Phospholipid Mediated Activation of Calcium Dependent Protein Kinase 1 (CaCDPK1) from Chickpea: A New Paradigm of Regulation

Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported here. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in Vmax of the enzyme activity by these phospholipids significantly decreased the Km indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K½ = 114 nM) compared to PA (K½ = 335 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response.

CDPKs exist as monomeric serine/threonine protein kinases consisting of four domains: an amino-terminal variable domain, a kinase domain, an auto-inhibitory domain and a regulatory domain (CaM-LD -calmodulin-like domain). Many CDPKs are predicted to have myristoylation and palmitoylation sites at their N-terminal domain which are essential for membrane anchorage [25,26].
In the absence of Ca 2+ , the auto-inhibitory domain acts as a pseudo-substrate which blocks the active site of the enzyme, thus keeping it in inactive state. However in presence of Ca 2+ , the enzyme undergoes conformational changes which remove the blocking and thus bringing the enzyme in active state [5].
The plasma membrane is a selective barrier between living cells and their environments and plays a key role in the perception and transmission of external information during stress condition. However, it can also serve as precursor for the generation of molecules like Inositol trisphosphate (IP3), Diacylgylcerol (DAG), Phosphatidylserine (PS), Phosphatidic acid (PA) etc. during stress exposure [27]. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes and exclusively present in membranes. Increase in PC concentration was found during drought, osmotic stress and cold stress [28,29] suggesting its possible increased turnover in response to stress.
PA is known to regulate activities of many kinases like MAPK [30], AtPDK1 [31] or phosphatases like ABI1 [32]. Involvement of PA in signaling and healing wounds was indicated by its binding to wound-specific ZmCPK11 [33]. Addition of phosphatidylinositol (PI), lysophosphatidylcholine (LysoPC) and crude phospholipids stimulated activities of an Oat CDPK and AtCPK1 [34,35] where as activity of recombinant DcCPK1 was stimulated by PA, PS, PI and phosphatidylethanolamine (PE) [36]. CPK11 from maize showed stimulated activities in presence of PA, PS and PI, but not in presence of PC, LysoPC, PE, diolein, cardiolipin [37]. All these studies indicate the possible role(s) of phospholipid in regulating activity of CDPKs.
Earlier we have reported isolation and characterization of CDPK1 gene from Cicer arietinum (designated as CaCDPK1) [38]. The expression of CaCDPK1 in leaves was enhanced in response to high salt stress and fungal infection suggesting its functional role in salt stress and fungal infection [39].
Since it is known that osmotic stress increased PA and PC concentration, we decided to look at the possible role(s) of these phospholipids in regulation of CaCDPK1 activity. We have also measured the catalytic parameters in presence of these phospholipids. In addition we also found that salt stress caused increase in concentration of PA in chickpea plants.
Crude phospholipid was isolated from egg yolk according to Bligh and Dyer method [40]. After extraction, phospholipids were concentrated by rotary evaporator and re-dissolved in chloroform: methanol (2:1v/v), and stored in 220uC. Before each experiment the appropriate amount of crude phospholipid was dried under vacuum, re-suspended in 50 mM of Tris-HCl pH 7.2 and sonicated for 10-15 min.

Expression and Purification of CaCDPK1
Over-expression and purification of CaCDPK1 was done as described previously [41]. In brief, the CaCDPK1 ORF was subcloned in pRSET A vector and was transformed in Escherichia coli BL21 strain. Cells were grown at 37uC with vigorous shaking until an absorbance of 0.6 at 600 nm was reached. After induction with 0.1 mM of isopropyl-3-D-thiogalactopyranoside, cells were grown further for 4 h at 25uC and then harvested by centrifugation.
Recombinant CaCDPK1 protein was purified under native conditions by affinity chromatography using Ni-NTA. Cells were re-suspended in lysis buffer pH 8.0 (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, 10% (v/v) glycerol, 1% (v/v) Triton X-100 containing 1 mM phenylmethylsulfonyl fluoride). Resuspended cells were lysed by adding lysozyme (1 mg/ml) followed by incubation for 30 min and sonication for 5-7 min in an icebath. Suspension was then centrifuged at 120006 g for 20 min. The supernatant was incubated with Ni -NTA slurry and mixed gently for 1 h at 4uC. The slurry was packed as a column and washed several times with wash buffer pH 8.0 (50 mM Tris-HCl, 300 mM NaCl, and 50 mM imidazole). Bound proteins were eluted with elution buffer pH 8.0 (50 mM Tris-HCl, 300 mM NaCl, and 300 mM imidazole). The protein elution was monitored by checking the fractions on 12% (w/v) SDS-polyacrylamide gel. Fractions containing the protein were pooled and dialyzed against the storage buffer (50 mM Tris -HCl pH 7.2, 150 mM NaCl, 1 mM DTT, and 10% glycerol) with a minimum of 4 changes. Protein concentrations were estimated by Bradford dyebinding method with BSA as the standard [42].

Protein kinase assay
Autophosphorylation and histone phosphorylation assays were carried out according to a previously reported protocol [41]. In brief, the assay mixture contained histone III-S (1 mg/ml), Ca 2+ / EGTA buffer (50 mM Tris-HCl, pH 7.2, 10 mM MgCl 2 and 1 mM EGTA) and 50 ng of the purified recombinant CaCDPK1 in presence or absence of 1.2 mM CaCl 2 . Unless mentioned, in all assays calcium is used as Ca 2+ /EGTA buffer. Autophosphorylation assays were done in same conditions as substrate phosphorylation, except 500 ng of CaCDPK1 was used and histone III-S was omitted. c 32 -P ATP stock was prepared by mixing the 1 mM cold ATP with 32 P labeled ATP. Reactions were initiated by addition of c 32 -P ATP (300 cpm/pmole) and incubated for 10 min at 37uC. After 10 min the reactions were terminated by spotting it on P81 phosphocellulose papers. P81 papers were immediately washed three times with 150 mM H 3 PO 4 , and once with acetone. Papers were dried and 32 P incorporation was counted in a liquid scintillation counter (Beckman Counter). For autoradiography the reactions were stopped by addition of 16 SDS loading dye and were separated on a 12% (w/v) SDS-PAGE and subjected to autoradiography.
To determine initial velocities in presence of phospholipids, protein kinase assays were carried out as described above using histone III-S at concentrations ranging from 1 to 200 mM, for 10 min presence of 200 mM of PA or PC. K m and V max were calculated from Lineweaver-Burk (1/V Vs 1/S) plot.

Handling of lipids
Appropriate amounts of phospholipids were dissolved in chloroform/methanol (9:1 v/v) and dried under vacuum and lipids were re-suspended in 50 mM of Tris-HCl pH 7.2 and sonicated till turbidity of lipid samples attains a constant value (10-15 min). The solutions were kept at room temperature for 30 min and then used in CaCDPK1 assay.
In vivo -32 P labeling and salt treatment of chickpea Chickpea (Cicer arietinum L. cv. Kabuli) seeds were sterilized and grown on wet paper towel for 5 days in dark. Salt treatment experiments were done according Darwish et al [43] with minor modifications. Equally grown 5 days old seedlings were transferred to 2.5 mM MES buffer (pH 5.7) and 10 mM KCl. For phospholipids labeling, 5 mCi 32 P-H 3 PO 4 was added per tube and incubated for 15 min. Salt treatments were started by transferring the seedlings to 2.5 mM MES buffer containing 300 mM NaCl for 15 min. Reaction was stopped by freezing the seedling in liquid nitrogen. Seedlings were crushed and 400 ml of chloroform/methanol/HCl (50:100:1, (v:v:v)) was added to the mixture, followed by vigorous shaking for 10 min. Two phases were induced by addition of 400 ml CHCl 3 and 200 ml of 0.9% (w/v) NaCl. The organic phase was collected and mixed with 400 ml chloroform/methanol/HCl (3:48:47, (v:v:v)). Repeated shaking, spinning and removing the upper phase yielded a purified organic phase. The organic phase was dried by vacuum centrifuge and re-suspended in minimal amount of CHCl 3 . The phospholipids were separated on TLC using solvent system chloroform:acetone:methanol:acetic acid:H 2 O(10:4:3:2:1(v/v)). Labeled PA was identified by co-migrating standard PA. Radioactivity was visualized by autoradiography and quantified by phosphoimaging. The difference in the amount of PA formed was calculated by subtracting the radioactivity of treated cells by that of non-treated seedlings.

Fluorescence studies
Fluorescence emission spectra were recorded at 25uC in a Perkin-Elmer luminescence spectrophotometer. Intrinsic spectrum of CaCDPK1 protein was recorded (1 mM) in buffer containing 50 mM Tris-HCl (pH 7.2), 150 mM NaCl and 1 mM DTT using 280 nm as excitation wavelength (slit 5 nm) and 300-420 nm (slit 5 nm) as emission range. CaCDPK1was titrated with increasing amount of PA or PC vesicles. Changes in fluorescence at 341 nm (F 0 -F i ) was plotted against phospholipid vesicle concentrations where F 0 is fluorescence intensity at zero concentration of phospholipid vesicles and F i is fluorescence intensity at given concentration of phospholipid vesicles. K 1/2 was calculated from this graph. Care was taken to avoid scattering or inner filter effect. K 1/2 values were calculated as the concentration of phospholipid required for a half-maximal change in fluorescence.

Activation of CaCDPK1 by phospholipids
Using histone as exogenous substrate, kinase activity of CaCDPK1 was measured in presence or absence of crude phospholipids. Crude phospholipid stimulated the kinase activity of the enzyme by 80 % indicating the requirement of phospholipid for maximum activity (Fig. 1).
PA stimulated autophoshorylation activity as well as histone phoshorylation activity (   the presence PC autophosphorylation ( Fig. 2A lane 4) and histone phosphorylation activities (Fig. 2 A and B lane 4) were stimulated to a higher degree than other phospholipids tested.

Role of calcium and phospholipids in CaCDPK1 activity
The activity of CaCDPK1 was enhanced by PA and PC only in presence of calcium, as PA and PC failed to stimulate CaCDPK1 activity in absence of calcium ( Figure 2C and D). Adding N-(6aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7), a calmodulin antagonist, in assays, affected the calcium dependent activation as well as phospholipid dependent activation of CaCDPK1 ( Figure 2C and D). Together the results obtained indicated that CaCDPK1 required Ca 2+ , for its enzyme activity, and the Ca 2+ -dependent activity was further enhanced by phospholipids.
PA stimulated autophosphorylation and substrate phosphorylation activities in a dose dependent manner (Fig. 3 A and B). Maximum activity was observed at a concentration 200 mM of PA. At this concentration, 58% stimulation in autophosphorylation activity and 62 % stimulation in histone phoshorylation activity  were observed. At higher concentrations, however, PA seems to be inhibitory for the both activities of the enzyme. Kinetics parameters were measured in presence of PA (200 mM) using histone as substrate (Fig. 3C). The values of V max and Km were found to be 123 nmoles/min/mg protein and 16 mM in the presence of PA and 13.2 nmoles/min/mg proteins and 34.3 mM in the absence of any phospholipid [41], respectively (Table 1). Thus, addition of PA decreased the K m value by 2 fold and increased the V max value by 9-fold.
Both activities of autophosphorylation and substrate phosphorylation were stimulated by PC in a dose dependent manner (Fig. 4  A and B). About 60% activation was observed at optimum concentration of PC. Catalytic parameters of the enzyme were calculated using histone as the substrate in presence of 200 mM of PC (Fig. 4C). The values of V max and K m of the enzyme were found to be 285 nmoles/min/mg protein and 10.5 mM (Table 1).
In the presence of PC, 3.2-fold decrease in K m and 21-fold increase in V max were observed.
It is of interest to note that another important membrane phospholipid, PE, failed to stimulate CaCDPK1 activity (Fig. 5 A). This indicated the specificity of PA and PC for activation of the enzyme.
Lack of effect of diacylglycerol on CaCDPK1 activity DAG stimulated the activity of CaCDPK1 only at high, unphysiological concentrations (Fig. 5B). At concentrations between 50-400 mM, comparable to those used for PC and PA, diacylgylcerol failed to stimulate the CaCDPK1 activity (Fig. S1). The foregoing data demonstrate that PC is the most effective activator of this plant enzyme, CaCDPK1, when compared to PA and DAG

P incorporation into phosphatidic acid during salt stress
Increase in phosphatidic acid content in response to various environmental stress conditions is known in plants [43]. We investigated the response in 5-day old chickpea seedlings subjected to salt stress for 15 min by radio labeling PA with 32 P-phosphate. Under the stress condition, incorporation of 32 P into PA increased by about 2-fold (Fig. 6).

Binding of phospholipid vesicles to CaCDPK1
CaCDPK1 exhibited an emission maximum of 341 nm showing the presence of tryptophan residues exposed to aqueous environment. Binding of PA and PC vesicles to CaCDPK1 was monitored by recording fluorescence emission spectra in the presence of calcium with varying phospholipid concentrations. Changes in fluorescence intensity as the function of PA and PC concentration at a fixed CaCDPK1 concentration are shown in Fig. 7A and B, respectively. Quenching of fluorescence emission of CaCDPK1 on addition of phospholipid vesicles indicated re-localization of the tryptophan residues into a relatively more hydrophobic environment. Binding studies of phospholipid vesicles to CaCDPK1 had to be carried out in a limited concentration range as lower concentrations did not induce significant change in fluorescence emission and higher concentrations caused scattering. Half maximal change in fluorescence intensity with PC (K K = 114 nM) was lower than with PA (K K = 335 nM) indicating more efficient binding of PC to CaCDPK1, correlating with its higher activity (Fig. 7C).
Fluorescence emission spectroscopy showed quenching in fluorescence emission after binding to PA vesicles indicating that PA physically interacts with CaCDPK1 and showed K K of 335 nM. Several PA-binding proteins have been identified but there is no consensus sequence of the binding site. Different amino acid residues participate in PA binding in different proteins. Deletion of a KKR motif in the Opi1 transcription factor abolished PA binding to the protein. CaCDPK1 protein also contains a KKR motif (Fig. S2) and its involvement in PA binding will require further studies on deletion of this motif.
Plants utilize one of their two major pathways for PA production, via phospholipase D (PLD) or phospholipase C/ diacylgycerol kinase (PLC/DGK), depending on the nature of stress or signal [47]. Salt stress causes accumulation of PA by PLC/DGK pathway. Generation of PA by PLC/DGK, a fast  reaction, occurs in min after imposing stress on the plant, and is usually monitored by the widely used method of incorporation of labeled 32 P into diacylglycerol by DAK to produce PA. Our study confirmed increased incorporation of 32 P in PA during salt stress indicating increased availability of PA.
PC is the most abundant phospholipid in eukaryotic membranes and exclusively present in membranes. Some studies showed that PC synthesis increased during salt stress, drought and cold stress [29] indicating its increased turnover during stress conditions, particularly the salt stress. PC is presently not considered signaling molecule. Till now not many CDPKs are known to be activated by PC. Pure PC did not significantly stimulate the kinase activity of AtCPK1 [35] and ZmCDPK11 [33], and only small stimulation was found in case of Oat CDPK [34]. To best of our knowledge this is the first time we are reporting strong stimulation of CDPK by PC. Moreover, it was also reported that AtPLAs are regulated by CDPK which enhanced AtPLAs activities on phosphatidylcholine indicating involvement of CDPKs in PLA medicated signaling [48]. In view of selective and efficient activation of CaCDPK1 by PC shown here, we propose that it may be involved in membraneanchoring this protein in fully activated form. Activation of kinase and binding to CaCDPK1 of PC was found to be stronger than PA hinting a specific role in regulating CaCDPK1 activity. Indeed inter-conversion of PC and PA might afford the required modulation of the activity within a range of the already calciumactivated form of the enzyme and give an insight into lipid signaling during stress conditions.

Conclusions
We demonstrate in the present study activation of CaCDPK1 by PC and PA, but not by PE or diacylglycerol. Both phospholipids were able to bind to CaCDPK1 and increased its V max and affinity towards the exogenous substrate, histone. Figure S1 CaCDPK1 activity in presence of diacylgylcerol (50-400 mM). Kinase activity was measured in presence of diacylglycerol. The reaction mixture contained 50 ng of CaCDPK1 in 50 mM Tris-HCl buffer (pH 7.2), 1.2 mM CaCl 2 , 1 mM EGTA, 10 mM MgCl 2 , 1 mg/ml histone and indicated amounts of DAG. Reactions were stopped by spotting the reaction mixtures on P81 phosphocellulose papers and were immediately processed as described in ''Material and methods''. (TIF)