Calcium (Ca2+) is a versatile and ubiquitous second messenger in all eukaryotes including plants. In response to various stimuli, cytosolic calcium concentration ([Ca2+]cyt) is increased, leading to activation of Ca2+ sensors including Arabidopsis calcineurin B-like proteins (CBLs). CBLs interact with CBL-interacting protein kinases (CIPKs) to form CBL–CIPK complexes and transduce the signal downstream in the signalling pathway. Although there are many reports on the regulation of downstream targets by CBL–CIPK module, knowledge about the regulation of upstream components by individual CIPKs is inadequate. In the present study, we have carried out a detailed biochemical characterization of CIPK9, a known regulator of K+ deficiency in Arabidopsis, with its interacting CBLs. The present study suggests that CIPK9 specifically interacts with four CBLs, i.e. CBL1, CBL2, CBL3 and CBL9, in yeast two-hybrid assays. Out of these four CBLs, CBL2 and CBL3, specifically enhance the kinase activity of CIPK9, while the CBL1 and CBL9 decrease it as examined by in vitro kinase assays. In contrast, truncated CIPK9 (CIPK9ΔR), without the CBL-interacting regulatory C-terminal region, is not differentially activated by interacting CBLs. The protein phosphorylation assay revealed that CBL2 and CBL3 serve as preferred substrates of CIPK9. CBL2– and CBL3–CIPK9 complexes show altered requirement for metal cofactors when compared with CIPK9 alone. Moreover, the autophosphorylation of constitutively active CIPK9 (CIPK9T178D) and less active CIPK9 (CIPK9T178A) in the presence of CBL2 and CBL3 was further enhanced. Our study suggests that CIPK9 differentially phosphorylates interacting CBLs, and furthermore, the kinase activity of CIPK9 is also differentially regulated by specific interacting CBLs.
A typical plant cell responds to environmental stress conditions by elevation of Ca2+ in the cytosol through activation of Ca2+ channels/transporters. Owing to low mobility, the [Ca2+] elevation is often localized in microdomains in a time-dependent manner, formulating spatial and temporal Ca2+ changes in the cell called ‘Ca2+ signatures’ . The specific Ca2+ signatures generated are sensed and relayed by Ca2+ sensors such as calmodulin (CaM), CaM-like protein, Ca2+-dependent protein kinase and calcineurin B-like protein (CBL) in various signalling pathways in Arabidopsis and other plants [2–6]. Our study here focuses on CBLs that, like other sensors, have Ca2+-binding EF-hand motifs, and specifically interact with a family of Ser–Thr protein kinases, known as CBL-interacting protein kinases (CIPKs) to convert the signal into phosphorylation events [3,4,7].
CIPKs have an N-terminal kinase domain and a C-terminal regulatory domain [8,9]. The regulatory domain of CIPK has two specific motifs — asparagine-alanine-phenylalanine (NAF) motif and protein phosphatase interaction (PPI) motif [3,7–10]. The NAF motif of CIPKs is responsible for physical interaction with CBLs, while the PPI motif is required for interaction with 2C-type protein phosphatases (PP2Cs) [8–12]. It is hypothesized that binding of CBLs to the NAF motif of CIPKs releases the autoinhibition of kinase and thus switches on the kinase activity [8,9]. This activation may further be enhanced by transphosphorylation of the activation loop in the kinase domain by other kinases [9,13–15]. Several CBL–CIPK signalling pathways are well characterized and their physiological implications have been well documented in Arabidopsis, such as the SOS3–SOS2–SOS1 (CBL4–CIPK24–Na+/H+ antiporter) module for salt tolerance [16,17], the CBL1/CBL9–CIPK23–AKT1 module for potassium (K+) uptake  and the CBL1/CBL9–CIPK23–CHL1 for regulation of nitrate (NO3−) sensing and uptake .
K+deficiency in agricultural soil severely compromises yields and crop productivity . To improve crop productivity, an in-depth molecular understanding of the low-K+ response is required. The genetic analysis and biochemical studies have identified CIPK23, which interacts with CBL1 and CBL9. These CBL1/9–CIPK23 complexes regulate the activity of a shaker-type K+-channel, AKT1, under low-K+ conditions [21–23]. Another study identified a PP2C-type phosphatase, AKT1-interacting PP2C1 (AIP1), as a negative regulator of AKT1 activity , assembling an activation–inactivation cycle of the AKT1 regulation by reversible phosphorylation. Interestingly, another member of the CIPK family, CIPK9, was also found to be involved in plant adaptation to K+ starvation . Transcripts of CIPK9 were highly inducible under the low-K+ condition and cipk9, the loss-of-function mutant, showed impaired seedling and root growth under K+-deficient conditions . Under prolonged K+ deficiency, the cipk9 mutant displayed tolerance in the shoots (less chlorosis) . CIPK9 is expressed in all root cell types and CIPK9 protein has been found to interact with CBL2 and CBL3 at the tonoplast . It has been hypothesized that the CBL3–CIPK9 complex might regulate an unidentified vacuolar K+ transport system to maintain cytosolic K+ homeostasis under low-K+ conditions [25,26]. Further investigation is required to understand the role of the CBL2/3-CIPK9 complex in K+ homeostasis, independent of CBL1/9–CIPK23–AKT1-mediated K+ uptake.
It has been established that specific CBLs physically interact with multiple CIPKs in diverse signalling pathways. It is recognized that CIPKs, after interacting with CBLs, may phosphorylate the downstream targets such as channels/transporters involved in plant nutrition and stress adaptations [19,21–23,27–34]. More recent works show that CIPKs phosphorylate their upstream signalling components, i.e. CBLs, which may provide an additional mechanism in the regulation of signal transduction pathways [32,35–37]. It has been observed that CBL1 and CBL9 are phosphorylated by CIPK23 and these phosphorylated CBL1/CIPK23 and CBL9/CIPK23 protein complexes modulate AKT1 activation . It was proposed that phosphorylation of the CBL increases the activity of the CBL–CIPK complex towards its substrate by an unknown mechanism [32,37]. One hypothesis is that phosphorylation of CBLs by CIPKs leads to an increase in the stability of the CBL–CIPK complex .
Previously, it has been shown that Arabidopsis CBL1 binds to Ca2+ and interacts with CIPK1 in a Ca2+-dependent manner by the Ca2+–EGTA-based pull-down assay . Moreover, earlier structural studies of CBL–CIPKs revealed their binding with Ca2+, their interaction strength and activation of interacting kinase. This type of study has been carried out only for a few CBLs, CIPKs and CBL–CIPK complexes. These are SOS3, SOS2, SOS3–SOS2 complex, CBL2 and CBL2–CIPK14 and CIPK23 [38–40]. The structural analysis of the SOS3–SOS2 complex revealed that Ca2+ binds to only two EF-hands, and the addition of Ca2+-chelating agent initiates dissociation of the SOS3–SOS2 complex . In case of the CBL2–CIPK14 module, four Ca2+ bind, while the free form of CBL2 could bind only two Ca2+ . The mutagenesis and low-resolution structural studies show that the formation of the CBL2–CIPK14 complex is Ca2+-independent . The available structural data suggest that the Ca2+ binding to CBLs promotes a sudden conformational change in the Ca2+ sensor, which triggers both the FISL-mediated CIPK binding and the activation of their interacting kinase. However, structural studies of other CBLs and their complex formation with CIPKs are not known and open future directions to investigate the role of Ca2+ in this differential interaction and phosphorylation of CBLs and CIPKs.
In this report, we show that one of the CIPKs, CIPK9 interacts with a subset of four CBLs, including CBL1, 2, 3 and 9, but CIPK9 interacts more strongly with CBL2 and 3 compared with CBL1 and 9. Consistent with interaction data, CIPK9 also preferentially phosphorylates CBL2 and 3. Furthermore, CBL2 and CBL3 activate autophosphorylation of CIPK9 more strongly as well as its transphosphorylation activity towards other substrates. These results suggest that specificity in CBL–CIPK interaction facilitates not only the activation of CIPKs by CBLs but also phosphorylation of CBLs by the CIPKs. The present study provides a biochemical basis for dissection of the physiological significance of CBL phosphorylation by CIPKs in planta, which will further increase our understanding of CBL–CIPK signalling mechanisms.
Materials and methods
Yeast two-hybrid interaction analysis
To examine the physical interaction between the CIPK9 and all 10 Arabidopsis CBLs, the yeast two-hybrid system was adopted. CIPK9 cDNA was cloned into the yeast two-hybrid prey vector, pGAD.GH (activation-domain vector) and all the 10 CBLs were cloned into bait vector, pGBT9 (DNA-binding domain vector). Each of 10 BD-CBLs along with AD-CIPK9 was co-transformed into yeast strain AH109. The yeast co-transformants were selected on synthetic complete medium lacking leucine and tryptophan (SD–L-W). For serial dilution, transformed yeast colonies were allowed to grow in liquid medium SD–L-W at 28°C (at 200 rpm overnight). The respective cultures were diluted to obtain an OD600 of 0.5. From three 10-fold serially diluted cultures, 5 µl was used for spotting on synthetic complete medium lacking leucine, tryptophan (SD–L-W), SD-leucine, tryptophan and histidine (SD–L-W-H) and SD–L-W-H medium supplemented with 1 mM 3-amino-1,2,4-triazole (Sigma, Saint Louis) to score growth as an indicator of the protein–protein interaction. Plates were incubated at 28°C for 3 days. The sequences of primers used are listed in Supplementary Table S1 and previously published constructs are mentioned in Supplementary Table S2. For qualitative β-galactosidase expression analysis of CBLs and CIPK9 interaction, the co-transformed cells were grown on SD–HLTW for 4 days and a chloroform overlay assay was performed . Quantification of β-galactosidase activity was performed in triplicate by using ortho-nitrophenyl-β-galactopyranoside (ONPG) as the substrate . Briefly, transformed cells were grown overnight in SD–LW media. The secondary culture was harvested at 1.0 at OD600. The pelleted cells were washed and resuspended in Z buffer. The resuspended cells were subjected to three freeze/thaw (liquid nitrogen/1 min and water bath 37°C/3 min) cycles. After pelleting the cells, the supernatant was collected. Freshly prepared 200 µl of ONPG (4 mg/ml) was added to the supernatant and kept in the dark at 30°C until the colour developed. To stop the reaction, 600 µl of 1 M Na2CO3 was added to the mixture. OD420 was recorded and activity was calculated .
Cloning of genes in bacterial expression vectors
To validate the phosphorylation of CIPK9 and CBLs, the cDNA of CIPK9, CIPK9ΔR and CBLs was cloned into bacterial recombinant protein expression pGEX4T-1, a GST-tag containing vector. The constructs were further confirmed by restriction enzyme digestion and sequencing. The restriction sites, primer sequence and other details are mentioned in Supplementary Table S1 and previously published constructs are mentioned in Supplementary Table S2.
Site-directed mutagenesis of CIPK9
A plasmid of CIPK9/pGEX4T-1 was used as a PCR template for mutagenesis. All primers designed to introduce the site-directed mutations are listed in Supplementary Table S1 and all mutagenized positions are underlined. A 50 µl PCR was carried out with 50 ng of template, 125 ng of primer pair, 200 µM dNTPs and 1 unit of PrimeSTAR HS DNA Polymerase (Takara, Japan). The extension reaction was initiated by preheating the reaction mixture to 98°C for 4 min, 18 cycles of 98°C for 1 min, 68°C for 1 min and 72°C for 6 min according to the length of the template; followed by final extension at 72°C for 10 min. The PCR products were treated with restriction enzyme DpnI (NEB, U.S.A.). An aliquot of 1 µl of amplified PCR product was transformed into DH5-α electro-competent cells and plated on an LB plate containing 100 µg/ml ampicillin. A total of 10 colonies were selected and their plasmids were isolated by mini-prep. The different mutations as mentioned in Supplementary Table S1 in CIPK9/pGEX4T-1 were confirmed by sequencing.
Recombinant protein induction and purification
CIPK9, CIPK9ΔR and CBLs were cloned in pGEX4T-1 vector and expressed in Escherichia coli as a GST-tag fusion protein. Recombinant protein expression was induced at OD600 0.5 with 0.1 mM IPTG at 28–30°C, 200 rpm for 4 h. To solubilize induced protein, 50 ml of induced pellet was resuspended in 10 ml of ice-cold lysis buffer [50 mM Tris (pH 8), 10 mM MgCl2, 150 mM NaCl and 0.1% Triton X-100, pH 8.0] along with protease inhibitor cocktail and 1 mM phenylmethanesulfonyl. Cells were lysed with lysozyme (0.5–1.0 mg/ml buffer) for 30 min and sonicated for soluble proteins. The total cell lysate was clarified by centrifugation at 15 000 g for 30 min at 4°C and the supernatant was collected for protein purification. The soluble fraction was incubated with Glutathione–Sepharose beads (GE Health Care) for 4 h at 4°C on an end-to-end rotatory shaker. The protein-bound Glutathione–Sepharose beads were loaded onto the purification column (Bio-Rad, U.S.A.). The protein-bound beads were washed with lysis buffer. Proteins were eluted in multiple fractions with elution buffer (50 mM Tris, 10 mM MgCl2, 150 mM NaCl and 10 mM reduced glutathione, pH 8.0). The purity of purified proteins was analysed on 10% SDS–PAGE gel and recombinant proteins were visually quantified using the known concentration of bovine serum albumin (BSA) protein.
In vitro kinase assay
Purified proteins of GST–CIPK9, GST–CIPK9ΔR and GST–CBLs were gel-quantified with a known concentration of purified BSA. For in vitro kinase assay, 0.5–2.0 µg of proteins were used. An in vitro assay was performed in standard buffer containing 20 mM Tris (pH 7.2), 2.5 mM MnCl2, 0.5 mM CaCl2, 1 mM DTT, 10 µM ATP and 2–10 µCi 32γP, with different combinations of purified proteins in a 30 µl reaction. The reaction was done at 30°C for 30 min and terminated by 2× SDS–PAGE loading buffer. The reactions were resolved on 10% SDS–PAGE and gels were CBB-stained, destained and dried. The autoradiography using KODAK (Biomax Transcreen, HE, Intensifying Screen) was carried out to detect the signals.
Arabidopsis CIPK9 interacts with 4 out of the 10 CBLs tested
Although studies suggest a role of CIPK9 in low-K+ response in Arabidopsis, the mechanism and physiological function of CIPK9 remain unclear [23,26]. As a step towards understanding the function of CIPK9 and its interacting CBLs, we took a biochemical approach to dissect the regulation of CIPK9 by its interacting CBLs. First, we identified the CIPK9-interacting CBLs by yeast two-hybrid assays and found that CIPK9 specifically interacted with CBL1, CBL2, CBL3 and CBL9 based on the growth criteria on a stringent growth assay on selection medium, i.e. SC-HLW + 1 mM 3-AT (Figure 1A). To determine the strength of CBL–CIPK9 interaction, the β-galactosidase assay was performed (Figure 1B,C). Both the assays indicated differential interaction between CIPK9 and different CBLs (Figure 1B,C), especially CBL2 and CBL3 interaction was stronger than that of CBL1 and CBL9.
CIPK9 differentially interacts with different CBLs.
Domain organizations of CIPK9 and its interacting CBLs
CIPK9 is a Ser/Thr kinase possessing an N-terminal kinase domain and a C-terminal regulatory domain (Supplementary Figure S1A). The T178 residue in the activation loop of CIPK9 (also referred to as PKS6 in a previous report)  is responsible for autophosphorylation-mediated activation [22,28,43] (Supplementary Figure S1A). Based on previous studies of CIPKs, it was predicted that Lys48 residue in the ATP-binding pocket of the CIPK9 kinase domain should be critical for ATP binding and hence for its kinase activity [22,28,43]. The regulatory domain has two motifs, namely NAF and PPI. The NAF motif is sufficient for interaction with CBLs and the PPI motif is essential for interaction with PP2C-type phosphatases (Supplementary Figure S1A). On the basis of N-terminal region, CBLs can be divided into two groups. The first group comprises CBL1, CBL4, CBL5, CBL8 and CBL9 with shorter N-terminal and lipid modification sites crucial for targeting these CBLs to the membrane. CBL1 and CBL9 of this group interacted with CIPK9 in our assay. The N-terminal MGXXX(S/T) motifs of these CBLs are modified by myristoylation/prenylation for association with the plasma membrane [44–47]. Both CBL1 and CBL9 consist of 213 amino acid residues, four Ca2+-binding EF-hand motifs and PFPF motif, which are phosphorylated by CIPK23. They show more than 90% sequence homology with each other  (Supplementary Figure S1B,C).
The second group includes CBL2, CBL3, CBL6, CBL7 and CBL10, which have a longer N-terminal tail [12,45,47]. CBLs of this group do not undergo lipid modification at their N-terminal domain. Our data suggest that CIPK9 interacted with both CBL2 (226 AA) and CBL3 (230 AA) that have more than 90% sequence homology with each other (Supplementary Figure S1B,C).
CIPK9 is an active kinase and exhibits autophosphorylation
The wild-type CIPK9 and CIPK9ΔR (a variant of CIPK9 with only kinase domain) were expressed in E. coli as GST fusion proteins and purified by affinity chromatography on Glutathione–Sepharose beads (Supplementary Figure S2A,B). Apparent molecular masses of ∼76 kDa for CIPK9-GST fusion and 66 kDa for CIPK9ΔR-GST fusion were observed. By performing an in vitro kinase assay, we tested the kinase activity of CIPK9 by commonly used artificial protein substrates, such as myelin basic protein (MBP), histone-type III-S and BSA. Consistent with results in another study , CIPK9 shows autophosphorylation activity, also previously reported for other CIPKs  (Figure 2A). In addition, CIPK9 phosphorylated MBP protein, but not histone-type III-S or BSA (Figure 2A).
CIPK9 shows autophosphorylation and requires Mn2+ as a metal cofactor for its kinase activity.
The kinase activity of CIPKs varies with the concentration of cofactors such as Ca2+, Mn2+ and Mg2+ [28,43]. As reported earlier, CIPK9 requires metal ions such as Mn2+ and Mg2+ for kinase activity and also has the ability to phosphorylate P1, P2, P3 artificial peptides with different affinity . Increasing concentration of CaCl2 did not influence the autophosphorylation of either CIPK9 or CIPK9ΔR (Figure 2B,C). It was observed that both CIPK9 and CIPK9ΔR show autophosphorylation, and this autophosphorylation activity did not increase with additional Ca2+ in the in vitro kinase assay. CIPK9 shows maximum autophosphorylation at 10 mM Mn2+, while the Mg2+ did not influence kinase activity (Figure 2D,E). These results are similar to a previous study, showing that CIPK9 requires Mn2+ as a specific metal cofactor for its kinase activity .
CIPK9 differentially phosphorylates its interacting CBLs
Recent studies show that CBLs are phosphorylated by interacting CIPKs. This appears to be an additional regulatory mechanism in CBL–CIPK signalling pathways, although the physiological relevance remains unclear [32,35–37]. To test whether CIPK9 can phosphorylate its interacting CBLs, we expressed and purified several CBLs in E. coli including CBL1, 2, 3 and CBL9 that interacted with CIPK9 along with CBL4, which did not interact with CIPK9 (as a negative control) (Supplementary Figure S2C–G).
As mentioned earlier, the CBL1, CBL9 and CBL4 belong to the first group of CBLs localized to the plasma membrane; and CBL2 and CBL3 belong to the second group localized to the vacuolar membrane [12,45,47]. Interestingly, we observed that CIPK9 phosphorylated CBLs from both groups. CIPK9 specifically phosphorylated its interacting CBLs (CBL1, CBL2, CBL3 and CBL9), but did not phosphorylate CBL4 or GST protein (Figure 3A). Furthermore, CIPK9 weakly phosphorylated CBL1 and CBL9, but the autophosphorylation status of CIPK9 decreased in the presence of CBL1 or CBL9 when compared with CIPK9 alone (Figure 3A). CIPK9 strongly phosphorylated CBL2 and CBL3, and the autophosphorylation status of CIPK9 was dramatically enhanced in the presence of CBL2 and CBL3 when compared with CIPK9 alone (Figure 3A). The ImageJ quantification of the phosphorylation status of CIPK9 shows that CBL2 and CBL3 enhanced the autophosphorylation of CIPK9 drastically, whereas its activity was reduced in the presence of either CBL1 or CBL9 (Figure 3B). The kinase activity of CIPK9 with CBL2/CBL3 increased more than double in comparison with either alone or in the presence of CBL1 and CBL9. The complete absence of any detectable radioactivity in the lanes in which only CBL proteins were incubated in the kinase reaction buffer excludes non-specific phosphorylation to these CBLs. Therefore, CIPK9 kinase activity was differentially modulated by its interacting CBLs that are, in turn, differentially phosphorylated by CIPK9.
CIPK9 shows differential autophosphorylation and transphosphorylation in the presence of its interacting CBLs.
CIPK9ΔR lacks regulatory region containing an NAF domain and hence should not interact with CBLs. Indeed, contrary to full-length CIPK9, CIPK9ΔR failed to interact with CBLs in the yeast two-hybrid assay, but surprisingly it still phosphorylated CBLs, (CBL1, CBL2, CBL3 and CBL9) (Figure 3C). Full-length CIPK9 did not interact with CBL4. Moreover, neither CIPK9ΔR nor full-length CIPK9 phosphorylated CBL4 (Figure 3C). In contrast with the full-length CIPK9, the presence of interacting CBLs did not affect CIPK9ΔR autophosphorylation (Figure 3C). Here, we also noticed that autophosphorylation of CIPK9ΔR in the presence of CBLs was reduced when compared with autophosphorylation of CIPK9ΔR alone (Figure 3C). It was also observed that autophosphorylation of CIPK9ΔR in the presence of CBL2 was similar to CIPK9ΔR alone, suggesting an important role of the regulatory region, but this phenomenon cannot be explained at present and requires further experimental input. Moreover, differential autophosphorylation of CIPK9ΔR in the presence of CBLs diminished in contrast with full-length CIPK9, which indicated the key role of this regulatory region in kinase activation and substrate (CBLs) phosphorylation.
The autophosphorylation of CIPK9 was significantly increased in the presence of CBL2/CBL3 while the CIPK9 differentially transphosphorylated CBL2 and CBL3 among the four interacting CBLs. Increasing concentration of CIPK9 had little effect on phosphorylation of CBL1/CBL9, while the CBL4 was not phosphorylated by CIPK9 (Figure 4A,D,E). We observed that the autophosphorylation of CIPK9 was reduced when CBL1 and CBL9 were present in the assay. In contrast, autophosphorylation of CIPK9 in the presence of CBL2/CBL3 was at least double in comparison with CIPK9 alone (Figure 4B,C). The above results were further validated by the kinase assay in which an increasing amount of CBLs was used while keeping the amount of CIPK9 constant (Figure 4F–J). To show the specificity, the low exposure of blots (Figure 4B,C,G,H) are further shown in Supplementary Figure S4A–D. We again noticed that increasing concentrations of CBL1/CBL9 reduced the autophosphorylation of CIPK9, indicating that CBL1/CBL9 might negatively regulate the CIPK9 activity (Figure 4F,I). CIPK9 did not phosphorylate CBL4, but increasing concentration of CBL4 slightly reduced the autophosphorylation of CIPK9 (Figure 4J). Increasing concentration of CBL2/CBL3 further enhanced the kinase activity of equally loaded CIPK9 (Figure 4G–H). Therefore, it seems that CBL2 and CBL3 act as effective substrates for CIPK9. This assay implied that CBL1/CBL9 inhibits, while CBL2/CBL3 enhances the kinase activity of CIPK9.
The kinase activity of CIPK9 is enhanced by CBL2 and CBL3.
CBL2/CBL3 interaction alters cofactor requirement of CIPK9
The kinase activity of various CIPKs is known to be modulated by various metal cofactors such as Ca2+, Mn2+ and Mg2+ [8,9,43,48]. Previously, it has been shown that CIPK9 lacking the NAF domain (PKS6DF) exhibited a strong preference for Mn2+ over Mg2+ as a divalent cofactor for substrate phosphorylation (P3 peptide) . In this study, we have shown that CBL2/CBL3 enhanced autophosphorylation of full-length CIPK9 but not that of the CIPK9ΔR. To test the effect of these known cofactors on the optimum kinase activity of CIPK9 and CIPK9ΔR in the presence of CBL2/CBL3, we carried out further kinase assays in the presence of various metal cofactors. We found that a low level of Ca2+ was required for autophosphorylation of CIPK9 and CIPK9ΔR in the absence of CBLs (Figure 2B,C). Higher concentrations of Ca2+ (0.5–2.5 mM) increased the autophosphorylation of CIPK9 in the presence of CBL2/CBL3 and also increased the CBL2/CBL3 transphosphorylation (Figure 5A,D). The Ca2+ concentration beyond 2.5 mM becomes inhibitory for the kinase activity of CIPK9 in the presence of CBL2/CBL3 (Figure 5A,D). On the other hand, the increasing concentration of Ca2+ failed to increase the autophosphorylation of CIPK9ΔR in the presence of CBL2/CBL3 and did not significantly change the status of CBL2/CBL3 transphosphorylation (Supplementary Figure S4E). This indicated that the regulatory domain is critical for activation of CIPK9 by CBL2/CBL3 in the presence of Ca2+.
The metal cofactor requirement of the kinase, CIPK9, is altered in the presence of CBL2 and CBL3.
The effect of Mn2+ and Mg2+ on autophosphorylation of CIPK9 in the presence of CBL2/CBL3 was also tested. The results suggest that Mn2+ and Mg2+ are essential cofactors for activation of CIPK9 because in the presence of metal chelator EDTA or in the absence of Mn2+/Mg2+ (0 mM), CIPK9 autophosphorylation was completely abolished (Figure 5B–F). At 2.5 mM MnCl2, CIPK9 shows maximum autophosphorylation and transphosphorylation of CBL2/CBL3 and a further increase in MnCl2 seems inhibitory for CIPK9 kinase activity (Figure 5B,E). In the absence of CBL2/CBL3, CIPK9 required 10 mM Mn2+ for optimal kinase activity (Figure 2D). However, in the presence of CBL2/CBL3, the requirement of Mn2+ is lowered for autophosphorylation of CIPK9 and transphosphorylation of CBL2/CBL3. Similarly, in the absence of CBL2/CBL3, the autophosphorylation of CIPK9 was negligible even at 25 mM Mg2+ (Figure 2E) while in the presence of CBL2/CBL3, CIPK9 shows enhanced autophosphorylation and transphosphorylation of CBL2/CBL3 at a lower concentration of Mg2+. At the same time, a further increase (10 mM or more) in MgCl2 seems inhibitory for kinase activity as well as for CBL3 phosphorylation (Figure 5C,F). This result indicated that, in the presence of CBL2/CBL3, Mg2+ could serve as a major cofactor for activation of these complexes.
Prolonged kinase activity of CIPK9 in the presence of CBL3
Our kinase assays showed that CIPK9 is able to phosphorylate MBP, an artificial substrate. We performed a kinase assay and found that MBP phosphorylation by CIPK9 was enhanced with an increasing concentration of CBL3 (Figure 6A). We then performed the kinase assay in which CIPK9 was pre-incubated with CBL3 for 30 min, followed by addition of MBP, and the reaction was extended for another 30 min (Figure 6B). Based on the results, it was clearly observed that MBP phosphorylation was much higher when MBP was incubated simultaneously with CIPK9 and CBL3 (Figure 6B, lane 5), while the MBP phosphorylation was reduced when it was added after 30 min (Figure 6B, lane 7). This decrease in MBP phosphorylation may be due to prolonged exposure and hence reduced kinase activity. In another assay, CIPK9 was simultaneously incubated with MBP and after 30 min, CBL3 was added and the reaction was extended for an additional 30 min. Antithetical to the above, MBP phosphorylation was increased when CBL3 was added post 30 min of MBP incubation with CIPK9 (Figure 6C, lane 9), while the MBP phosphorylation was lesser when it was simultaneously incubated with CIPK9 and CBL3 (Figure 6C, lane 5). This leads us to conclude that possibly CBL3 further enhances kinase activity of CIPK9 and hence more phosphorylation of MBP. No such effects were observed when MBP was incubated with CBL3 and CIPK9ΔR. Therefore, it might be possible that CBL3 facilitates sustained kinase activity of CIPK9 for a longer period of time in the in vitro phosphorylation assay.
Prolong kinase activity of CIPK9 in the presence of CBL3.
CBL2/CBL3 enhances the activity of constitutively active CIPK9 and makes it a super-active kinase
According to previous reports, NAF/FISL motif of the regulatory domain of CIPK9 (PKS6) may interact with the activation loop and thus confer an autoinhibitory effect on its kinase activity in the absence of interacting CBLs . It has also been shown that T178D substitution in the activation loop of CIPK9 mimics phosphorylation and this version of mutated kinase becomes more active than the wild-type . It is believed that Thr178 in the activation loop serves as a target site for autophosphorylation or phosphorylation by the upstream kinase in the process of activation .
Several issues remain unresolved concerning the effect of CBLs on the activation process. If CBL interaction is to remove the autoinhibitory effect, then the constitutively active form of CIPK9 (T178D) may not require CBLs interaction for its activation. In parallel, we assume that the transphosphorylation of CBLs by the mutated CIPK9 may also be altered. To address these issues, the GST–CIPK9 construct was subjected to PCR-based site-directed mutagenesis to produce two mutations, CIPK9T178D and CIPK9T178A (Supplementary Figure S5A). Additionally, a dead CIPK9 kinase mutant was also generated where critical ATP-binding Lys (K) residue at the 48th amino acid position was changed into Asn (N) amino acid and named as CIPK9K48N (Supplementary Figure S5A). The CIPK9 variants CIPK9T178D, CIPK9178A and CIPK9K48N were expressed in E. coli BL21 strain as GST fusion proteins and purified by Glutathione–Sepharose beads-based affinity chromatography (Supplementary Figure S2I–L). In the kinase assay, CIPK9 and its variants showed autophosphorylation except for the dead kinase CIPK9K48N (Supplementary Figure S5B). As expected, CIPK9T178D was more active and its autophosphorylation was higher than the CIPK9-WT and CIPK9T178A variants. Surprisingly, CIPK9T178A (an inactive variant of the kinase) also showed autophosphorylation, albeit lower activity than CIPK9-WT (Supplementary Figure S5B). This finding suggests that T178 is not the only residue responsible for autophosphorylation of CIPK9, but other sites may also be phosphorylated and therefore might be involved in the kinase activation. CIPK9 and its variants did not phosphorylate GST protein (Supplementary Figures S3 and S5B).
Furthermore, the effect of CBL2/3 on the autophosphorylation of CIPK9 and its variants was investigated. The active mutated kinase CIPK9T178D showed higher autophosphorylation than the wild-type CIPK9 (Supplementary Figure S5B); hence, we used lesser amount (one-fifth) of CIPK9T178D protein (shown by *) in the kinase assay (Figure 7A,D). Interestingly, autophosphorylation of CIPK9T178D was further enhanced in the presence of CBL2/CBL3 (Figure 7A,C). The quantitative estimation of phosphorylation status by the ImageJ software also clearly shows that the addition of CBL2/CBL3 increased basic autophosphorylation of these variants. The kinase activity exhibited by CIPK9T178D was much higher than other variants in the presence of CBL2/CBL3 (Figure 7B,E). The phosphorylation status of CBL2/CBL3 was also measured in the presence of these variants, which indicated that the extent of CBL2/CBL3 phosphorylation by CIPK9T178D is higher than by other variants (Figure 7C,F). These results suggest that CIPK9 might have other phosphorylation sites besides T178 for its activation. It is also possible that there might be intramolecular conformational change and/or constitutive active kinase may also play a critical role in enhancement of autophosphorylation and substrate phosphorylation. It is reported that S164 in the putative activation loop of CIPK9 shows conservation with other CIPKs . It could be one of the probable phosphorylation sites for CIPK9 activation. This interesting observation was further confirmed by enhanced autophosphorylation of the CIPK9T178A variant in the presence of CBL2/CBL3 (Figure 7A,C). The dead kinase variant CIPK9K48N did not have kinase activity and hence ruled out the possibility of its activation by CBL2/CBL3. These results suggest that CIPK9 might be activated in the in vivo condition through CBL interaction, CBL phosphorylation and activation by phosphorylation at T178 and other sites in the activation loop. However, further detailed studies are required to understand the regulatory mechanism of CIPK9 autoactivation in the presence of CBLs, CBL phosphorylation by CIPKs and their downstream target activation under the in vivo condition. The present study also highlights how CIPK9 and its interacting CBLs have the potential to activate multiple signalling pathways by interacting with multiple CBLs through differential autoactivation and multiple substrates’ (CBLs) phosphorylation.
Constitutively active CIPK9 turns into super-active kinase in the presence of CBL2 and CBL3, indicating the presence of other phosphorylation sites.
The CBL–CIPK-mediated K+ uptake and homeostasis is a well-characterized signalling pathway in Arabidopsis. In the last decade, the Ca2+ sensor–kinase–channel complex, i.e. CBL1/CBL9–CIPK23–AKT1, was identified and characterized for K+ uptake [19,21,22]. Experiments with K+-selective microelectrodes depict that under variable extracellular K+-concentrations, [K+]ext, root cells maintain a constant K+ level by the release of vacuolar K+ . Thus, the stored vacuolar K+-pool is used as a flexible store for the maintenance of the cytosolic K+ level in the cells with the help of several tonoplastic K+-permeable channels under K+-deficient conditions . Another member of the CIPK family, CIPK9, was found to be up-regulated under K+-deficient condition, and mutation in CIPK9 shows hypersensitive growth under very low-K+ conditions (0.0–0.05 mM) . CIPK9 interacts with CBL2/CBL3 and localizes to the vacuolar membrane . It might be possible that CIPK9 regulates vacuolar K+-channels/transporters, thereby linking the CBL–CIPK9-transporter/channel via a cytoplasmic Ca2+ signal in response to fluctuation in the external K+ level.
CIPKs are activated by interaction with CBLs, which relieves the self-inhibition imposed by the regulatory domain on the kinase activity [3,8,9,39,43]. Recently, it was shown that along with CIPK activation via CBL binding, phosphorylation of the activation loop is required and these two steps are synergistic processes, indicating that the release of both the activation loop and the NAF motif from the catalytic domain is coupled [39,50]. Previously, it has been shown that CIPKs/PKS protein kinases phosphorylate the PFPF motif of interacting Ca2+ sensors CBLs/SCaBP [32,36,37]. In the biochemical analyses, PKS5 and PKS24 phosphorylate SCaBP1; CIPK23 phosphorylates CBL1/CBL9, whereas CIPK1 phosphorylates CBL1; and CIPK24 phosphorylates CBL4 [32,37]. In this study, we report for the first time how a single CIPK gets differentially activated in the presence of its interacting CBLs. The differential interaction of CIPK9 with CBLs prompted us to dissect the kinase activity as well as the phosphorylation status of interacting CBLs. Regulation of kinase activity generally results from protein phosphorylation by an upstream kinase(s), self-phosphorylation and/or by the involvement of regulatory domains [51,52]. CIPK9 phosphorylates all its interacting CBLs, but interestingly its effective substrates are CBL2/CBL3, not CBL1/CBL9 (Figure 3A). Surprisingly, we observed that CBL1 and CBL9 reduced the kinase activity of CIPK9. This type of differential activation of CIPK by specific CBLs has not been recognized previously.
To understand the specificity in CBL-mediated activation of CIPK9, we generated CIPK9ΔR (lacking the autoinhibitory domain of CIPK9). We found that the kinase became more active, and more importantly, it lost CBL-dependent activation. Interestingly, phosphorylation of interacting CBLs was still observed with the kinase domain only (lacking C-terminal regulatory domain) albeit to a lesser extent, suggesting that the C-terminal regulatory region is essential for differential activation of the kinase by CBLs but less so for substrate phosphorylation (Figure 3C–D).
Previous studies suggest that CBL phosphorylation by CIPKs may stabilize the CBL–CIPK complexes, as supported by the finding that mutation of Ser-201 (CIPK phosphorylation site) to Ala in CBL1 or CBL9 significantly decreased the interaction between CBL1/CBL9 and CIPK23 . It was also reported that CBL4 is phosphorylated by full-length CIPK24 but not by C-terminal-truncated CIPK24 . It is generally concluded that interacting CBLs activate CIPKs through physical interaction with the regulatory domain of CIPKs. However, some specific CBLs can still serve as substrates of partner CIPKs without the interacting domains. In other words, phosphorylation of CBLs by CIPKs may not require physical interaction between the CBLs and the C-terminal region of CIPKs.
CIPK9 preferred Mn2+ as a metal cofactor over Mg2+ for its optimal kinase activity (Figure 2D–E), consistent with the result in the original work on CIPK1 . An Arabidopsis receptor-like protein kinase, RLK5 , and several other CIPKs also prefer Mn2+ over Mg2+ for their autokinase activity [9,28,43,48,53]. In most of these studies, the kinase activity is examined in the absence of interacting CBLs. One important question arises — Do CIPKs have an altered cofactor requirement in the presence of CBLs? Indeed, here we show that lower levels of Mn2+ and Mg2+ are required for CIPK9 in the presence of CBL2/CBL3. In particular, the Mg2+ concentration required for CIPK9 activity falls into the physiological range of low mM, suggesting that CBL2/CBL3–CIPK9 complexes use Mg-ATP as a cofactor in the cell. Another interesting finding is that, in the presence of CBL2/CBL3, the autophosphorylation of CIPK9 and phosphorylation of these CBLs are Ca2+-dependent (Figure 5A,B). However, when the CBL-interacting domain of the CIPK9 was deleted (CIPK9ΔR), such Ca2+-dependent autophosphorylation or substrate phosphorylation disappeared (Supplementary Figure S4E). This indicates that CIPKs are not regulated by Ca2+ directly but become Ca2+-dependent when CBLs come into play, consistent with the general theme in which CBLs serve as Ca2+ sensors, while the CIPKs are effectors.
Previously, one of the important regulators of the low-K+ signalling pathway, i.e. CIPK23, was found to interact with CBL1, CBL2, CBL3 and CBL9 [21,24]. CIPK23 phosphorylates CBL1 and CBL9, but phosphorylation of CBL2 and CBL3 was not investigated . Moreover, CIPK23 does not show differential autophosphorylation in the presence of CBL1 and CBL9 . The kinase assay of CIPK23 with their interacting CBL2 and CBL3 will further provide more regulatory insights into AKT1-mediated K+ signalling in plants.
In a quest to identify the targets of CIPK9, we have identified a protein phosphatase 2C, AP2C1, which interacts with and dephosphorylates CIPK9 in vitro and negatively regulates CIPK9 activity . Because the ap2c1 mutant is more tolerant to the low-K condition, it suggests that in vivo association of CIPK9 with AP2C1 may negatively regulate CIPK9 under K+-deficient conditions in Arabidopsis. Thus, the CBL2/3–CIPK9–AP2C1 module might be acting as an alternate pathway to the established CBL1/9–CIPK23–AKT1–AIP1  module for improved K+ uptake and homeostasis under K+-deficient conditions. Moreover, a few other targets of CIPK9 have been identified. These include plasma membrane-localized Ca2+-ATPase, ACA8, which can be phosphorylated by CIPK9 and CIPK14 in a kinase assay . The same study also shows that CBL1–CIPK9-mediated phosphorylation of ACA8 shapes the cytosolic Ca2+ transients induced by mechanical injury of leaves . In two studies from different angles, CIPK9 and other three CIPKs including CIPK3, CIPK23 and CIPK26 are required for high Mg2+ tolerance [56,57]. While one study identified these CIPKs as interacting partners for ABA signalling components SnRK2s , the other study tracks these quartet CIPKs as targets of CBL2 and CBL3, which target the partner CIPKs to the tonoplast where the CBL–CIPK network activates a vacuolar transport system to sequester excessive Mg2+ under a high Mg2+ condition . The transporters/channels await further characterization.
In conclusion, this study has made several findings that may be further helpful in functional dissection of the CBL–CIPK pathways. First of all, although CIPK9 interacts with multiple CBLs including CBL1, CBL2, CBL3 and CBL9 in yeast two-hybrid assays, only CBL2 and CBL3 may be physiologically relevant partners because they significantly activate the partner kinase. Earlier studies observed the interaction of CIPKs with multiple CBLs, but differential regulation of CIPKs by CBLs has not been studied before. Secondly, differential phosphorylation of CBLs by CIPKs may be functionally relevant although genetic analysis will be required for understanding its functional implications. Thirdly, we found that the interaction with CBLs alters cofactor requirement for CIPKs. This is critical for the functional analysis of CIPKs in the future to avoid the artefact reported earlier when CIPKs were assayed without CBLs. Because CBL2 and CBL3 have been shown to be located at the tonoplast while the CIPK9 appears to function in several stress responses including high Mg2+ , low-K+ tolerance  and wounding , we hypothesize that CIPK9 may function downstream from CBLs activated by calcium signatures in response to these signals (Figure 8). Further in planta investigation will be required to understand these regulatory pathways.
A hypothetical model for CBLs-CIPK9 signalling in Arabidopsis.
bovine serum albumin
calcineurin B-like proteins
CBL-interacting protein kinases
myelin basic protein
2C-type protein phosphatases
protein phosphatase interaction
G.K.P. conceived and design the research plans. A.K.Y. and S.K.J. performed most of the experiments. S.K.S. performed site-directed mutagenesis. A.K.Y., S.L. and G.K.P. analysed the data. G.K.P., S.L. and A.K.Y. wrote the article.
This work is partially supported by the Department of Biotechnology (DBT), the Department of Science and Technology (DST), and University Grant Commission (DSRII grant) India to G.K.P. A.K.Y. thanks CSIR for a junior/senior research fellowship and S.K.J. acknowledges DST for an Inspire Fellowship. S.L. is supported by the National Science Foundation.
We are thankful to Dr Kailash C. Pandey (National Institute for Research in Environmental Health, Indian Council of Medical Research, Bhopal, India) for critical reading.
The Authors declare that there are no competing interests associated with the manuscript.
Present address: Sri Murli Manohar Town P.G. College, Ballia, Uttar Pradesh 277001, India.