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Identification and characterization of protein kinases that interact with the CBL3 calcium sensor in Arabidopsis
Plant Science 169 (2005) 1125–1135 www.elsevier.com/locate/plantsci
Identification and characterization of protein kinases that interact with the CBL3 calcium sensor in Arabidopsis Hye Jin Jeong, Nam-Soo Jwa, Kyung-Nam Kim * Department of Molecular Biology, Sejong University, Seoul 143-747, Republic of Korea Received 4 May 2005; received in revised form 24 June 2005; accepted 18 July 2005 Available online 15 August 2005
Abstract Calcineurine B-like proteins (CBLs) and their interacting protein kinases (CIPKs) in Arabidopsis relay the Ca2+ signals elicited by a variety of stresses. Because distinct CBL–CIPK complexes, respectively, play a role in a different signaling pathway, understanding of the entire network of the CBL–CIPK association is essential to unravel the Ca2+-mediated stress responses. In this study, we have identified 9 CIPKs that interact with the CBL3 Ca2+ sensor using the yeast two-hybrid system. They all contained a highly conserved region, spanning 32 amino acids in length, in the nonkinase domain. Further analyses with CIPK11, one of the CBL3-interacting CIPKs, revealed that the Cterminal region containing the conserved domain was required and sufficient for interaction with CBL3. In vitro interaction assays demonstrated that the CBL3–CIPK11 complex formed in a Ca2+-dependent manner. Expression of the CIPK11 gene was detected in the roots, the shoot apex, the axils of cauline leaves, and the anthers of the flowers. Taken together, our findings suggest that CIPK11 may associate with CBL3 in vivo, thereby mediating the Ca2+ signals downstream. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Calcium; CBL; CIPK; Signaling
1. Introduction Calcium ion (Ca2+) plays a role in diverse cellular and developmental processes, such as guard-cell turgor control, pollen-tube growth, and root-hair elongation [1–3]. In addition, Ca2+ also serves as a second messenger in plant response to a variety of extracellular stimuli, including light, plant hormones, pathogen attack, and abiotic stresses [4–6]. It is an intriguing question how a simple non-protein messenger Ca2+ can mediate such a number of disparate signal transduction pathways, and, thereby allow plants to exhibit a stimulus-specific response. Recent progresses in this subject suggest that the specificity of Ca2+ signaling pathways can be achieved at multiple levels. First, the Ca2+ signal itself is so complex that it can convey diverse information. It is known that the Ca2+ signature is represented not only by the concentration of * Corresponding author. Fax: +82 2 3408 3647. E-mail address: [email protected] (K.-N. Kim).
Ca2+ but also the temporal and spatial parameters, which include frequency, duration, and subcellular localization of the transient increases in the cytosolic Ca2+ concentration [6–8]. Due to such complexity of the Ca2+ parameters, it is possible that different stimuli can generate distinct Ca2+ signatures in the cytoplasm of plant cells. Second, plant cells contain several families of Ca2+binding proteins that sense and interpret the changes in the Ca2+ parameters. To date, three major families of Ca2+ sensors have been identified in plants: the family of calmodulins (CaMs) and CaM-related proteins contains four EF-hand motifs responsible for Ca2+ binding [9,10]. The family of Ca2+-dependent protein kinases (CDPKs) possesses both the Ca2+-binding EF-hand motifs and a protein kinase domain. Therefore, binding of Ca2+ to the EF-hand motifs activates the protein kinase activity of CDPKs [11,12]. Most recently identified is the family of calcineurin B-like (CBL) Ca2+-binding proteins, which is similar to the regulatory B subunit of the protein phosphatase calcineurin in animals.
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.07.016
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Third, each of the Ca2+ sensors interacts with different target proteins that can modulate discrete cellular processes. Because CaMs have no enzymatic activities, they act by interacting with diverse target proteins, including NAD kinase, glutamate decarboxylase, Ca2+-ATPase, and protein kinases [9,13–16]. Such interactions induce changes in three-dimensional structures of their target proteins, which in turn results in alteration of the biochemical activities [9]. In the case of the CDPK family, individual members appear to phosphorylate different substrates in vivo, including sucrose phosphate synthase, nitrate reductase, and ERlocalized Ca2+-ATPase [6,17,18]. The CBL family, which does not have enzymatic activities like CaMs, associates mainly with a group of serine/threonine protein kinases referred to as CIPKs (CBL-interacting protein kinases) [19– 22]. Overall, it is conceivable that specificity in the Ca2+ signal transduction pathways can be largely determined by a specific Ca2+ signature generated by a particular stimulus, and the availability of a distinct set of Ca2+ sensors and their target proteins, which is controlled by expression patterns and subcellular localizations. The CBL–CIPK pathway is a newly emerging Ca2+ signaling system that mediates a diverse array of external stimuli, such as cold, drought, salinity, and ABA [23–26]. In the Arabidopsis genome, 10 CBLs and 25 CIPKs are present and it seems that individual CBL members specially interact with only a few members of the CIPK family [10,20,27,28]. Such interaction specificity makes it possible to produce a number of distinct CBL–CIPK complexes, which can, respectively, play a role in different signaling pathways. In fact, biochemical and genetic analyses demonstrated that CBL4/SOS3 and its associating partner CIPK24/SOS2 are involved in mediating the salt stress-induced Ca2+ signals, thereby rendering salt tolerance [29]. However, little is known about the role of other CBL–CIPK complexes. Currently, we do not even know which CBL–CIPK complex really forms in plant cells. Therefore, in order to fully understand the CBL–CIPK Ca2+ signaling pathways, it is essential to dissect the entire network of the CBL–CIPK association and then unravel the role of the individual CBL–CIPK complexes. In this study, as an effort to gain insight into the interacting networks between CBL-type Ca2+ sensors and their target CIPK proteins, we screened an Arabidopsis cDNA library via a yeast two-hybrid system using CBL3 as bait and identified new CBL3-interacting CIPK proteins.
was carried out as described previously [20]. Briefly, the yeast Y190 cells harboring the pGBT.CBL3 plasmid were transformed with the plasmid library and then plated onto the synthetic medium that lacks leucine, tryptophan, and histidine (SC-Leu-Trp-His) to isolate positive colonies. Plasmids were isolated from the positive colonies and subjected to DNA sequencing. For yeast two-hybrid interaction assays, genes of interest were cloned into the activation domain (pGAD.GH) and the DNA-binding domain (pGBT9.BS) vectors, respectively, and then both plasmids were co-transformed into the yeast strain Y190. Yeast cells carrying both plasmids were selected on the synthetic medium lacking leucine and tryptophan (SC-Leu-Trp). 2.2. Cloning of the full-length CIPK11 cDNA For isolation of the full-length cDNAs encoding CIPK1, Arabidopsis cDNA libraries (CD4-7 and CD4-15) obtained from Arabidopsis Biological Resource Center (Columbus, OH) were screened essentially as described by Kim et al. [20]. Partial CIPK11 cDNA prepared from a CBL3interacting clone was labeled with 32P and used as a probe. Plasmids were isolated from the positive plaques, and their DNA sequences were determined and analyzed with the DNASTAR software (Madison, WI, USA). 2.3. Real-time RT-PCR analysis Real-time quantitative RT-PCR was performed on the Rotor-Gene Real-Time Centrifugal DNA Amplification System (Corbett Research, Australia) using the QuantiTect SYBR Green RT-PCR Kit (Qiagen) as described by the manufacturer. Briefly, total RNAs from plant tissues were isolated using the TRIzol reagent (Invitrogen) and 1 mg of total RNA was served as a template in a 20 mL reaction containing 0.25 mM each primer, 2 QuantiTect SYBR Green RT-PCR Master Mix and QuantiTect RT Mix. Specificity of the amplified transcripts was verified by monitoring melting curves generated after each run. CIPK11 primers (forward, 50 -CGCATTCGCCAAAGTCTTCCACGCA-30 ; reverse, 50 -TTTGATTTTGTCGCCATTACTTCGT-30 ) were designed to produce a 192-bp PCR fragment from the cDNA template. Part of the housekeeping gene Actin2 mRNA, 208 bp in length, was always coamplified by a pair of primers (forward, 50 -TGAGGATATTCAGCCACTTGTCTGT-30 ; reverse, 50 -GATTGGATACTTCAGAGTGAGGATA-30 ) and used as an internal standard.
2. Materials and methods 2.4. Analysis of CIPK11 promoter-GUS expression 2.1. Yeast two-hybrid screening and assays The Arabidopsis l-ACT cDNA expression library (CD422) provided by Arabidopsis Biological Resource Center (Columbus, OH) was used to produce the activation plasmid library by in vivo excision. The yeast two-hybrid screening
The CIPK11 promoter-GUS construct (pBI.CIPK11) was transformed into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis plants by the floral dip method [30]. Transformants were selected as described previously [25]. Histochemical GUS assays of the transgenic
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plants were performed according to the protocol described by Jefferson et al. [31]. 2.5. Assays of b-galactosidase Filter-lift assays for blue color development were performed as described by Breeden and Nasmyth [32]. Activity of b-galactosidase was quantitatively measured according to Kim et al. [20]. 2.6. Purification of GST-fusion proteins Glutathione S-transferase (GST)-fusion proteins were purified essentially according to the protocols described in the GST gene fusion system (Amersham Biosciences). Briefly, E. coli BL21 cells carrying a GST-fusion construct were grown at 37 8C overnight and were subcultured until the OD600 reached 0.5–0.6. Following 3 h induction with 0.3 mM Isopropyl-b-D-thiogalactopyranoside (IPTG) at 20 8C, cells were harvested by centrifugation, resuspended in ice-cold lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM PMSF, 5 mM DTT, 5 mM EDTA, and 1 mM EGTA), and lysed by sonication. Triton X-100 was added to a final concentration of 1%. After 1 h incubation on ice, the cell lysate was centrifuged at 10,000 g for 10 min at 4 8C. Glutathion-Sepharose 4B beads were added to the supernatant and incubated with gentle shaking for 45 min at 4 8C. The beads were washed with six times with ice-cold washing buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl). The GSTfusion protein was eluted with 10 mM glutathione in washing buffer from the beads.
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incubated at 4 8C with prey proteins lacking the GST protein in the binding buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 0.05% Tween 20, 1 mM PMSF) supplemented with either 0.2 mM CaCl2 or 1 mM EGTA. The beads were washed six times with the binding buffer and eluted with 40 ml of 10 mM reduced glutathione. Samples pulled down were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore). The membranes were probed with anti-CBL3 antibody and bound antibodies were detected with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and a chemiluminescence kit (Amersham Biosciences). 2.9. Affinity purification of CBL3 from Arabidopsis total protein extract Total protein preparation and affinity purification were performed as described previously [21]. Briefly, total proteins were prepared from 2-week-old Arabidopsis seedlings overexpressing the CBL3 protein (ecotype Columbia-0) with extraction buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 0.05% Tween 20, 0.2 mM CaCl2, 1 mM PMSF, 5 mg/mL leupeptin, and 5 mg/mL aprotinin). The GST-CIPK11 protein immobilized on the glutathioneSepharose 4B beads was mixed with the total protein extract. After 4h of incubation, the beads were washed six times with the binding buffer described above. The affinitypurified products were subject to immunoblot analysis to detect the Arabidopsis CBL3 protein. 2.10. Construction of plasmids
2.7. Preparation of polyclonal antibodies against CBL3 The CBL3 protein was expressed in E. coli and purified with glutathion-Sepharose 4B beads. After thrombin cleavage to remove the GST protein, the purified CBL3 protein was separated by SDS-PAGE and visualized with Coomassie staining. The CBL3 protein band was carved out, washed extensively with deionized water, and used to immunize a rabbit (New Zealand white/male). For immunization, the rabbit was injected four times with 0.5 mg of the protein at an interval of 2 weeks. Following incubation at 37 8C to inactivate complements, the rabbit whole blood collected from the celiac artery and heart was centrifuged to produce the blood serum. The polyclonal antibodies were purified from the blood antiserum by affinity chromatography using the antigen coupled to cyanogen bromide-activated Sepharose 4B (Amersham Biosciences). 2.8. Pull-down assays and immunoblot analysis Pull-down assays and western blot analysis were performed as described previously [21]. Briefly, GST-fusion proteins attached the glutathion-Sepharose 4B beads were
The pGBT.CBL3 plasmid was constructed as described previously [20]. To create plasmids pGAD.CBL3 and pGEX.CBL3, the coding region of the CBL3 was released from the pGBT.CBL3 by EcoRI/SalI digestion and the resulting restriction fragment was ligated into pGAD.GH and pGEX-4T-3, respectively. To generate plasmids pGBT.CIPK11, pGAD.CIPK11, and pGEX.CIPK11, the coding region of the CIPK11 cDNA was PCR-amplified with a pair of primers CK11-1 and CK11-2. The PCR product was digested with EcoRI/SalI and cloned into pGBT9.BS, pGAD.GH, pGEX-4T-3, respectively. The plasmids pGAD.CIPK11N, which lacks 135 amino acid residues in the C-terminal region, was constructed by cloning the PCR product amplified with CK11-1 and CK11-3 primers into the EcoRI/SalI sites of the pGAD.GH plasmid. Primers CK11-4 and CK11-2 were used to PCR-amplify the C-terminal 135 amino acids of CIPK1. The amplified PCR product was digested with EcoRI and SalI and cloned into pGAD.GH to produce pGAD.CIPK11C. To make the pBI.CIPK11 plasmid, we performed PCR on the Arabidopsis (Col-0) genomic DNA with CK11-P1 and CK11-P2 primers in order to amplify the 50 flanking DNA sequences between 1032 and 1 relative to the translation start codon (ATG) of the
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CIPK11 gene. After digestion with SalI and BamHI, the resulting 997-bp PCR fragment was cloned into the pBI101.1 binary vector (Clontech, Palo Alto, CA). All the PCRs were carried out using Pfu DNA polymerase (Stratagene, La Jolla, CA) to enhance fidelity. All the constructs above were verified by DNA sequencing. 2.11. Oligonucleotide primers used in the plasmid construction Primers used in the plasmid construction were listed below, with restriction enzyme sites underlined. Three additional bases, which were chosen randomly by considering their effect on Tm and on dimer and stem-loop formation, were included at 50 -end of the primers for efficient digestion by restriction enzymes: CK11-1, 50 -TATGAATTCCATGCCAGAGATCGAGATTG30 ; CK11-2, 50 -AATGTCGACTTAAATAGCCGCGTTTGTTG30 ; CK11-3, 50 -ATTGTCGACTGACGATTCCACTTTCTGAT30 ; CK11-4, 50 -TTAGAATTCACTAGAAGCTGTGAAGAGTT- 30 ; CK11-P1, 50 -CGCGTCGACTTGTTCCACCGTTCGGATA30 ; CK11-P2, 50 -ATAGGATCCGATTGATGAATCCAGAGATT-30 .
3. Results 3.1. Identification of new CIPK members that associate with CBL3 We performed yeast two-hybrid screening in order to identify new target proteins of the CBL3 Ca2+ sensor. The complete coding region of CBL3 cDNA was cloned in-frame into the GAL4 DNA binding domain vector (pGBT9.BS) and it was used as a bait to screen the Arabidopsis ACT cDNA expression library. Sequence analysis revealed that a large proportion of the positive clones represented the CIPK family genes. In addition to the previously known CBL3 interactors, such as CIPK1, CIPK2, CIPK3, and CIPK6, this yeast two-hybrid screen has identified new target proteins that include CIPK9, CIPK11, CIPK12, CIPK15, and CIPK21 [27]. As depicted in Fig. 1, alignment of the deduced amino acid sequences of the 5 newly identified CIPK clones showed that they all have, in the N-terminal, the conserved amino acid residues and the 11 subdomains that are characteristic of the Ser/Thr protein kinase domain [33]. They also contained 10 conserved amino acids in the C-terminal regions, which significantly overlap with the NAF domain responsible for interaction with the CBL family proteins [19].
For further analysis, we selected the CIPK11 gene among the CBL3-interacting protein kinases and isolated a fulllength cDNA clone that contains an open reading frame (ORF) of 1308 bp. The ORF encodes a polypeptide of 435 amino acid residues with an estimated molecular mass of 49 kDa and a calculated pI of 8.21. According to information derived from the Arabidopsis genomic sequence database, CIPK11 is a single-copy gene located on chromosome 2. Comparison between the sequences of the cDNA and the genomic region revealed that the CIPK11 gene does not possess any introns. To gain first insights into possible roles of CIPK11 in the plant development, we initially investigated expression patterns of the CIPK11 gene with real-time RT-PCR analysis using total RNAs prepared from the various organs of 5-weekold Arabidopsis wild-type plants (ecotype Col-0). As shown in Fig. 2A, significantly higher levels of CIPK11 transcripts were detected in the stems and roots than in the flowers. In contrast, the CIPK11 mRNA was barely detectable in the leaves. To analyze the expression patterns of the CIPK11 gene in more detail, we created transgenic Arabidopsis plants carrying a chimeric construct, which consists of the putative promoter region of CIPK11 fused to the b-glucuronidase (GUS) reporter gene in pBI101. Histochemical assays of the transgenic plants showed that GUS activity began to appear in the radicles of the 2-day-old seedlings and continued to be detectable in the roots of the mature Arabidopsis plants (Fig. 2B). The hypocotyls of the 1-week-old seedlings also exhibited GUS activity. Other parts that express the GUS protein include the shoot apex, the axils of cauline leaves, and the anthers of the flowers. In contrast, GUS activity was not detected in the germinating seeds, mature siliques, and leaves. 3.2. CIPK11 specifically interacts with CBL3 Because all the CIPK11 clones obtained by the yeast twohybrid screen were not full-length cDNAs, we investigated whether the complete form of CIPK11 can still interact with CBL3. To do this, we created the pGBT.CIPK11 and pGAD.CIPK11 constructs by cloning the complete coding region of CIPK11 cDNA into yeast expression vectors containing a DNA binding domain and an activation domain, respectively. We also produced the pGBT.CBL3 and pGAD.CBL3 plasmids. As shown in Fig. 3, both CIPK11 and CBL3 failed to activate expression of the nutritional reporter gene HIS3 when co-transformed into the yeast strain Y190 with an empty vector. In contrast, the yeast cells carrying either pGBT.CIPK11 and pGAD.CBL3 or pGAD.CIPK11 and pGBT.CBL3 grew well on the selection medium (SC-HLT), indicating expression of the HIS3 gene. The yeast cells also expressed the other reporter gene LacZ, which encodes b-galactosidase, as determined by the filterlift assay. These results indicate that the full-length CIPK11 protein specifically interacts with CBL3 in the yeast twohybrid system and the interaction is independent of the vectors expressing the proteins.
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Fig. 1. Amino acid sequence comparison of CBL3-interacting CIPKs. CIPK9 (GenBank protein identification No. AAK16684), CIPK11 (AAK16686), CIPK12 (AAK16687), CIPK15 (AAK16692), and CIPK21 (AAK59696) were aligned. Residues with black background indicate amino acids conserved in at least three genes, and dashes represent gaps to maximize alignment. Arrowheads and Roman numerals above the sequences indicate the conserved amino acids and subdomains of Ser/Thr protein kinases, respectively. Asterisks indicate amino acids conserved in the C-terminal region of the five CIPKs.
3.3. Interaction domains in CIPK11 and CBL3 To delimit the CIPK11 region necessary for the interaction with CBL3, we generated two deletion constructs, pGAD.CIPK11N and pGAD.CIPK11C, by cloning the kinase and nonkinase domains of CIPK11 into the pGAD.GH vector, respectively. These pGAD constructs were used to transform the yeast strain Y190 carrying the pGBT.CBL3 plasmid. Interactions were determined by monitoring growth of the transformants on the selection medium and by measuring b-galactosidase activity. As depicted in Fig. 4A, CBL3 interacted with fulllength CIPK11 and the C-terminal 136-amino acid region (CIPK11C), but not with the N-terminal 300-amino acid kinase domain (CIPK11N). These results indicate that the C-terminal nonkinase region of the CIPK11 is required and sufficient for the interaction. It is noteworthy that the nonkinase domain alone interacted with the CBL3 protein at much higher strength than the full-length CIPK11 protein, suggesting that the N-terminal kinase domain
harbors inhibitory information hindering the interaction between the CIPK11 C-terminal region and CBL3. Using a series of CBL3 deletion mutants, we also dissected the structural requirement in the CBL3 protein for interaction with CIPK11. Interestingly, any deletions from either the N-terminal or the C-terminal end of the CBL3 protein completely abolished interaction with CIPK11 as shown in Fig. 4B. These findings suggest that the entire amino acid sequence of CBL3 is critical for interaction with CIPK11. 3.4. Different interaction affinity between CIPK11 and CBLs To investigate whether CIPK11 can interact with other members of the CBL family, we transformed the yeast cells carrying the pGAD.CIPK11 plasmid with various CBLs in the DNA-binding domain vector. Interactions between the baits and the prey were determined by monitoring the growth of co-transformed yeasts on the selection medium
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Fig. 2. Expression patterns of the CIPK11 gene. (A) Real-time RT-PCR analysis of CIPK11 transcript levels in different organs of Arabidopsis plants. Total RNA was isolated from various tissues (root, stem, leaf, and flower) of 5-week-old wild-type plants grown under long-day conditions. Real-time RT-PCR was performed with either CIPK11-specific primers or Actin2-specific primers. Bars indicate the relative CIPK11 transcript levels normalized to the housekeeping gene Actin2 transcript levels. Data present means of triplicate samples. (B) Histochemical GUS analysis of CIPK11 promoter-GUS transgenic plants. I, a germinating seed; II, a 2-day-old seedling; III, a 3-day-old seedling; IV, a 1-week-old seedling; V, a 2-week-old plant; VI, a 3-week-old plants; VII, flower buds and cauline leaves; VIII, a flower; IX, a leaf; X, a mature silique.
(SC-HLT). We also performed the b-galactosidase activity assay to measure the interaction strength. Among the CBL family members tested in this study, only CBL4/SOS3 did not interact with CIPK11 as shown in Table 1. Although CBL1 and CBL9 did interact with CIPK11, strength of their
interaction was significantly lower than that of the CBL3CIPK interaction. Such different interaction affinities imply that CIPK11 can preferentially forms a complex with CBL3, thereby being involved in CBL3-mediated calcium signaling pathway in Arabidopsis.
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Fig. 3. CIPK11 interacts with CBL3 in a yeast two-hybrid system. (A) The circle shows the arrangement of the Y190 yeast cells carrying the indicated pGBT and pGAD plasmids. (B) Yeast growth on synthetic complete media lacking leucine and tryptophan (SC-LT). (C) Yeast growth on synthetic complete media lacking histidine, leucine, and tryptophan (SC-HLT). (D) Filter-lift assay showing b-galactosidase activity.
Fig. 4. Identification of interaction domain in CIPK11 and CBL3. (A) The kinase (white box) and nonkinase (black box) domains were, respectively, cloned into the pGAD vector and transformed into the Y190 yeast cells carrying pGBT or pGBT.CBL3. (B) Different regions of CBL3 were, respectively, cloned into the pGBT vector [20] and transformed into the Y190 yeast cells carrying pGAD or pGAD.CIPK11. Yeast growth on the selection media (SC-HLT) was scored as growth (+) and no growth (). Numbers in the parentheses represent units of b-galactosidase activity. Numbers that flank the boxes indicate the beginning and the ending positions of each protein fragment.
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Table 1 Interaction of CIPK1 with CBLs in a yeast two-hybrid assay
Pgbt PGBT.CBL1 PGBT.CBL3 PGBT.CBL4 PGBT.CBL9
pGAD
PGAD.CIPK11
(<1) (<1) (<1) (<1) (<1)
(<1) +(<3.03) +(<45.29) (<1) +(<3.01)
Yeast growth on the selection media (SC-HLT) was scored as growth (+) and no growth (). Numbers in the parentheses represent units of b-galactosidase activity.
3.5. CIPK11 associates with CBL3 in a Ca2+-dependent manner To verify the interaction between the CBL3 calcium sensor and CIPK11 in vitro and to investigate the effect of Ca2+ on the CBL3-CIPK11 complex formation, we expressed and purified both CBL3 and CIPK11 proteins using the glutathion S-transferase (GST) gene fusion system. As shown in Fig. 5, CBL3 and CIPK11 were first expressed as GST fusion forms and subsequently cleaved and purified as 26 kDa and 49 kDa proteins, respectively. The cleaved CBL3 protein was used to produce polyclonal antibody from the rabbit. For the pull-down assay, we mixed GST-CIPK11 (bait) with the cleaved form of CBL3 (prey) in the presence or absence of Ca2+ and checked whether GST beads pulled down the prey using western blot analyses. The anti-CBL3 antibody purified with the CBL3 antigen was used as probe. As shown in Fig. 6, GST-CIPK11 did not pull down the CBL3 protein in the presence of EGTA that chelates Ca2+. The addition of micromolar concentration of Ca2+, however, allowed it to successfully retrieve CBL3. The GST protein, used a negative control, failed to pull down CBL3 regardless of Ca2+. Taken together, these results suggest that the CBL3 Ca2+ sensor physically associates with the CIPK11 protein
Fig. 5. Expression and purification of recombinant CBL3 and CIPK11 proteins. (A) GST-CBL3. (B) GST-CIPK11. Lanes 1–3 contain the GSTfused forms, the thrombin-digested forms, and purified forms, respectively. The proteins were analyzed by SDS-PAGE, and the gels were stained with Coomassie blue. The molecular masses of the proteins are indicated at left in kilodaltons.
Fig. 6. CBL3 physically interacts with CIPK11 in vitro. The GST-CIPK1 fusion protein was used as a bait to pull down the cleaved CBL3 protein in the presence of 1 mM EGTA () or 0.2 mM CaCl2 (+). The GST protein (GST) was used as a negative control. (A) An immunoblot probed with rabbit anti-CBL3 antibody. (B) A Coomassie blue-stained SDS-PAGE gel showing the amount of bait proteins used in each pull-down assay. The molecular masses of the proteins are indicated at right in kilodaltons.
in a Ca2+-dependent manner. This finding supports Shi et al.’s report [21], which demonstrated that CBL1–CIPK1 complex forms only in the presence of Ca2+. Such Ca2+dependent interaction between Ca2+ sensors and their target proteins was also well known in the case of calmodulin and calmodulin-binding proteins [9]. In contrast, Halfter et al. [22] reported that binding of CBL4/SOS3 with CIPK24/ SOS2 itself does not depend on Ca2+, although activation of the CIPK24/SOS2 activity by CBL4/SOS requires Ca2+. 3.6. CIPK11 affinity chromatography purifies CBL3 from Arabiopsis plants It is apparent that CBL3 specifically interacts with CIPK11 according to the results of both the yeast two-hybrid system and pull-down assays. To further confirm the interaction between these two proteins, we performed affinity-chromatography purification in the presence of Ca2+ by using CIPK11 as an affinity reagent to isolate CBL3 from an Arabidopsis total protein extract. As shown in Fig. 7, CIPK11 attached to the GST beads retrieved a 26-kDa
Fig. 7. Affinity purification of CBL3 by GST-CIPK11 from the Arabidopsis total protein extract. (A) An immunoblot probed with rabbit anti-CBL3 antibody. The proteins that were purified by the GST (lane 2) and GSTCIPK11 (lane 3) affinity beads from the plant extract were characterized by western blot analysis using anti-CIPK1 antibody as probe. Total protein extract (5 mg) from the CBL3-overexpressing Arabidopsis plants was included in lane 1 to show the size of the CBL3 protein. (B) A Coomassie blue-stained SDS-PAGE gel. Lane 1 contains 5 mg of total protein extract from the CBL3-overexpressing Arabidopsis plants. Lanes 2 and 3 show the GST and GST-CIPK11 proteins, respectively, which were used as affinity beads. The molecular masses of the proteins are indicated at left and right in kilodaltons.
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protein that was recognized by the anti-CBL3 antibody. The 26-kDa protein band is also found in the plant total protein extract and it is the expected size of CBL3. In contrast, the GST control did not produce any protein bands when the anti-CBL3 antibody was used as probe. These results strongly suggest that the CBL3 and CIPK11 proteins specifically associate in Arabidopsis plants.
4. Discussion Ca2+ plays a pivotal role in a variety of eukaryotic signal transduction pathways as a ubiquitous second messenger. Ca2+ signaling cascades typically begin with Ca2+-binding proteins that sense and transmit the Ca2+ signatures downstream by interacting with other signaling proteins [9]. Recently, a new family of Ca2+ sensor proteins have been identified from Arabidopsis and referred to as CBLs due to the substantial sequence similarity with the regulatory B subunit of calcineurin (CNB) in animals [34,35]. CBLs contain three typical EF-hand motifs that have been shown to bind calcium in vitro [35,36]. Although the animal CNB associates with a protein phosphatase (CNA), the plant CBLs mainly interact with a group of protein kinases called CIPKs or SnRKs [11,19–22]. CIPKs represent a new subclass of protein kinases, because they contain a unique regulatory domain in the C-terminal region, although the kinase domains of CIPKs are most related to those of the yeast SNF1 protein kinase (sucrose nonfermenting 1) and the mammalian AMPdependent protein kinase (AMPK) [35]. Several lines of evidence suggested that the CBL–CIPK complex play an important role in plant responses to a diverse array of external stimuli, including cold, drought, salinity, and ABA [23–26]. GenBank database search indicated that other plant species including monocots and dicots also contain genes, which encode polypeptides homologous to Arabidopsis CBLs or CIPKs. In particular, the rice (Oryza sativa) genome is currently predicted to have 10 CBLs and 30 CIPKs [27]. In fact, we have recently isolated a CIPK-like gene (designated OsCK1) from rice and demonstrated that OsCK1, which is involved in response to diverse signals including cold, light, and salts, interacts with Arabidopsis CBL3 in a yeast two-hybrid system. In addition, OsCK1 prefers Mn2+ to Mg2+ as cofactor like Arabidopsis CIPK1 [21,37]. Together, these findings suggest the CBL–CIPK Ca2+-signaling networks originally found in Arabdidopsis are probably also present in other plants. As mentioned above, the Arabidopsis genome appears to contain 10 CBLs and 25 CIPKs. Judging from the number of genes in the CBL and CIPK families, it is likely that some CBLs would have more than one interacting CIPK partner. In fact, Kolukisaoglu et al. [27] demonstrated that CBL1 and CBL9, respectively, interact with multiple CIPKs in the yeast two-hybrid system. Because an individual component of the CBL–CIPK complexes functions in a specific stress-signaling pathway [23–26,29], it is important to figure out the entire
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CBL–CIPK interacting networks which has not been fully established yet. As a step toward this goal, we sought to identify CIPKs that associate with the CBL3 protein in the present study. We have screened a yeast two-hybrid library using CBL3 as bait and identified nine CIPK family members that include CIPK1, CIPK2, CIPK3, CIPK6, CIPK9, CIPK11, CIPK12, CIPK15, and CIPK21. Our detailed analysis of the interaction between a pair of partner proteins, CBL3 and CIPK11, has demonstrated these two proteins physically interact in a Ca2+dependent manner (Fig. 6). This result is consistent with the previous finding that CBL1, another CBL family member, associates with CIPK1 only in the presence of Ca2+ [21]. Recently, the crystal structure analysis of CBL2, almost identical to CBL3 (92% identity), indicated that conformation of Ca2+-bound CBL2 was markedly different from the unbound form [38]. Taken together, these findings suggest that Ca2+-bound CBLs undergoes conformational change to associate with the target CIPKs, thereby modulating their kinase activity. We also investigated the structural basis for the interaction between CIPK11 and CBL3. Our deletion analysis indicated that the C-terminal region of CIPK11 (CIPK11C) is involved in the actual interaction with CBL3 and the kinase domain (CIPK11N) has a negative effect on the interaction (Fig. 4A). These results suggest that both the kinase and nonkinase domains exert their effect on specifying the final interaction affinity with CBL3. Regarding the structural requirement of CBL3, we found that the interaction with CIPK11 was completely abolished by removing short sequences from either the N- or C-terminal end of CBL3 (Fig. 4B). This finding suggests that both terminal sequences, most variable regions in the CBL family members, are necessary to interact with CIPK11. Considering the three conserved EF-hand motifs located in the center of CBL members, we reason that the diverse terminal sequences may play a critical role in determining the interaction affinity toward CIPKs and other target proteins. Our reasoning could explain why CBL5, CBL7, and CBL8, which have terminal sequences totally different from CIPK-associating CBLs, such as CBL1, CBL2, CBL3, CBL4, and CBL9, did not interact with any of the CIPKs tested in the study [20,27]. Although we cannot completely rule out the possibility that a group of unidentified CIPKs serve as targets for these CBL members, it is more likely that CBL5, CBL7, and CBL8 may have protein targets other than the CIPK family members. In this study, we identified nine CBL3-interacting CIPK members as potential CBL3 targets, supporting the previous finding that each CBL family member can have multiple CIPK partners [27]. Our study also suggests that each of the CIPK family members has the ability to interact with several CBLs, because three CBL family members including CBL1, CBL3, and CBL9 interacted, respectively, with CIPK11 in the yeast two-hybrid system (Table 1). However, their interaction affinities toward CIPK11 were significantly different among the three CBL isoforms. CIPK11 interacted
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with CBL3 at much higher strength than with CBL1 and CBL9, implying that CBL3 will out-compete CBL1 and CBL9 to produce the CBL3–CIPK11 complex. Therefore, it is likely that such different interaction affinities will contribute in part to determining final parings between the CBL and CIPK family members in plant cells. In addition, we speculate about other factors that can influence formation of a specific CBL–CIPK complex in plant cells. First, the temporal and spatial expression patterns of CBLs and CIPKs may vary so that different cells at any given time may have a distinct pattern of CBL–CIPK complexes. For example, the expression level of CBL1 dramatically increased in response to stressful conditions, such as cold, wounding, and drought, although other CBL family members, including CBL2, CBL3, and CBL4/SOS3, did not respond [35]. Similarly, the CIPK family members also exhibited different expression patterns. Both CIPK1 and CIPK3 were expressed in all organs including roots, leaves, stems, and flowers [21,25], whereas CIPK11 was not expressed in the flowers. Besides the difference in the spatial expression pattern, CIPK1 and CIPK3 differently responded to the stimuli, such as cold, NaCl, ABA, wounding, and drought. Although the stresses activated CIPK3 expression, they did not change the level of CIPK1 expression [21,25]. Second, different subcellular localization of CBLs and CIPKs may also play a role in generating specific CBL– CIPK complexes in plant cells. Among the 10 CBLs in the Arabidopsis genome, four CBLs that include CBL1, CBL4/ SOS3, CBL5, and CBL9 are known to possess the conserved N-myristoylation motif (MGXXXS/T) that usually targets proteins to cell membrane [27]. The other members lacking the N-myristoylation motif appear to lack a mechanism to become membrane associated. Ishitani et al. [39] demonstrated the importance of the N-myristoylation motif by showing that CBL4/SOS3 must have the N-myristoylation motif intact in order to function normally in the salt tolerant response. Unfortunately, however, little is known about subcellular localization of the CIPK family members to date.
Acknowledgements This work was supported by grants from the Plant Signaling Network Research Center, Korea Science and Engineering Foundation, and the BioGreen21 Program of the Rural Development Administration. It was also supported in part by the Special Grant Research Program in the Ministry of Agriculture and Forestry of the Korean Government.
[3]
[4] [5] [6] [7] [8] [9] [10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
References [1] M.R. Blatt, Cellular signaling and volume control in stomatal movements in plants, Annu. Rev. Cell Dev. Biol. 16 (2000) 221–241. [2] T.L. Holdaway-Clarke, J.A. Feijo, G.R. Hackett, J.G. Kunkel, P.K. Hepler, Pollen tube growth and the intracellular cytosolic calcium
[23]
[24]
gradient oscillate in phase while extracellular calcium influx is delayed, Plant Cell 9 (1997) 1999–2010. C.L. Wymer, T.N. Bibikova, S. Gilroy, Cytoplasmic free calcium distributions during the development of root hairs of Arabidopsis thaliana, Plant J. 12 (1997) 427–439. D.W. Ehrhardt, R. Wais, S.R. Long, Calcium spiking in plant root hairs responding to Rhizobium nodulation signals, Cell 85 (1996) 673–681. D. Sanders, C. Brownlee, J.F. Harper, Communicating with calcium, Plant Cell 11 (1999) 691–706. J.J. Rudd, V.E. Franklin-Tong, Unravelling response-specificity in Ca2+ signalling pathways in plant cells, New Phytol. 151 (2001) 7–33. D. Sanders, J. Pelloux, C. Brownlee, J.F. Harper, Calcium at the crossroads of signaling, Plant Cell 14 (Suppl.) (2002) S401–S417. N.H. Evans, M.R. McAinsh, A.M. Hetherington, Calcium oscillations in higher plants, Curr. Opin. Plant Biol. 4 (2001) 415–420. R.E. Zielinski, Calmodulin and calmodulin-binding proteins in plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 697–725. S. Luan, J. Kudla, M. Rodriguez-Concepcion, S. Yalovsky, W. Gruissem, Calmodulins and calcineurin B-like proteins: calcium sensors for specific signal response coupling in plants, Plant Cell 14 (Suppl.) (2002) S389–S400. E.M. Hrabak, C.W. Chan, M. Gribskov, J.F. Harper, J.H. Choi, N. Halford, J. Kudla, S. Luan, H.G. Nimmo, M.R. Sussman, M. Thomas, K. Walker-Simmons, J.K. Zhu, A.C. Harmon, The Arabidopsis CDPK-SnRK superfamily of protein kinases, Plant Physiol. 132 (2003) 666–680. S.H. Cheng, M.R. Willmann, H.C. Chen, J. Sheen, Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family, Plant Physiol. 129 (2002) 469–485. V.S. Reddy, G.S. Ali, A.S. Reddy, Genes encoding calmodulin-binding proteins in the Arabidopsis genome, J. Biol. Chem. 277 (2002) 9840– 9852. W.A. Snedden, H. Fromm, Calmodulin as a versatile calcium signal transducers in plants, New Phytol. 151 (2001) 35–66. W.A. Snedden, N. Koutsia, G. Baum, H. Fromm, Activation of a recombinant petunia glutamate decarboxylase by calcium/calmodulin or by a monoclonal antibody which recognizes the calmodulin binding domain, J. Biol. Chem. 271 (1996) 4148–4153. W.A. Snedden, H. Fromm, Calmodulin, calmodulin-related proteins and plant responses to the environment, Trends Plant Sci. 3 (1998) 299–304. P. Douglas, G. Moorhead, Y. Hong, N. Morrice, C. MacKintosh, Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase, Planta 206 (1998) 435–442. I. Hwang, H. Sze, J.F. Harper, A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis, Proc. Natl. Acad. Sci. 97 (2000) 6224–6229. V. Albrecht, O. Ritz, S. Linder, K. Harter, J. Kudla, The NAF domain defines a novel protein–protein interaction module conserved in Ca2+regulated kinases, EMBO J. 20 (2001) 1051–1063. K.N. Kim, Y.H. Cheong, R. Gupta, S. Luan, Interaction specificity of Arabidopsis calcineurin B-like calcium sensors and their target kinases, Plant Physiol. 124 (2000) 1844–1853. J. Shi, K.N. Kim, O. Ritz, V. Albrecht, R. Gupta, K. Harter, S. Luan, J. Kudla, Novel protein kinases associated with calcineurin B-like calcium sensors in Arabidopsis, Plant Cell 11 (1999) 2393–2405. U. Halfter, M. Ishitani, J.K. Zhu, The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3, Proc. Natl. Acad. Sci. 97 (2000) 3735–3740. Y.H. Cheong, K.N. Kim, G.K. Pandey, R. Gupta, J.J. Grant, S. Luan, CBL1, a calcium sensor that differentially regulates salt, drought, and cold responses in Arabidopsis, Plant Cell 15 (2003) 1833–1845. Y. Guo, Q.S. Qiu, F.J. Quintero, J.M. Pardo, M. Ohta, C. Zhang, K.S. Schumaker, J.K. Zhu, Transgenic evaluation of activated mutant alleles of SOS2 reveals a critical requirement for its kinase activity
H.J. Jeong et al. / Plant Science 169 (2005) 1125–1135
[25]
[26]
[27]
[28]
[29] [30]
[31]
and C-terminal regulatory domain for salt tolerance in Arabidopsis thaliana, Plant Cell 16 (2004) 435–449. K.N. Kim, Y.H. Cheong, J.J. Grant, G.K. Pandey, S. Luan, CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis, Plant Cell 15 (2003) 411–423. G.K. Pandey, Y.H. Cheong, K.N. Kim, J.J. Grant, L. Li, W. Hung, C. D’Angelo, S. Weinl, J. Kudla, S. Luan, The calcium sensor calcineurin B-like 9 modulates abscisic acid sensitivity and biosynthesis in Arabidopsis, Plant Cell 16 (2004) 1912–1924. U. Kolukisaoglu, S. Weinl, D. Blazevic, O. Batistic, J. Kudla, Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and rice CBL-CIPK signaling networks, Plant Physiol. 134 (2004) 43–58. O. Batistic, J. Kudla, Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network, Planta 219 (2004) 915–924. J.K. Zhu, Salt and drought stress signal transduction in plants, Annu. Rev. Plant Biol. 53 (2002) 247–273. S.J. Clough, A.F. Bent, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16 (1998) 735–743. R.A. Jefferson, T.A. Kavanagh, M.W. Bevan, GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants, EMBO J. 6 (1987) 3901–3907.
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[32] L. Breeden, K. Nasmyth, Regulation of the yeast HO gene, Cold Spring Harb. Symp. Quant. Biol. 50 (1985) 643–650. [33] S.K. Hanks, A.M. Quinn, Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members, Methods Enzymol. 200 (1991) 38–62. [34] J. Liu, J.K. Zhu, A calcium sensor homolog required for plant salt tolerance, Science 280 (1998) 1943–1945. [35] J. Kudla, Q. Xu, K. Harter, W. Gruissem, S. Luan, Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals, Proc. Natl. Acad. Sci. 96 (1999) 4718–4723. [36] M. Nagae, A. Nozawa, N. Koizumi, H. Sano, H. Hashimoto, M. Sato, T. Shimizu, Crystallization and preliminary X-ray characterization of a novel calcium-binding protein AtCBL2 from Arabidopsis thaliana, Acta Crystallogr. D Biol. Crystallogr. 59 (2003) 1079–1080. [37] K.N. Kim, J.S. Lee, H. Han, S.A. Choi, S.J. Go, I.S. Yoon, Isolation and characterization of a novel rice Ca2+-regulated protein kinase gene involved in responses to diverse signals including cold, light, cytokinins, sugars and salts, Plant Mol. Biol. 52 (2003) 1191–1202. [38] M. Nagae, A. Nozawa, N. Koizumi, H. Sano, H. Hashimoto, M. Sato, T. Shimizu, The crystal structure of the novel calcium-binding protein AtCBL2 from Arabidopsis thaliana, J. Biol. Chem. 278 (2003) 42240– 42246. [39] M. Ishitani, J. Liu, U. Halfter, C.S. Kim, W. Shi, J.K. Zhu, SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding, Plant Cell 12 (2000) 1667–1678.
Identification and characterization of protein kinases that interact with the CBL3 calcium sensor in Arabidopsis Hye Jin Jeong, Nam-Soo Jwa, Kyung-Nam Kim * Department of Molecular Biology, Sejong University, Seoul 143-747, Republic of Korea Received 4 May 2005; received in revised form 24 June 2005; accepted 18 July 2005 Available online 15 August 2005
Abstract Calcineurine B-like proteins (CBLs) and their interacting protein kinases (CIPKs) in Arabidopsis relay the Ca2+ signals elicited by a variety of stresses. Because distinct CBL–CIPK complexes, respectively, play a role in a different signaling pathway, understanding of the entire network of the CBL–CIPK association is essential to unravel the Ca2+-mediated stress responses. In this study, we have identified 9 CIPKs that interact with the CBL3 Ca2+ sensor using the yeast two-hybrid system. They all contained a highly conserved region, spanning 32 amino acids in length, in the nonkinase domain. Further analyses with CIPK11, one of the CBL3-interacting CIPKs, revealed that the Cterminal region containing the conserved domain was required and sufficient for interaction with CBL3. In vitro interaction assays demonstrated that the CBL3–CIPK11 complex formed in a Ca2+-dependent manner. Expression of the CIPK11 gene was detected in the roots, the shoot apex, the axils of cauline leaves, and the anthers of the flowers. Taken together, our findings suggest that CIPK11 may associate with CBL3 in vivo, thereby mediating the Ca2+ signals downstream. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Calcium; CBL; CIPK; Signaling
1. Introduction Calcium ion (Ca2+) plays a role in diverse cellular and developmental processes, such as guard-cell turgor control, pollen-tube growth, and root-hair elongation [1–3]. In addition, Ca2+ also serves as a second messenger in plant response to a variety of extracellular stimuli, including light, plant hormones, pathogen attack, and abiotic stresses [4–6]. It is an intriguing question how a simple non-protein messenger Ca2+ can mediate such a number of disparate signal transduction pathways, and, thereby allow plants to exhibit a stimulus-specific response. Recent progresses in this subject suggest that the specificity of Ca2+ signaling pathways can be achieved at multiple levels. First, the Ca2+ signal itself is so complex that it can convey diverse information. It is known that the Ca2+ signature is represented not only by the concentration of * Corresponding author. Fax: +82 2 3408 3647. E-mail address: [email protected] (K.-N. Kim).
Ca2+ but also the temporal and spatial parameters, which include frequency, duration, and subcellular localization of the transient increases in the cytosolic Ca2+ concentration [6–8]. Due to such complexity of the Ca2+ parameters, it is possible that different stimuli can generate distinct Ca2+ signatures in the cytoplasm of plant cells. Second, plant cells contain several families of Ca2+binding proteins that sense and interpret the changes in the Ca2+ parameters. To date, three major families of Ca2+ sensors have been identified in plants: the family of calmodulins (CaMs) and CaM-related proteins contains four EF-hand motifs responsible for Ca2+ binding [9,10]. The family of Ca2+-dependent protein kinases (CDPKs) possesses both the Ca2+-binding EF-hand motifs and a protein kinase domain. Therefore, binding of Ca2+ to the EF-hand motifs activates the protein kinase activity of CDPKs [11,12]. Most recently identified is the family of calcineurin B-like (CBL) Ca2+-binding proteins, which is similar to the regulatory B subunit of the protein phosphatase calcineurin in animals.
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.07.016
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Third, each of the Ca2+ sensors interacts with different target proteins that can modulate discrete cellular processes. Because CaMs have no enzymatic activities, they act by interacting with diverse target proteins, including NAD kinase, glutamate decarboxylase, Ca2+-ATPase, and protein kinases [9,13–16]. Such interactions induce changes in three-dimensional structures of their target proteins, which in turn results in alteration of the biochemical activities [9]. In the case of the CDPK family, individual members appear to phosphorylate different substrates in vivo, including sucrose phosphate synthase, nitrate reductase, and ERlocalized Ca2+-ATPase [6,17,18]. The CBL family, which does not have enzymatic activities like CaMs, associates mainly with a group of serine/threonine protein kinases referred to as CIPKs (CBL-interacting protein kinases) [19– 22]. Overall, it is conceivable that specificity in the Ca2+ signal transduction pathways can be largely determined by a specific Ca2+ signature generated by a particular stimulus, and the availability of a distinct set of Ca2+ sensors and their target proteins, which is controlled by expression patterns and subcellular localizations. The CBL–CIPK pathway is a newly emerging Ca2+ signaling system that mediates a diverse array of external stimuli, such as cold, drought, salinity, and ABA [23–26]. In the Arabidopsis genome, 10 CBLs and 25 CIPKs are present and it seems that individual CBL members specially interact with only a few members of the CIPK family [10,20,27,28]. Such interaction specificity makes it possible to produce a number of distinct CBL–CIPK complexes, which can, respectively, play a role in different signaling pathways. In fact, biochemical and genetic analyses demonstrated that CBL4/SOS3 and its associating partner CIPK24/SOS2 are involved in mediating the salt stress-induced Ca2+ signals, thereby rendering salt tolerance [29]. However, little is known about the role of other CBL–CIPK complexes. Currently, we do not even know which CBL–CIPK complex really forms in plant cells. Therefore, in order to fully understand the CBL–CIPK Ca2+ signaling pathways, it is essential to dissect the entire network of the CBL–CIPK association and then unravel the role of the individual CBL–CIPK complexes. In this study, as an effort to gain insight into the interacting networks between CBL-type Ca2+ sensors and their target CIPK proteins, we screened an Arabidopsis cDNA library via a yeast two-hybrid system using CBL3 as bait and identified new CBL3-interacting CIPK proteins.
was carried out as described previously [20]. Briefly, the yeast Y190 cells harboring the pGBT.CBL3 plasmid were transformed with the plasmid library and then plated onto the synthetic medium that lacks leucine, tryptophan, and histidine (SC-Leu-Trp-His) to isolate positive colonies. Plasmids were isolated from the positive colonies and subjected to DNA sequencing. For yeast two-hybrid interaction assays, genes of interest were cloned into the activation domain (pGAD.GH) and the DNA-binding domain (pGBT9.BS) vectors, respectively, and then both plasmids were co-transformed into the yeast strain Y190. Yeast cells carrying both plasmids were selected on the synthetic medium lacking leucine and tryptophan (SC-Leu-Trp). 2.2. Cloning of the full-length CIPK11 cDNA For isolation of the full-length cDNAs encoding CIPK1, Arabidopsis cDNA libraries (CD4-7 and CD4-15) obtained from Arabidopsis Biological Resource Center (Columbus, OH) were screened essentially as described by Kim et al. [20]. Partial CIPK11 cDNA prepared from a CBL3interacting clone was labeled with 32P and used as a probe. Plasmids were isolated from the positive plaques, and their DNA sequences were determined and analyzed with the DNASTAR software (Madison, WI, USA). 2.3. Real-time RT-PCR analysis Real-time quantitative RT-PCR was performed on the Rotor-Gene Real-Time Centrifugal DNA Amplification System (Corbett Research, Australia) using the QuantiTect SYBR Green RT-PCR Kit (Qiagen) as described by the manufacturer. Briefly, total RNAs from plant tissues were isolated using the TRIzol reagent (Invitrogen) and 1 mg of total RNA was served as a template in a 20 mL reaction containing 0.25 mM each primer, 2 QuantiTect SYBR Green RT-PCR Master Mix and QuantiTect RT Mix. Specificity of the amplified transcripts was verified by monitoring melting curves generated after each run. CIPK11 primers (forward, 50 -CGCATTCGCCAAAGTCTTCCACGCA-30 ; reverse, 50 -TTTGATTTTGTCGCCATTACTTCGT-30 ) were designed to produce a 192-bp PCR fragment from the cDNA template. Part of the housekeeping gene Actin2 mRNA, 208 bp in length, was always coamplified by a pair of primers (forward, 50 -TGAGGATATTCAGCCACTTGTCTGT-30 ; reverse, 50 -GATTGGATACTTCAGAGTGAGGATA-30 ) and used as an internal standard.
2. Materials and methods 2.4. Analysis of CIPK11 promoter-GUS expression 2.1. Yeast two-hybrid screening and assays The Arabidopsis l-ACT cDNA expression library (CD422) provided by Arabidopsis Biological Resource Center (Columbus, OH) was used to produce the activation plasmid library by in vivo excision. The yeast two-hybrid screening
The CIPK11 promoter-GUS construct (pBI.CIPK11) was transformed into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis plants by the floral dip method [30]. Transformants were selected as described previously [25]. Histochemical GUS assays of the transgenic
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plants were performed according to the protocol described by Jefferson et al. [31]. 2.5. Assays of b-galactosidase Filter-lift assays for blue color development were performed as described by Breeden and Nasmyth [32]. Activity of b-galactosidase was quantitatively measured according to Kim et al. [20]. 2.6. Purification of GST-fusion proteins Glutathione S-transferase (GST)-fusion proteins were purified essentially according to the protocols described in the GST gene fusion system (Amersham Biosciences). Briefly, E. coli BL21 cells carrying a GST-fusion construct were grown at 37 8C overnight and were subcultured until the OD600 reached 0.5–0.6. Following 3 h induction with 0.3 mM Isopropyl-b-D-thiogalactopyranoside (IPTG) at 20 8C, cells were harvested by centrifugation, resuspended in ice-cold lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM PMSF, 5 mM DTT, 5 mM EDTA, and 1 mM EGTA), and lysed by sonication. Triton X-100 was added to a final concentration of 1%. After 1 h incubation on ice, the cell lysate was centrifuged at 10,000 g for 10 min at 4 8C. Glutathion-Sepharose 4B beads were added to the supernatant and incubated with gentle shaking for 45 min at 4 8C. The beads were washed with six times with ice-cold washing buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl). The GSTfusion protein was eluted with 10 mM glutathione in washing buffer from the beads.
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incubated at 4 8C with prey proteins lacking the GST protein in the binding buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 0.05% Tween 20, 1 mM PMSF) supplemented with either 0.2 mM CaCl2 or 1 mM EGTA. The beads were washed six times with the binding buffer and eluted with 40 ml of 10 mM reduced glutathione. Samples pulled down were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore). The membranes were probed with anti-CBL3 antibody and bound antibodies were detected with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and a chemiluminescence kit (Amersham Biosciences). 2.9. Affinity purification of CBL3 from Arabidopsis total protein extract Total protein preparation and affinity purification were performed as described previously [21]. Briefly, total proteins were prepared from 2-week-old Arabidopsis seedlings overexpressing the CBL3 protein (ecotype Columbia-0) with extraction buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 0.05% Tween 20, 0.2 mM CaCl2, 1 mM PMSF, 5 mg/mL leupeptin, and 5 mg/mL aprotinin). The GST-CIPK11 protein immobilized on the glutathioneSepharose 4B beads was mixed with the total protein extract. After 4h of incubation, the beads were washed six times with the binding buffer described above. The affinitypurified products were subject to immunoblot analysis to detect the Arabidopsis CBL3 protein. 2.10. Construction of plasmids
2.7. Preparation of polyclonal antibodies against CBL3 The CBL3 protein was expressed in E. coli and purified with glutathion-Sepharose 4B beads. After thrombin cleavage to remove the GST protein, the purified CBL3 protein was separated by SDS-PAGE and visualized with Coomassie staining. The CBL3 protein band was carved out, washed extensively with deionized water, and used to immunize a rabbit (New Zealand white/male). For immunization, the rabbit was injected four times with 0.5 mg of the protein at an interval of 2 weeks. Following incubation at 37 8C to inactivate complements, the rabbit whole blood collected from the celiac artery and heart was centrifuged to produce the blood serum. The polyclonal antibodies were purified from the blood antiserum by affinity chromatography using the antigen coupled to cyanogen bromide-activated Sepharose 4B (Amersham Biosciences). 2.8. Pull-down assays and immunoblot analysis Pull-down assays and western blot analysis were performed as described previously [21]. Briefly, GST-fusion proteins attached the glutathion-Sepharose 4B beads were
The pGBT.CBL3 plasmid was constructed as described previously [20]. To create plasmids pGAD.CBL3 and pGEX.CBL3, the coding region of the CBL3 was released from the pGBT.CBL3 by EcoRI/SalI digestion and the resulting restriction fragment was ligated into pGAD.GH and pGEX-4T-3, respectively. To generate plasmids pGBT.CIPK11, pGAD.CIPK11, and pGEX.CIPK11, the coding region of the CIPK11 cDNA was PCR-amplified with a pair of primers CK11-1 and CK11-2. The PCR product was digested with EcoRI/SalI and cloned into pGBT9.BS, pGAD.GH, pGEX-4T-3, respectively. The plasmids pGAD.CIPK11N, which lacks 135 amino acid residues in the C-terminal region, was constructed by cloning the PCR product amplified with CK11-1 and CK11-3 primers into the EcoRI/SalI sites of the pGAD.GH plasmid. Primers CK11-4 and CK11-2 were used to PCR-amplify the C-terminal 135 amino acids of CIPK1. The amplified PCR product was digested with EcoRI and SalI and cloned into pGAD.GH to produce pGAD.CIPK11C. To make the pBI.CIPK11 plasmid, we performed PCR on the Arabidopsis (Col-0) genomic DNA with CK11-P1 and CK11-P2 primers in order to amplify the 50 flanking DNA sequences between 1032 and 1 relative to the translation start codon (ATG) of the
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CIPK11 gene. After digestion with SalI and BamHI, the resulting 997-bp PCR fragment was cloned into the pBI101.1 binary vector (Clontech, Palo Alto, CA). All the PCRs were carried out using Pfu DNA polymerase (Stratagene, La Jolla, CA) to enhance fidelity. All the constructs above were verified by DNA sequencing. 2.11. Oligonucleotide primers used in the plasmid construction Primers used in the plasmid construction were listed below, with restriction enzyme sites underlined. Three additional bases, which were chosen randomly by considering their effect on Tm and on dimer and stem-loop formation, were included at 50 -end of the primers for efficient digestion by restriction enzymes: CK11-1, 50 -TATGAATTCCATGCCAGAGATCGAGATTG30 ; CK11-2, 50 -AATGTCGACTTAAATAGCCGCGTTTGTTG30 ; CK11-3, 50 -ATTGTCGACTGACGATTCCACTTTCTGAT30 ; CK11-4, 50 -TTAGAATTCACTAGAAGCTGTGAAGAGTT- 30 ; CK11-P1, 50 -CGCGTCGACTTGTTCCACCGTTCGGATA30 ; CK11-P2, 50 -ATAGGATCCGATTGATGAATCCAGAGATT-30 .
3. Results 3.1. Identification of new CIPK members that associate with CBL3 We performed yeast two-hybrid screening in order to identify new target proteins of the CBL3 Ca2+ sensor. The complete coding region of CBL3 cDNA was cloned in-frame into the GAL4 DNA binding domain vector (pGBT9.BS) and it was used as a bait to screen the Arabidopsis ACT cDNA expression library. Sequence analysis revealed that a large proportion of the positive clones represented the CIPK family genes. In addition to the previously known CBL3 interactors, such as CIPK1, CIPK2, CIPK3, and CIPK6, this yeast two-hybrid screen has identified new target proteins that include CIPK9, CIPK11, CIPK12, CIPK15, and CIPK21 [27]. As depicted in Fig. 1, alignment of the deduced amino acid sequences of the 5 newly identified CIPK clones showed that they all have, in the N-terminal, the conserved amino acid residues and the 11 subdomains that are characteristic of the Ser/Thr protein kinase domain [33]. They also contained 10 conserved amino acids in the C-terminal regions, which significantly overlap with the NAF domain responsible for interaction with the CBL family proteins [19].
For further analysis, we selected the CIPK11 gene among the CBL3-interacting protein kinases and isolated a fulllength cDNA clone that contains an open reading frame (ORF) of 1308 bp. The ORF encodes a polypeptide of 435 amino acid residues with an estimated molecular mass of 49 kDa and a calculated pI of 8.21. According to information derived from the Arabidopsis genomic sequence database, CIPK11 is a single-copy gene located on chromosome 2. Comparison between the sequences of the cDNA and the genomic region revealed that the CIPK11 gene does not possess any introns. To gain first insights into possible roles of CIPK11 in the plant development, we initially investigated expression patterns of the CIPK11 gene with real-time RT-PCR analysis using total RNAs prepared from the various organs of 5-weekold Arabidopsis wild-type plants (ecotype Col-0). As shown in Fig. 2A, significantly higher levels of CIPK11 transcripts were detected in the stems and roots than in the flowers. In contrast, the CIPK11 mRNA was barely detectable in the leaves. To analyze the expression patterns of the CIPK11 gene in more detail, we created transgenic Arabidopsis plants carrying a chimeric construct, which consists of the putative promoter region of CIPK11 fused to the b-glucuronidase (GUS) reporter gene in pBI101. Histochemical assays of the transgenic plants showed that GUS activity began to appear in the radicles of the 2-day-old seedlings and continued to be detectable in the roots of the mature Arabidopsis plants (Fig. 2B). The hypocotyls of the 1-week-old seedlings also exhibited GUS activity. Other parts that express the GUS protein include the shoot apex, the axils of cauline leaves, and the anthers of the flowers. In contrast, GUS activity was not detected in the germinating seeds, mature siliques, and leaves. 3.2. CIPK11 specifically interacts with CBL3 Because all the CIPK11 clones obtained by the yeast twohybrid screen were not full-length cDNAs, we investigated whether the complete form of CIPK11 can still interact with CBL3. To do this, we created the pGBT.CIPK11 and pGAD.CIPK11 constructs by cloning the complete coding region of CIPK11 cDNA into yeast expression vectors containing a DNA binding domain and an activation domain, respectively. We also produced the pGBT.CBL3 and pGAD.CBL3 plasmids. As shown in Fig. 3, both CIPK11 and CBL3 failed to activate expression of the nutritional reporter gene HIS3 when co-transformed into the yeast strain Y190 with an empty vector. In contrast, the yeast cells carrying either pGBT.CIPK11 and pGAD.CBL3 or pGAD.CIPK11 and pGBT.CBL3 grew well on the selection medium (SC-HLT), indicating expression of the HIS3 gene. The yeast cells also expressed the other reporter gene LacZ, which encodes b-galactosidase, as determined by the filterlift assay. These results indicate that the full-length CIPK11 protein specifically interacts with CBL3 in the yeast twohybrid system and the interaction is independent of the vectors expressing the proteins.
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Fig. 1. Amino acid sequence comparison of CBL3-interacting CIPKs. CIPK9 (GenBank protein identification No. AAK16684), CIPK11 (AAK16686), CIPK12 (AAK16687), CIPK15 (AAK16692), and CIPK21 (AAK59696) were aligned. Residues with black background indicate amino acids conserved in at least three genes, and dashes represent gaps to maximize alignment. Arrowheads and Roman numerals above the sequences indicate the conserved amino acids and subdomains of Ser/Thr protein kinases, respectively. Asterisks indicate amino acids conserved in the C-terminal region of the five CIPKs.
3.3. Interaction domains in CIPK11 and CBL3 To delimit the CIPK11 region necessary for the interaction with CBL3, we generated two deletion constructs, pGAD.CIPK11N and pGAD.CIPK11C, by cloning the kinase and nonkinase domains of CIPK11 into the pGAD.GH vector, respectively. These pGAD constructs were used to transform the yeast strain Y190 carrying the pGBT.CBL3 plasmid. Interactions were determined by monitoring growth of the transformants on the selection medium and by measuring b-galactosidase activity. As depicted in Fig. 4A, CBL3 interacted with fulllength CIPK11 and the C-terminal 136-amino acid region (CIPK11C), but not with the N-terminal 300-amino acid kinase domain (CIPK11N). These results indicate that the C-terminal nonkinase region of the CIPK11 is required and sufficient for the interaction. It is noteworthy that the nonkinase domain alone interacted with the CBL3 protein at much higher strength than the full-length CIPK11 protein, suggesting that the N-terminal kinase domain
harbors inhibitory information hindering the interaction between the CIPK11 C-terminal region and CBL3. Using a series of CBL3 deletion mutants, we also dissected the structural requirement in the CBL3 protein for interaction with CIPK11. Interestingly, any deletions from either the N-terminal or the C-terminal end of the CBL3 protein completely abolished interaction with CIPK11 as shown in Fig. 4B. These findings suggest that the entire amino acid sequence of CBL3 is critical for interaction with CIPK11. 3.4. Different interaction affinity between CIPK11 and CBLs To investigate whether CIPK11 can interact with other members of the CBL family, we transformed the yeast cells carrying the pGAD.CIPK11 plasmid with various CBLs in the DNA-binding domain vector. Interactions between the baits and the prey were determined by monitoring the growth of co-transformed yeasts on the selection medium
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Fig. 2. Expression patterns of the CIPK11 gene. (A) Real-time RT-PCR analysis of CIPK11 transcript levels in different organs of Arabidopsis plants. Total RNA was isolated from various tissues (root, stem, leaf, and flower) of 5-week-old wild-type plants grown under long-day conditions. Real-time RT-PCR was performed with either CIPK11-specific primers or Actin2-specific primers. Bars indicate the relative CIPK11 transcript levels normalized to the housekeeping gene Actin2 transcript levels. Data present means of triplicate samples. (B) Histochemical GUS analysis of CIPK11 promoter-GUS transgenic plants. I, a germinating seed; II, a 2-day-old seedling; III, a 3-day-old seedling; IV, a 1-week-old seedling; V, a 2-week-old plant; VI, a 3-week-old plants; VII, flower buds and cauline leaves; VIII, a flower; IX, a leaf; X, a mature silique.
(SC-HLT). We also performed the b-galactosidase activity assay to measure the interaction strength. Among the CBL family members tested in this study, only CBL4/SOS3 did not interact with CIPK11 as shown in Table 1. Although CBL1 and CBL9 did interact with CIPK11, strength of their
interaction was significantly lower than that of the CBL3CIPK interaction. Such different interaction affinities imply that CIPK11 can preferentially forms a complex with CBL3, thereby being involved in CBL3-mediated calcium signaling pathway in Arabidopsis.
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Fig. 3. CIPK11 interacts with CBL3 in a yeast two-hybrid system. (A) The circle shows the arrangement of the Y190 yeast cells carrying the indicated pGBT and pGAD plasmids. (B) Yeast growth on synthetic complete media lacking leucine and tryptophan (SC-LT). (C) Yeast growth on synthetic complete media lacking histidine, leucine, and tryptophan (SC-HLT). (D) Filter-lift assay showing b-galactosidase activity.
Fig. 4. Identification of interaction domain in CIPK11 and CBL3. (A) The kinase (white box) and nonkinase (black box) domains were, respectively, cloned into the pGAD vector and transformed into the Y190 yeast cells carrying pGBT or pGBT.CBL3. (B) Different regions of CBL3 were, respectively, cloned into the pGBT vector [20] and transformed into the Y190 yeast cells carrying pGAD or pGAD.CIPK11. Yeast growth on the selection media (SC-HLT) was scored as growth (+) and no growth (). Numbers in the parentheses represent units of b-galactosidase activity. Numbers that flank the boxes indicate the beginning and the ending positions of each protein fragment.
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Table 1 Interaction of CIPK1 with CBLs in a yeast two-hybrid assay
Pgbt PGBT.CBL1 PGBT.CBL3 PGBT.CBL4 PGBT.CBL9
pGAD
PGAD.CIPK11
(<1) (<1) (<1) (<1) (<1)
(<1) +(<3.03) +(<45.29) (<1) +(<3.01)
Yeast growth on the selection media (SC-HLT) was scored as growth (+) and no growth (). Numbers in the parentheses represent units of b-galactosidase activity.
3.5. CIPK11 associates with CBL3 in a Ca2+-dependent manner To verify the interaction between the CBL3 calcium sensor and CIPK11 in vitro and to investigate the effect of Ca2+ on the CBL3-CIPK11 complex formation, we expressed and purified both CBL3 and CIPK11 proteins using the glutathion S-transferase (GST) gene fusion system. As shown in Fig. 5, CBL3 and CIPK11 were first expressed as GST fusion forms and subsequently cleaved and purified as 26 kDa and 49 kDa proteins, respectively. The cleaved CBL3 protein was used to produce polyclonal antibody from the rabbit. For the pull-down assay, we mixed GST-CIPK11 (bait) with the cleaved form of CBL3 (prey) in the presence or absence of Ca2+ and checked whether GST beads pulled down the prey using western blot analyses. The anti-CBL3 antibody purified with the CBL3 antigen was used as probe. As shown in Fig. 6, GST-CIPK11 did not pull down the CBL3 protein in the presence of EGTA that chelates Ca2+. The addition of micromolar concentration of Ca2+, however, allowed it to successfully retrieve CBL3. The GST protein, used a negative control, failed to pull down CBL3 regardless of Ca2+. Taken together, these results suggest that the CBL3 Ca2+ sensor physically associates with the CIPK11 protein
Fig. 5. Expression and purification of recombinant CBL3 and CIPK11 proteins. (A) GST-CBL3. (B) GST-CIPK11. Lanes 1–3 contain the GSTfused forms, the thrombin-digested forms, and purified forms, respectively. The proteins were analyzed by SDS-PAGE, and the gels were stained with Coomassie blue. The molecular masses of the proteins are indicated at left in kilodaltons.
Fig. 6. CBL3 physically interacts with CIPK11 in vitro. The GST-CIPK1 fusion protein was used as a bait to pull down the cleaved CBL3 protein in the presence of 1 mM EGTA () or 0.2 mM CaCl2 (+). The GST protein (GST) was used as a negative control. (A) An immunoblot probed with rabbit anti-CBL3 antibody. (B) A Coomassie blue-stained SDS-PAGE gel showing the amount of bait proteins used in each pull-down assay. The molecular masses of the proteins are indicated at right in kilodaltons.
in a Ca2+-dependent manner. This finding supports Shi et al.’s report [21], which demonstrated that CBL1–CIPK1 complex forms only in the presence of Ca2+. Such Ca2+dependent interaction between Ca2+ sensors and their target proteins was also well known in the case of calmodulin and calmodulin-binding proteins [9]. In contrast, Halfter et al. [22] reported that binding of CBL4/SOS3 with CIPK24/ SOS2 itself does not depend on Ca2+, although activation of the CIPK24/SOS2 activity by CBL4/SOS requires Ca2+. 3.6. CIPK11 affinity chromatography purifies CBL3 from Arabiopsis plants It is apparent that CBL3 specifically interacts with CIPK11 according to the results of both the yeast two-hybrid system and pull-down assays. To further confirm the interaction between these two proteins, we performed affinity-chromatography purification in the presence of Ca2+ by using CIPK11 as an affinity reagent to isolate CBL3 from an Arabidopsis total protein extract. As shown in Fig. 7, CIPK11 attached to the GST beads retrieved a 26-kDa
Fig. 7. Affinity purification of CBL3 by GST-CIPK11 from the Arabidopsis total protein extract. (A) An immunoblot probed with rabbit anti-CBL3 antibody. The proteins that were purified by the GST (lane 2) and GSTCIPK11 (lane 3) affinity beads from the plant extract were characterized by western blot analysis using anti-CIPK1 antibody as probe. Total protein extract (5 mg) from the CBL3-overexpressing Arabidopsis plants was included in lane 1 to show the size of the CBL3 protein. (B) A Coomassie blue-stained SDS-PAGE gel. Lane 1 contains 5 mg of total protein extract from the CBL3-overexpressing Arabidopsis plants. Lanes 2 and 3 show the GST and GST-CIPK11 proteins, respectively, which were used as affinity beads. The molecular masses of the proteins are indicated at left and right in kilodaltons.
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protein that was recognized by the anti-CBL3 antibody. The 26-kDa protein band is also found in the plant total protein extract and it is the expected size of CBL3. In contrast, the GST control did not produce any protein bands when the anti-CBL3 antibody was used as probe. These results strongly suggest that the CBL3 and CIPK11 proteins specifically associate in Arabidopsis plants.
4. Discussion Ca2+ plays a pivotal role in a variety of eukaryotic signal transduction pathways as a ubiquitous second messenger. Ca2+ signaling cascades typically begin with Ca2+-binding proteins that sense and transmit the Ca2+ signatures downstream by interacting with other signaling proteins [9]. Recently, a new family of Ca2+ sensor proteins have been identified from Arabidopsis and referred to as CBLs due to the substantial sequence similarity with the regulatory B subunit of calcineurin (CNB) in animals [34,35]. CBLs contain three typical EF-hand motifs that have been shown to bind calcium in vitro [35,36]. Although the animal CNB associates with a protein phosphatase (CNA), the plant CBLs mainly interact with a group of protein kinases called CIPKs or SnRKs [11,19–22]. CIPKs represent a new subclass of protein kinases, because they contain a unique regulatory domain in the C-terminal region, although the kinase domains of CIPKs are most related to those of the yeast SNF1 protein kinase (sucrose nonfermenting 1) and the mammalian AMPdependent protein kinase (AMPK) [35]. Several lines of evidence suggested that the CBL–CIPK complex play an important role in plant responses to a diverse array of external stimuli, including cold, drought, salinity, and ABA [23–26]. GenBank database search indicated that other plant species including monocots and dicots also contain genes, which encode polypeptides homologous to Arabidopsis CBLs or CIPKs. In particular, the rice (Oryza sativa) genome is currently predicted to have 10 CBLs and 30 CIPKs [27]. In fact, we have recently isolated a CIPK-like gene (designated OsCK1) from rice and demonstrated that OsCK1, which is involved in response to diverse signals including cold, light, and salts, interacts with Arabidopsis CBL3 in a yeast two-hybrid system. In addition, OsCK1 prefers Mn2+ to Mg2+ as cofactor like Arabidopsis CIPK1 [21,37]. Together, these findings suggest the CBL–CIPK Ca2+-signaling networks originally found in Arabdidopsis are probably also present in other plants. As mentioned above, the Arabidopsis genome appears to contain 10 CBLs and 25 CIPKs. Judging from the number of genes in the CBL and CIPK families, it is likely that some CBLs would have more than one interacting CIPK partner. In fact, Kolukisaoglu et al. [27] demonstrated that CBL1 and CBL9, respectively, interact with multiple CIPKs in the yeast two-hybrid system. Because an individual component of the CBL–CIPK complexes functions in a specific stress-signaling pathway [23–26,29], it is important to figure out the entire
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CBL–CIPK interacting networks which has not been fully established yet. As a step toward this goal, we sought to identify CIPKs that associate with the CBL3 protein in the present study. We have screened a yeast two-hybrid library using CBL3 as bait and identified nine CIPK family members that include CIPK1, CIPK2, CIPK3, CIPK6, CIPK9, CIPK11, CIPK12, CIPK15, and CIPK21. Our detailed analysis of the interaction between a pair of partner proteins, CBL3 and CIPK11, has demonstrated these two proteins physically interact in a Ca2+dependent manner (Fig. 6). This result is consistent with the previous finding that CBL1, another CBL family member, associates with CIPK1 only in the presence of Ca2+ [21]. Recently, the crystal structure analysis of CBL2, almost identical to CBL3 (92% identity), indicated that conformation of Ca2+-bound CBL2 was markedly different from the unbound form [38]. Taken together, these findings suggest that Ca2+-bound CBLs undergoes conformational change to associate with the target CIPKs, thereby modulating their kinase activity. We also investigated the structural basis for the interaction between CIPK11 and CBL3. Our deletion analysis indicated that the C-terminal region of CIPK11 (CIPK11C) is involved in the actual interaction with CBL3 and the kinase domain (CIPK11N) has a negative effect on the interaction (Fig. 4A). These results suggest that both the kinase and nonkinase domains exert their effect on specifying the final interaction affinity with CBL3. Regarding the structural requirement of CBL3, we found that the interaction with CIPK11 was completely abolished by removing short sequences from either the N- or C-terminal end of CBL3 (Fig. 4B). This finding suggests that both terminal sequences, most variable regions in the CBL family members, are necessary to interact with CIPK11. Considering the three conserved EF-hand motifs located in the center of CBL members, we reason that the diverse terminal sequences may play a critical role in determining the interaction affinity toward CIPKs and other target proteins. Our reasoning could explain why CBL5, CBL7, and CBL8, which have terminal sequences totally different from CIPK-associating CBLs, such as CBL1, CBL2, CBL3, CBL4, and CBL9, did not interact with any of the CIPKs tested in the study [20,27]. Although we cannot completely rule out the possibility that a group of unidentified CIPKs serve as targets for these CBL members, it is more likely that CBL5, CBL7, and CBL8 may have protein targets other than the CIPK family members. In this study, we identified nine CBL3-interacting CIPK members as potential CBL3 targets, supporting the previous finding that each CBL family member can have multiple CIPK partners [27]. Our study also suggests that each of the CIPK family members has the ability to interact with several CBLs, because three CBL family members including CBL1, CBL3, and CBL9 interacted, respectively, with CIPK11 in the yeast two-hybrid system (Table 1). However, their interaction affinities toward CIPK11 were significantly different among the three CBL isoforms. CIPK11 interacted
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with CBL3 at much higher strength than with CBL1 and CBL9, implying that CBL3 will out-compete CBL1 and CBL9 to produce the CBL3–CIPK11 complex. Therefore, it is likely that such different interaction affinities will contribute in part to determining final parings between the CBL and CIPK family members in plant cells. In addition, we speculate about other factors that can influence formation of a specific CBL–CIPK complex in plant cells. First, the temporal and spatial expression patterns of CBLs and CIPKs may vary so that different cells at any given time may have a distinct pattern of CBL–CIPK complexes. For example, the expression level of CBL1 dramatically increased in response to stressful conditions, such as cold, wounding, and drought, although other CBL family members, including CBL2, CBL3, and CBL4/SOS3, did not respond [35]. Similarly, the CIPK family members also exhibited different expression patterns. Both CIPK1 and CIPK3 were expressed in all organs including roots, leaves, stems, and flowers [21,25], whereas CIPK11 was not expressed in the flowers. Besides the difference in the spatial expression pattern, CIPK1 and CIPK3 differently responded to the stimuli, such as cold, NaCl, ABA, wounding, and drought. Although the stresses activated CIPK3 expression, they did not change the level of CIPK1 expression [21,25]. Second, different subcellular localization of CBLs and CIPKs may also play a role in generating specific CBL– CIPK complexes in plant cells. Among the 10 CBLs in the Arabidopsis genome, four CBLs that include CBL1, CBL4/ SOS3, CBL5, and CBL9 are known to possess the conserved N-myristoylation motif (MGXXXS/T) that usually targets proteins to cell membrane [27]. The other members lacking the N-myristoylation motif appear to lack a mechanism to become membrane associated. Ishitani et al. [39] demonstrated the importance of the N-myristoylation motif by showing that CBL4/SOS3 must have the N-myristoylation motif intact in order to function normally in the salt tolerant response. Unfortunately, however, little is known about subcellular localization of the CIPK family members to date.
Acknowledgements This work was supported by grants from the Plant Signaling Network Research Center, Korea Science and Engineering Foundation, and the BioGreen21 Program of the Rural Development Administration. It was also supported in part by the Special Grant Research Program in the Ministry of Agriculture and Forestry of the Korean Government.
[3]
[4] [5] [6] [7] [8] [9] [10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
References [1] M.R. Blatt, Cellular signaling and volume control in stomatal movements in plants, Annu. Rev. Cell Dev. Biol. 16 (2000) 221–241. [2] T.L. Holdaway-Clarke, J.A. Feijo, G.R. Hackett, J.G. Kunkel, P.K. Hepler, Pollen tube growth and the intracellular cytosolic calcium
[23]
[24]
gradient oscillate in phase while extracellular calcium influx is delayed, Plant Cell 9 (1997) 1999–2010. C.L. Wymer, T.N. Bibikova, S. Gilroy, Cytoplasmic free calcium distributions during the development of root hairs of Arabidopsis thaliana, Plant J. 12 (1997) 427–439. D.W. Ehrhardt, R. Wais, S.R. Long, Calcium spiking in plant root hairs responding to Rhizobium nodulation signals, Cell 85 (1996) 673–681. D. Sanders, C. Brownlee, J.F. Harper, Communicating with calcium, Plant Cell 11 (1999) 691–706. J.J. Rudd, V.E. Franklin-Tong, Unravelling response-specificity in Ca2+ signalling pathways in plant cells, New Phytol. 151 (2001) 7–33. D. Sanders, J. Pelloux, C. Brownlee, J.F. Harper, Calcium at the crossroads of signaling, Plant Cell 14 (Suppl.) (2002) S401–S417. N.H. Evans, M.R. McAinsh, A.M. Hetherington, Calcium oscillations in higher plants, Curr. Opin. Plant Biol. 4 (2001) 415–420. R.E. Zielinski, Calmodulin and calmodulin-binding proteins in plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 697–725. S. Luan, J. Kudla, M. Rodriguez-Concepcion, S. Yalovsky, W. Gruissem, Calmodulins and calcineurin B-like proteins: calcium sensors for specific signal response coupling in plants, Plant Cell 14 (Suppl.) (2002) S389–S400. E.M. Hrabak, C.W. Chan, M. Gribskov, J.F. Harper, J.H. Choi, N. Halford, J. Kudla, S. Luan, H.G. Nimmo, M.R. Sussman, M. Thomas, K. Walker-Simmons, J.K. Zhu, A.C. Harmon, The Arabidopsis CDPK-SnRK superfamily of protein kinases, Plant Physiol. 132 (2003) 666–680. S.H. Cheng, M.R. Willmann, H.C. Chen, J. Sheen, Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family, Plant Physiol. 129 (2002) 469–485. V.S. Reddy, G.S. Ali, A.S. Reddy, Genes encoding calmodulin-binding proteins in the Arabidopsis genome, J. Biol. Chem. 277 (2002) 9840– 9852. W.A. Snedden, H. Fromm, Calmodulin as a versatile calcium signal transducers in plants, New Phytol. 151 (2001) 35–66. W.A. Snedden, N. Koutsia, G. Baum, H. Fromm, Activation of a recombinant petunia glutamate decarboxylase by calcium/calmodulin or by a monoclonal antibody which recognizes the calmodulin binding domain, J. Biol. Chem. 271 (1996) 4148–4153. W.A. Snedden, H. Fromm, Calmodulin, calmodulin-related proteins and plant responses to the environment, Trends Plant Sci. 3 (1998) 299–304. P. Douglas, G. Moorhead, Y. Hong, N. Morrice, C. MacKintosh, Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase, Planta 206 (1998) 435–442. I. Hwang, H. Sze, J.F. Harper, A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis, Proc. Natl. Acad. Sci. 97 (2000) 6224–6229. V. Albrecht, O. Ritz, S. Linder, K. Harter, J. Kudla, The NAF domain defines a novel protein–protein interaction module conserved in Ca2+regulated kinases, EMBO J. 20 (2001) 1051–1063. K.N. Kim, Y.H. Cheong, R. Gupta, S. Luan, Interaction specificity of Arabidopsis calcineurin B-like calcium sensors and their target kinases, Plant Physiol. 124 (2000) 1844–1853. J. Shi, K.N. Kim, O. Ritz, V. Albrecht, R. Gupta, K. Harter, S. Luan, J. Kudla, Novel protein kinases associated with calcineurin B-like calcium sensors in Arabidopsis, Plant Cell 11 (1999) 2393–2405. U. Halfter, M. Ishitani, J.K. Zhu, The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3, Proc. Natl. Acad. Sci. 97 (2000) 3735–3740. Y.H. Cheong, K.N. Kim, G.K. Pandey, R. Gupta, J.J. Grant, S. Luan, CBL1, a calcium sensor that differentially regulates salt, drought, and cold responses in Arabidopsis, Plant Cell 15 (2003) 1833–1845. Y. Guo, Q.S. Qiu, F.J. Quintero, J.M. Pardo, M. Ohta, C. Zhang, K.S. Schumaker, J.K. Zhu, Transgenic evaluation of activated mutant alleles of SOS2 reveals a critical requirement for its kinase activity
H.J. Jeong et al. / Plant Science 169 (2005) 1125–1135
[25]
[26]
[27]
[28]
[29] [30]
[31]
and C-terminal regulatory domain for salt tolerance in Arabidopsis thaliana, Plant Cell 16 (2004) 435–449. K.N. Kim, Y.H. Cheong, J.J. Grant, G.K. Pandey, S. Luan, CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis, Plant Cell 15 (2003) 411–423. G.K. Pandey, Y.H. Cheong, K.N. Kim, J.J. Grant, L. Li, W. Hung, C. D’Angelo, S. Weinl, J. Kudla, S. Luan, The calcium sensor calcineurin B-like 9 modulates abscisic acid sensitivity and biosynthesis in Arabidopsis, Plant Cell 16 (2004) 1912–1924. U. Kolukisaoglu, S. Weinl, D. Blazevic, O. Batistic, J. Kudla, Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and rice CBL-CIPK signaling networks, Plant Physiol. 134 (2004) 43–58. O. Batistic, J. Kudla, Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network, Planta 219 (2004) 915–924. J.K. Zhu, Salt and drought stress signal transduction in plants, Annu. Rev. Plant Biol. 53 (2002) 247–273. S.J. Clough, A.F. Bent, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16 (1998) 735–743. R.A. Jefferson, T.A. Kavanagh, M.W. Bevan, GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants, EMBO J. 6 (1987) 3901–3907.
1135
[32] L. Breeden, K. Nasmyth, Regulation of the yeast HO gene, Cold Spring Harb. Symp. Quant. Biol. 50 (1985) 643–650. [33] S.K. Hanks, A.M. Quinn, Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members, Methods Enzymol. 200 (1991) 38–62. [34] J. Liu, J.K. Zhu, A calcium sensor homolog required for plant salt tolerance, Science 280 (1998) 1943–1945. [35] J. Kudla, Q. Xu, K. Harter, W. Gruissem, S. Luan, Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals, Proc. Natl. Acad. Sci. 96 (1999) 4718–4723. [36] M. Nagae, A. Nozawa, N. Koizumi, H. Sano, H. Hashimoto, M. Sato, T. Shimizu, Crystallization and preliminary X-ray characterization of a novel calcium-binding protein AtCBL2 from Arabidopsis thaliana, Acta Crystallogr. D Biol. Crystallogr. 59 (2003) 1079–1080. [37] K.N. Kim, J.S. Lee, H. Han, S.A. Choi, S.J. Go, I.S. Yoon, Isolation and characterization of a novel rice Ca2+-regulated protein kinase gene involved in responses to diverse signals including cold, light, cytokinins, sugars and salts, Plant Mol. Biol. 52 (2003) 1191–1202. [38] M. Nagae, A. Nozawa, N. Koizumi, H. Sano, H. Hashimoto, M. Sato, T. Shimizu, The crystal structure of the novel calcium-binding protein AtCBL2 from Arabidopsis thaliana, J. Biol. Chem. 278 (2003) 42240– 42246. [39] M. Ishitani, J. Liu, U. Halfter, C.S. Kim, W. Shi, J.K. Zhu, SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding, Plant Cell 12 (2000) 1667–1678.