, 2004.
1 This work was supported in part by the National Natural Science Foundation of China (grant no. 30230050/30170449) and Major State Basic Research Program of China (grant no. 2002CCA00100 to Y.-T.L.), and by a grant from the National Science Foundation to (R.L.J. and Y-T.L.). 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041970. * Corresponding author; e-mail yingtlu{at}whu.edu.cn; fax 86–27–87666380.
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ABSTRACT
RESULTS
ZAPII and poly(A) RNA purified from tobacco cells (cv Wisconsin-38) was screened with 35S-labeled CaM as a ligand probe for the presence of CaM-binding proteins. A candidate recombinant phage was excised in vivo into recombinant pBluescript SK– plasmid pNtCaMK1 and the insert sequenced. 
) or EGTA (
) or Ca2+ (
). EGTA was added to the reaction mixture containing Ca2+/CaM after 30 s of incubation for initiation of NtCaMK1 autophosphorylation to a final concentration of 2 mM (
). The data are averages of three determinations done in duplicate. C, Time course of phosphorylation of histone IIIs by NtCaMK1 in the presence of Ca2+/CaM (
-amidocaproic acid-N-hydroxysuccinimide ester (Sigma, St. Louis) dissolved in N,N-dimethylformamide at a final concentration of 1 mM. The incubation was performed for 2 h at 4°C with constant stirring. The biotinylated CaMs were further dialyzed in 0.1 M phosphate buffer (pH 7.4) for 48 h at 4°C and stored in 20% glycerol at –20°C. The amount of labeled NtCaM1, NtCaM3, and NtCaM13 proteins were measured as described (Bradford, 1976
-32P]ATP (5,000 Ci/mM) plus 1 mM CaCl2 or 1 mM CaCl2/1 µM CaM or 2 mM EGTA at 30°C. For time course assays, 0.2 µg of NtCaMK1 was used in a 100-µL reaction mixture. Aliquots for the zero time point were taken immediately after the addition of NtCaMK1 to initiate the reaction, and the reactions were terminated by adding one-fifth volume 5x SDS-PAGE sample buffer. All aliquots were separated on 10% SDS-PAGE. After staining with 0.1% Coomassie Brilliant Blue, gels were vacuum-dried and exposed to x-ray film at –80°C. The amount of phosphates transferred to the enzyme was determined by counting the radioactivities of the excised NtCaMK1 bands in a liquid scintillation counter (Beckman LS 6500). The experiments were repeated three times in duplicate. 
