Journal of Experimental Botany, Vol. 52, No. 357, pp. 691-700,
April 15, 2001
© 2001 Oxford University Press
Original Papers |
Zea mays CCaMK: autophosphorylation-dependent substrate phosphorylation and down-regulation by red light
1 School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
2 International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India
Received 29 March 2000; Accepted 15 October 2000
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The role of protein kinases has been extensively studied in various signal transduction pathways and they are one of the most important components that link the signal perception to the final response. However, not many studies have been reported, especially from the plant systems, that show the regulation pattern of the kinase itself under different conditions. A calcium/calmodulin-dependent protein kinase has already been purified and characterized from etiolated maize coleoptiles. In this paper a detailed study of how the kinase itself is regulated at the autophosphorylation level is provided. Evidence is also given that the autophosphorylation of kinase effects its activity towards substrate phosphorylation. It is further shown that the kinase is an important component of the light signalling pathway as the level of the kinase itself decreases by red light irradiation.
Key words: Autophosphorylation, Ca2+/CaM-dependent protein kinase, red light, Zea mays.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant cells can respond to various signals in a precise and specific manner due to the presence of a complex but efficient perception and signal transduction apparatus. Following perception, a signal is decoded via a change in concentration of a plethora of second messengers such as inositol phosphates, cADPR, diacyl glycerol, and calcium. Calcium ions are the ubiquitous signal molecules and their role is central to the theme of signalling in both plants and animal systems. They are the most widely accepted second messengers, which couple a large number of diverse stimuli to their characteristic responses (Trewavas and Malho, 1997
; Wu et al., 1996, 1997; Pandey et al., 2000).
The information encoded in the calcium signals can be de-coded downstream generally by two distinct signal transduction pathways, i.e. one involving calmodulin, which is an universal calcium receptor and other involving calcium-dependent protein kinases. Both these pathways are inter-linked and they also interact with a large number of other factors down the signal transduction cascade, working either independently or jointly. Thus all these components, i.e. CaM, CaM-binding proteins and calcium-dependent protein kinases interact with each other at different levels and form a very complex network (Poovaiah and Reddy, 1993; Bush, 1995). Calcium-dependent protein kinases are one of the most important components of this signalling network as they transduce different signals mediated by calcium in a specific and precise manner (Stone and Walker, 1995).
Calcium-dependent protein kinases can be divided into two major categories based on the components they require for their activation, i.e. Ca2+/phospholipid-dependent protein kinases and Ca2+/calmodulin-dependent protein kinases. Ca2+/CaM-dependent kinases are the main transducers of various calcium signals in animal systems. Some of them are multifunctional, for example, CaMK II, CaMK IV and CaMK Ia/Ib as they phosphorylate a variety of substrates in vivo whereas others are more dedicated (Hanson and Schulman, 1992). In plants, the main transducers of calcium signals known to date are kinases from the CDPK family, but recent evidence for the presence of calmodulin-dependent kinases (Watillon et al., 1995; Lu et al., 1996; Takezawa et al., 1996), suggest the possibility of involvement of these CaM kinases in a novel signalling pathway parallel to the CDPK-mediated signalling.
It is very well known that most of the Ca2+ signal transduction processes are mediated by a change in the phosphorylation/dephosphorylation pattern of related proteins in plants (Huber et al., 1994; Monroy et al., 1997), but only in a few cases have the Ca2+-dependent kinases been identified and characterized. Most of the studies are based on the observations that transcript levels of the protein kinase homologues change in response to a particular stimulus. (Anderberg and Walker-Simmons, 1992; Sano and Youssefien, 1994; Botella et al., 1996). Various stress signals which act through the modulation of [Ca2+]cyt level, have also been shown to act by modulating CDPKs (Sheen, 1996). Light-mediated responses have been shown to be accompanied by changes in cytoplasmic calcium concentration (Tretyn et al., 1991; Chory, 1994, 1997). Red light-mediated responses have been studied in maize coleoptiles and mesophyll protoplasts of wheat and a fast transient increase in calcium levels have been observed (Shacklock et al., 1992). In Chlamydomonas both calcium channel blockers and calmodulin antagonists affect the induction of the gsa gene suggesting that calcium and calmodulin are involved in a signal transduction pathway linking the blue light perception and induction of the gene (Im et al., 1996). Ca2+ and CaM have been shown to be directly involved in the regulation of gene expression in phytochrome-mediated responses (Neuhaus et al., 1993; Bowler et al., 1994; Wu et al., 1996; Mustilli and Bowler, 1997; Neuhaus et al., 1997), but so far no report for direct modulation of a kinase level in response to light has been observed.
Earlier, the biochemical characterization of a Ca2+/CaM-dependent protein kinase from etiolated maize coleoptiles was reported (Pandey and Sopory, 1998). This kinase (ZmCCaMK) has turned out to be novel, showing similarity to both animal system CaM kinases and plant CDPKs. The autophosphorylation of ZmCCaMK was studied in detail, and compared with the autophosphorylation activity of other known kinases as well as with its own substrate phosphorylation activity. Ca2+/CaM-stimulated autophosphorylation is a prominent characteristic of multifunctional CaM kinases in animal systems and it has been shown conclusively to play a role in the regulation of kinase activity (Kwiatkowski et al., 1988). In plants, there are few studies related to the regulation of a kinase activity by its autophosphorylation. In this study, it is shown that autophosphorylation of ZmCCaMK regulates the kinase activity towards its substrate phosphorylation. In order to elucidate the possible functional role(s) played by this kinase in signalling, its expression level was studied under different developmental conditions. It is shown for the first time that the ZmCCaMK level itself changes in response to red light, indicating that this kinase may be involved in the light-dependent signal transduction pathway.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Materials
-32P ATP (specific activity 3000 Ci mM-1) and 45Ca2+(specific activity 29.08 mCi mg-1) was obtained from Bhabha Atomic Research Centre, India. DEAE Sephacel and MW markers were obtained from Pharmacia. Histone IIIS, syntide-2, BSA, chlorpromazine (CPZ), trifluopromazine (TFP), compound 48/80, W7, H7, staurosporine, KN-62, PMSF (phenyl methyl sulphonyl fluoride), aprotinin, dithiothreitol (DTT), HEPES, EGTA, EDTA, CaM, CaM Sepharose, BCIP/NBT (5-bromo-4-chloro-3-indocyl phosphate/nitroblue tetrazolium), and alkaline phosphatase linked anti-rabbit IgG were from Sigma chemical Co. All the other chemicals were of AR grade.
Plant material and light treatments
Maize seeds (Zea mays var. Ganga 5) were grown at 25±2 °C for 8 d in dark on moist germination paper. Etiolated coleoptiles were harvested in dark under green safe light and frozen immediately in liquid N2.
Different light treatments were given to 8-d-old plants, grown in continuous darkness. Red (
max.600 nm), far red (max. 730 nm) and blue light (max. 427 nm) of desired intensities were obtained from respective light emitting diodes (Quantrum devices, Wisconsin USA). White light was obtained from fluorescent tubes (intensity 1200 µW cm-2). All the treatments were given under green safe light obtained from a white fluorescent tube covered with several layers of green cellophane paper (max. 500 nm).
Protein purification
The kinase was purified from 8-d-old, etiolated, maize coleoptiles essentially as reported earlier (Pandey and Sopory, 1998). Briefly, the tissue was ground in the presence of liquid N2 to a fine powder and mixed with 3 vols of extraction buffer (20 mM HEPES, pH 7.5, 5 mM EGTA, 2 mM EDTA, 2 mM PMSF, 5 mM DTT, and 10% glycerol v/v). The slurry was centrifuged at 15 000 g, 4 °C for 45 min and the pellet of cell debris was discarded. The supernatant was ultracentrifuged at 100 000 g, 4 °C for 1 h to remove the membranes and undissolved material. Crude protein extract thus obtained was further fractionated by 40–50% ammonium sulphate precipitation. Precipitated proteins were proceeded for ion exchange chromatography.
A DEAE Sephacel column was pre-equilibrated with equilibration buffer (10 mM HEPES, pH 7.5, 5 mM EGTA, 2 mM EDTA, and 10% glycerol v/v). The proteins after binding were washed extensively with washing buffer (equilibration buffer with 10 mMß-mercaptoethanol) and eluted with a linear gradient of 0–0.4 M KCl. Alternate fractions were assayed for Ca2+-dependent protein kinase activity. The active fractions were pooled and proceeded for affinity chromatography using CaM Sepharose.
Pooled proteins were dialysed extensively against 40 vols of dialysis buffer (10 mM HEPES, pH 7.5, 200 mM CaCl2, 100 mM NaCl, 1 mM DTT, 0.2 mM PMSF, 10 mg ml-1 aprotinin, and 10% glycerol v/v) and bound on a CaM Sepharose column pre-equilibrated with the same dialysis buffer. Non-specifically bound proteins were removed by washing the column with 1 M NaCl. Specifically bound proteins were step eluted with one column volume of elution buffer containing 1–4 mM EGTA and assayed for Ca2+/CaM-dependent kinase activity.
Active fractions were run on 10% SDS-PAGE and all the four polypeptides were excised separately. Gel containing the 72 kDa polypeptides was cut into small pieces and transferred to elution buffer (20 mM HEPES, pH 7.5, 5 mM DTT, 2 mM PMSF, 10 mg ml-1 aprotinin, and 20% v/v glycerol). After overnight incubation at 4 °C, the slurry was centrifuged and supernatant containing eluted protein was run on 12% SDS-PAGE to check the purity and used for further characterization and for raising antibodies as described earlier (Pandey and Sopory, 1998).
Protein kinase assay
Protein kinase assay was performed as reported earlier (Pandey and Sopory, 1998). Purified enzyme (0.2 ng µl-1) was incubated in 30 mM HEPES (pH 7.5) buffer containing 5 mM MgCl2, 0.5 mM DTT and 100 µM of -32P-ATP with syntide-2 (20 µM) in a total reaction volume of 100 µl. Assay was performed at 30 °C for 5 min. The reaction was stopped either by spotting 10 µl of reaction mixture on P81 phospho-cellulose paper (Whatman) or by adding equal volume of SDS sample buffer to it. P81 paper was washed thoroughly with 75 mM phosphoric acid for 45 min with frequent changes. The paper was dried under a lamp and placed in scintillation vial containing cocktail-O and counts were recorded in a liquid scintillation counter (Beckman). When the reaction was stopped by the addition of SDS sample buffer, the mixture was boiled at 100 °C for 5 min and run on a 10% SDS-PAGE. The gel was dried and exposed for autoradiography. For inhibitor studies various Ca2+/CaM inhibitors were incubated with the reaction mixture with proper controls. To check the effect of different pH buffers, the reaction was performed in presence of 0.25 M citrate buffer (for pH 3–6) and 0.25 M TRIS buffer (for pH 7–10).
Autophosphorylation
Purified protein (5 µg) was incubated in autophosphorylation buffer (20 mM HEPES, pH 7.5, 3 mM MnCl2, 10 µM ZnCl2, 50 µM DTT, 0.2% NP-40, 10 mg ml-1 aprotinin). To start the reaction, 10 µM of -32P-ATP was added and the reaction mixture was incubated at 30 °C for 5 min. The reaction was stopped by adding equal volume of SDS sample buffer; boiled for 5 min, and run on 10% SDS-PAGE. The gel was dried and exposed for autoradiography (Dasgupta, 1994). For inhibitor studies, the inhibitors were included in the reaction mixture and to check the effect of pH the reaction was performed in the presence of buffers of different pH values as in the case of substrate phosphorylation.
To check the effect of autophosphorylation on substrate phosphorylation activity of ZmCCaMK, purified kinase was autophosphorylated, and then acetone precipitated. The protein was air-dried and then used for substrate phosphorylation studies in the presence or absence of 2 mM EGTA.
For kinetic studies, tubes containing both autophosphorylation and substrate phosphorylation reaction mixes were simultaneously incubated at 30 °C and small aliquots were taken out on P81 paper, at different time intervals, starting from 1 min up to 30 min. Counts incorporated were determined using scintillation counter.
Phospho amino acid analysis
Phospho amino acid analysis of phosphorylated proteins was performed as described previously (Cooper et al., 1983) to determine which amino acid residue was getting phosphorylated. Briefly the phosphorylated band was cut from the gel and digested with 5.7 N HCl at 110 °C for 2 h. Digested protein was dried under vacuum and run on an ascending paper chromatogram using propionic acid, 1 M NH4OH, isopropanol (45:17.5:17.5, by vol.) as solvent, along with phosphoserine, phosphothreonine and phosphotyrosine as standards. Standards were visualized by ninhydrin reagent and the chromatogram containing phosphorylated proteins was autoradiographed.
Preparation of total protein extract and Western blotting
For preparation of total protein extract, the tissue was ground in the presence of liquid N2 to a fine powder and mixed with 3 vols of extraction buffer (20 mM HEPES, pH 7.5, 5 mM EGTA, 2 mM EDTA, 2 mM PMSF, 5 mM DTT, and 10% glycerol v/v). The slurry was centrifuged at 15000 g, 4 °C for 45 min and the pellet of cell debris was discarded. The clear supernatant obtained was used for Western blotting. For Western blotting 20 µg of total protein was separated on 10% SDS-PAGE and electro-blotted on nitro-cellulose membrane using Semi-dry transfer apparatus for 1.5 h. Membrane was blocked with 3% BSA in TBS (150 mM NaCl and 10 mM TRIS buffer, pH 7.4) for 1 h at 37 °C with shaking and probed with anti-kinase antibodies at a dilution of 1:20000 in TBS containing 1% BSA and 0.05% Tween-20 for 1 h at 37 °C. Alkaline phosphatase linked anti-rabbit IgG (Sigma Chemical Co.) was used as secondary antibody at a dilution of 1:30000 in TBS containing 1% BSA and incubated for 1 h at 37 °C. Blot was developed with BCIP/NBT solution till a purple-blue colour appeared.
Other methods
Protein estimation was done by using BSA as a standard (Bradford, 1976). SDS-PAGE was done according to Laemmli (Laemmli, 1970) and silver staining of proteins was done according to Blum et al. (Blum et al., 1986).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Autophosphorylation of Zea mays CCaMK is at threonine residue(s) whereas substrate phosphorylation is at serine residue(s)
Purified ZmCCaMK was autophosphorylated by incubating it in the presence of 10 µmol of free calcium as described in the Materials and methods. As shown in Fig. 1
, the purified kinase autophosphorylated, resulting in the presence of a 72 kDa band on the autoradiogram. To determine which amino acid was phosphorylated, phospho amino acid analysis was done with purified ZmCCaMK, as well as with substrates such as histone IIIS and syntide-2. As seen in Fig. 1, both the substrates were phosphorylated at serine residue(s); ZmCCaMK autophosphorylated at threonine residue(s) showing this kinase belongs to the serine/threonine family of protein kinases.
|
Autophosphorylation and substrate phosphorylation activities of Zea mays CCaMK are differentially regulated in response to various physiological factors
It has been shown earlier that purified ZmCCaMK undergoes a calcium-dependent but CaM-independent autophosphorylation. In this study it is further shown that calcium is the only physiological factor that affects the autophosphorylation activity of ZmCCaMK. The purified ZmCCaMK was autophosphorylated in the presence of various exogenous physiological factors. The purified protein kinase was also used to phosphorylate syntide-2 under similar conditions. As evident from the autoradiogram (Fig. 2A), the autophosphorylation activity of the ZmCCaMK was fully calcium-dependent and no activity could be detected in the presence of EGTA (200 µmol). The addition of 10 µmol calcium optimally stimulated the kinase activity. Autophosphorylation was unaffected by the addition of calmodulin at all the concentrations tested. But the syntide-2 phosphorylation activity, which is also fully calcium dependent, showed a further 2.5-fold stimulation with calmodulin. CaM alone had no effect on autophosphorylation, similar to the substrate phosphorylation activity. Other factors tested such as PS and PMA also failed to affect both the autophosphorylation as well as the substrate phosphorylation activity of the ZmCCaMK. The data show that calcium is the only exogenous factor that affects autophosphorylation of ZmCCaMK. Furthermore, CaM which affects the substrate phosphorylation activity of the same kinase has no effect on autophosphorylation. The activity of this kinase thus shows a distinct dual regulation by calcium and CaM. This is in contrast to other known plant CDPKs where the activity is only calcium dependent, and CaM has no effect. The ZmCCaMK is also distinct from the lily anther kinase, which showed an inhibition of autophosphorylation activity in the presence of CaM (Takezawa et al., 1996).
|
As autophosphorylation showed absolute calcium dependency, the effect of various calcium and kinase inhibitors on the activity of the kinase was determined. To confirm further if CaM has any role during autophosphorylation, the effect of various CaM antagonists on autophosphorylation activity of ZmCCaMK was also analysed. Syntide-2 phosphorylation using purified kinase was also performed under similar conditions. As evident from autoradiogram (Fig. 2B
), EGTA inhibited both the autophosphorylation and syntide-2 phosphorylation activities almost completely. Substrate phosphorylation activity was inhibited to different extents with different CaM antagonists and kinase inhibitors. But in sharp contrast, CaM antagonists as CPZ, TFP and W7, as well as various kinase inhibitors such as 48/80, H7, KN-62, and staurosporine had no effect on autophosphorylation activity of the kinase. These data give further evidence that the autophosphorylation and substrate phosphorylation activities of the kinase are differentially regulated.
The effect of pH on autophosphorylation and substrate phosphorylation activity of ZmCCaMK was determined by performing the reaction under different pH conditions. As is clear from Fig. 2C, both the autophosphorylation and substrate phosphorylation activities were pH-dependent, but each showed a different optimum pH value. The autophosphorylation activity could not be detected below pH 5 and above pH 8, with an optimum activity at pH 6, whereas syntide-2 was phosphorylated with a pH optimum of 7.5, showing that the two activities are differentially regulated by pH.
Autophosphorylation precedes the substrate phosphorylation and makes the Zea mays CCaMK Ca2+-independent towards substrate phosphorylation
Time kinetic studies of both substrate phosphorylation and autophosphorylation activities were performed with ZmCCaMK. Figure 3A shows that the autophosphorylation activity of the kinase was faster, attaining the saturation level at approximately 1 min and then going down slightly over a period of 30 min. The substrate phosphorylation activity, however, was comparatively slower, attaining the saturation level at 4 min and remained constant over a period of 30 min. This indicated that the autophosphorylation activity of the kinase is preceded by the substrate phosphorylation activity. It is known from animal systems, that autophosphorylation modulates the activity of CaM kinases and makes them calcium-independent (Lai et al., 1986; Miller and Kennedy, 1986; Hanson et al., 1994). Since ZmCCaMK showed many properties similar to the animal system CaM kinases, it was determined whether autophosphorylation could induce calcium independence towards substrate phosphorylation activity. Using non-autophosphorylated kinase for the assay, no substrate phosphorylation could be achieved in the presence of 2 mM EGTA. If autophosphorylated kinase was used for the assay, even in the presence of 2 mM EGTA, histone IIIS phosphorylation could be achieved (Fig. 3B). This shows that autophosphorylation induced calcium-independence in ZmCCaMK activity.
|
Expression of Zea mays CCaMK is tissue specific and development-dependent
As ZmCCaMK was purified from etiolated coleoptiles of maize plants, its presence was determined in other plant parts as well. Western blot analysis of protein extracts from leaf, hypocotyl, stem and roots of maize showed that the kinase has differential expression (Fig. 4A). Leaves showed the maximum expression of kinase while the level was minimum in roots. The stem portion and hypocotyl also expressed a lesser amount of protein compared to leaf. Although the level of expression of this kinase was different in various plant parts, it was not localized specifically to a particular tissue or organ. Expression of ZmCCaMK was also studied with different ages of maize seedlings. Probing total protein extracts of maize seeds (taken as 0 d), 3 d, 5 d, 8 d, and 10-d-old maize coleoptiles with the antibodies showed an age-dependent expression of ZmCCaMK. Seeds had very low level of protein, followed by 3-d-old coleoptiles. By 5 d the level of protein was high and it remained constant for up to 10 d (Fig. 4B).
|
Zea mays CCaMK expression level is down-regulated by red light
Light is one of the main environmental signals, which affect the growth and development of plants at every stage, mostly via phosphorylation/dephosphorylation. Since ZmCCaMK was initially purified from etiolated tissues, the effect of different light radiation on expression levels of the kinase was sought. Eight-day-old etiolated cut coleoptiles were given different light treatments (as described in the Materials and methods) and non-treated etiolated, cut coleoptiles served as the control. Equal quantities of total protein extract of all the treatments were separated on SDS-PAGE, blotted and probed with the ZmCCaMK antibodies. The blot showed a clear difference in expression level of the kinase with different light treatments (Fig. 5). Red light treatment for 5 min decreased the kinase level in comparison to dark-grown (control) coleoptiles. Red light treatment followed by far-red light treatment did not restore the level of kinase Far red light alone had no effect on the level of the kinase. Incubating coleoptiles treated with 5 min red light in the dark for 2 h decreased the kinase level even further. White light treatment for 5 min also down-regulated the ZmCCaMK level slightly.
|
As red light decreased the ZmCCaMK level, a concentration and time kinetics of red light treatment was performed. Etiolated maize coleoptiles were treated with red light for 5 min, 15 min, 30 min, and 2 h continuously; dark-grown, non-treated plants served as the control. Blots containing equal amounts of total protein probed with the ZmCCaMK antibodies showed that red light lowered the level of kinase with time (Fig. 6A
) and very little amount of the ZmCCaMK could be detected after 2 h of red light treatment.
|
To check the intensity of red light required for this effect, the etiolated coleoptiles were treated with 25, 50, 75, and 100 µmol min-1 of red light. As seen in Fig. 6B
, red light at 75 and 100 µmol min-1 intensity decreased the level of kinase in a concentration-dependent manner.
Because red light lowered the level of ZmCCaMK, but no far-red reversibility was seen, the role of blue light was tested on the expression level of this kinase. A blue light kinetics was performed starting from 5 min to 15 min, 30 min and 2 h similar to red light kinetics. Equal quantities of protein separated on SDS-PAGE, blotted and probed with the ZmCCaMK antibodies showed no effect of blue light treatment (Fig. 7A) and the level remained same in dark and blue light-treated plants. No difference in the level of kinase could be seen at any of the intensities of blue light (Fig. 7B) confirming that the response is specifically mediated at a particular intensity of red light.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Autophosphorylation is an important property of kinases reported both from the plants as well as animal systems. Ca2+ and CaM affect the autophosphorylation of kinases in different ways. In animal systems CaM kinases, both Ca2+ and CaM are required for autophosphorylation and this activity is inhibited by EGTA as well as by various CaM inhibitors. In plants, the calcium-dependent protein kinases (CDPKs) undergo autophosphorylation in a fully calcium-dependent manner. Although CDPK autophosphorylation activity is not affected by the addition of exogenous CaM, it can be inhibited by EGTA as well as various CaM inhibitors possibly due to the presence of CaMLD in these kinases (Harmon et al., 1987
; Putnam-Evans et al., 1990; Dasgupta, 1994). The known CaM kinases from plants have some unique characteristics. In CCaMK from lily anthers, autophosphorylation is Ca2+-dependent/stimulated but, interestingly, CaM inhibits this activity in a concentration-dependent manner (Takezawa et al., 1996).
During purification of ZmCCaMK, the property of autophosphorylation was used to identify this kinase from the protein fractions eluted from a CaM Sepharose column (Pandey and Sopory, 1998). The kinase also showed autophosphorylation in a Ca2+-dependent manner (optimum concentration 10 µM free calcium) like CDPKs. But in contrast to CDPKs and substrate phosphorylation activity of this CCaMK itself, CaM had no effect on autophosphorylation. Various CaM and kinase inhibitors tested on ZmCCaMK autophosphorylation activity also showed no effect. These data show that the autophosphorylation and substrate phosphorylation activities of ZmCCaMK are differentially regulated. Autophosphorylation of the kinase was at threonine residue(s), similar to animal CaM kinases and lily anther CCaMK whereas in CDPKs, autophosphorylation has been reported to occur at both serine and threonine residues (Putnam-Evans et al., 1990; Saha and Singh, 1995).
Like the animal system CaM kinases, ZmCCaMK substrate phosphorylation activity is regulated by its own phosphorylation. As clear by the time kinetics of both the reactions, the autophosphorylation is preceded by substrate phosphorylation activity. Autophosphorylated ZmCCaMK could also phosphorylate substrate in the presence of EGTA, showing no calcium requirement and pointing towards the possible mechanism of regulation of the kinase activity. ZmCCaMK is active in its autophosphorylated form for which calcium is the only requirement. After its activation with calcium, it can phosphorylate different substrates without the requirement of calcium. CaM probably binds to the autophosphorylated form of ZmCCaMK and either stabilize it or somehow enhance the substrate availability. Further studies are required in this direction to discover how calcium and CaM are regulating the autophosphorylation and the 2-step substrate phosphorylation.
Apart from showing an interesting dual regulation of the activity by calcium and CaM, ZmCCaMK also showed a very specific regulation by light. Red light treatment for 5 min decreased the level of kinase protein, which could not be reversed by far-red light treatment. This loss of far-red light reversibility could either be due to the very fast nature of the response which is somehow escaping the reversal by far red light or it may be a Phy B-mediated phenomenon. Red light treatment for different time periods decreased the level of the kinase in a time-dependent manner. The possibility that the long duration of red light treatment had a deleterious effect was ruled out; 5 min of red light immediately followed by 2 h dark incubation showed same level of the ZmCCaMK as 30 min continuous red light treatment.
In in vivo systems, in response to any signal such as light, the kinases and phosphatases play in concert with each other; affecting either of them could bring about the same response. The phytochrome (Pr form) after absorbing the red light gets converted to the Pfr form and the phytochrome itself has recently been shown to be present in a phosphorylated form (Elich and Chory, 1997, and references therein). The phytochrome on absorbing light could either affect kinases or phosphatases to bring the same response, i.e. up-regulation of phosphorylation could either be due to an increase in activity/level of kinases or due to a decrease in activity/level of phosphatases and vice versa (Sopory and Munshi, 1998). Many pathways, which show an increase in phosphorylation, are reported, but the existence of a kinase that is down-regulated by red light shows the possibility of existence of other complex regulatory pathways as well.
Though the level of the ZmCCaMK decreases with red light, blue light had no effect on its level at all the time points and intensities studied. Low levels of ZmCCaMK in response to red light treatment could possibly be explained in the following ways. Light has been shown to affect the expression of a number of genes in both positive as well as negative ways (Neuhaus et al., 1997). Such responses are generally very fast and a change in transcript level could be detected within 1–5 min. So this kinase could be one such example showing down-regulation by light and thus might be involved in the light-signalling pathway. Other possibilities could be a very fast turnover of the protein or by some kind of relocalization in response to red light. Recently, a phospho-protein regulatory factor was shown to migrate to the nucleus following light irradiation (Wellmer et al., 1999). At present there is no proof in support of either of the possibilities and further studies are required to confirm anything conclusively.
|
Acknowledgments |
|---|
This work was financially supported by the Department of Biotechnology, Government of India. SP was supported by a senior research fellowship from the University Grants Commission, New Delhi, India.
|
Notes |
|---|
3 Present address and to whom correspondence should be sent: 208 Mueller Laboratory, Biology Department, The Pennsylvania State University, University Park, PA 16802, USA. Fax: +1 814 865 9131. E-mail: sxp49{at}psu.edu
|
Abbreviations |
|---|
BSA, bovine serum albumin; CaM, calmodulin; CaMK, calmodulin kinase; CCaMK, calcium/calmodulin kinase; CDPK, calcium-dependent protein kinase; EDTA, (ethylenedinitrilo)-tetra acetic acid; EGTA, ethylene glycol-bis-(ß-aminoethylether) N,N,N'N'-tetra acetic acid; H-7, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine; HEPES, (N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulphonic acid]); PMA, phorbol 12-myristate 13-acetate; PS, phosphatidyl serine; W-7, N-(6-aminohexyl)5-chloro-1-naphthalenesulphonamide hydrochloride; ZmCCaMK, Zea mays calcium/calmodulin-dependent protein kinase\..
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Anderberg RJ, Walker-Simmons MK.1992. Isolation of a wheat cDNA clone from an abscisic acid-inducible transcript with homology to protein kinases. Proceedings of the National Academy of Sciences, USA 89, 10183–10187.
Blum H, Bier H, Gross HJ.1987. The best silver stain. Electrophoresis 8, 93–99.[Web of Science]
Botella JR, Arteca JM, Somodevilla M, Arteca RN.1996. Calcium-dependent protein kinase gene expression in response to physical and chemical stimuli in mung bean (Vigna radiata). Plant Molecular Biology 30, 1129–1137.[Web of Science][Medline]
Bowler C, Neuhaus G, Yamagata H, Chua NH.1994. Cyclic GMP and calcium mediate phytochrome phototransduction. Cell 77, 73–81.[Web of Science][Medline]
Bradford MM.1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.[Web of Science][Medline]
Bush DS.1995. Calcium regulation in plant cells and its role in signalling. Annual Review in Plant Physiology and Plant Molecular Biology 46, 95–122.[Web of Science]
Chory J.1994. Plant phototransduction: phytochrome signal transduction. Current Biology 4, 844–846.[Web of Science][Medline]
Chory J.1997. Light modulation of vegetative development. The Plant Cell 9, 1225–1234.[Web of Science][Medline]
Cooper JA, Sefton BM, Hunter T.1983. Detection and quantitation of phosphotyrosine in proteins. Methods in Enzymology 99, 387–401.[Web of Science][Medline]
Dasgupta M.1994. Characterization of a calcium-dependent protein kinase from Arachis hypogea (ground nut) seeds. Plant Physiology 104, 961–969.[Abstract]
Elich TD, Chory J.1997. Phytochrome: if it looks and smells like a histidine kinase, is it a histidine kinase? Cell 91, 713–716.[Web of Science][Medline]
Hanson PI, Schulman H.1992. Neuronal Ca2+/calmodulin-dependent protein kinases. Annual Reviews in Biochemistry 61, 559–601.[Web of Science][Medline]
Hanson PI, Meyer T, Stryer L, Schulman H.1994. Dual role of calmodulin in autophosphorylation of multifunctional CaM kinase may underlie decoding of calcium signals. Neuron 12, 943–956.[Web of Science][Medline]
Harmon AC, Putnam-Evans C, Cormier MJ.1987. A calcium-dependent but calmodulin-independent protein kinase from soybean. Plant Physiology 83, 830–837.
Huber SC, Huber JL, McMichael RW.1994. Control of plant enzyme activity by reversible protein phosphorylation. International Reviews in Cytology 149, 47–98.
Im CS, Matters GL, Beale SL.1996. Calcium and calmodulin are involved in blue light induction of the gsa gene for a early chlorophyll biosynthetic step in Chlamydomonas. The Plant Cell 8, 2245–2253.[Abstract]
Kwiatkowski AP, Shell DJ, King MM.1988. Role of autophosphorylation in activation of the type II calmodulin dependent protein kinase. Journal of Biological Chemistry 263, 6484–6486.
Laemmli UK.1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[Medline]
Lai Y, Nairn AC, Greengard P.1986. Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II. Proceedings of the National Academy of Sciences, USA 83, 4253–4257.
Lu YT, Hidaka H, Feldman LJ.1996. Characterization of a calcium/calmodulin protein kinase homologue from maize roots showing light-regulated gravitropism. Planta 199, 18–24.[Web of Science][Medline]
Miller SG, Kennedy MB.1986. Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch. Cell 44, 861–870.[Web of Science][Medline]
Monroy AF, Labbe E, Dhindsa RS.1997. Low temperature perception in plants: effects of cold on protein phosphorylation in cell-free extracts. FEBS Letters 410, 206–209.[Web of Science][Medline]
Mustilli AC, Bowler C.1997. Turning into the signals controlling photoregulated gene expression in plants. EMBO Journal 16, 5801–5806.[Web of Science][Medline]
Neuhaus G, Bowler C, Hiratsuka K, Yamagata H, Chua NH.1997. Phytochrome-regulated repression of gene expression requires calcium and cGMP. EMBO Journal 16, 2552–2564.
Neuhaus G, Bowler C, Kern R, Chua NH.1993. Calcium/ calmodulin-dependent and independent phytochrome signal transduction pathways. Cell 73, 937–952.[Web of Science][Medline]
Pandey S, Sopory SK.1998. Biochemical evidence for a calmodulin-stimulated calcium-dependent protein kinase in maize. European Journal of Biochemistry 255, 718–726.[Web of Science][Medline]
Pandey S, Tiwari SB, Upadhyaya KC, Sopory SK.2000. Calcium signalling: linking environmental signals to cellular functions. Critical Reviews in Plant Sciences 19, 291–318.
Poovaiah BW, Reddy ASN.1993. Calcium and signal transduction in plants. Critical Reviews in Plant Sciences 12, 185–211.[Medline]
Putnam-Evans CL, Harmon AC, Coromier MJ.1990. Purification and characterization of a novel calcium-dependent protein kinase from soybean. Biochemistry 29, 2488–2495.[Medline]
Saha P, Singh M.1995. Characterization of a winged bean (Psophocarpus tetragonolobus) protein kinase with calmodulin like domain: regulation by autophosphorylation. Biochemical Journal 305, 205–210.
Sano H, Youssefian S.1994. Light and nutritional regulation of transcripts encoding a wheat protein kinase homologue is mediated by cytokinins. Proceedings of the National Academy of Sciences, USA 91, 2582–2586.
Shacklock PS, Read ND, Trewavas AJ.1992. Cytosolic free calcium mediates red light-induced photomorphogenesis. Nature 358, 753–755.[Web of Science]
Sheen J.1996. Ca2+-dependent protein kinases and stress signal transduction in plants. Science 274, 1900–1902.
Stone JM, Walker JC.1995. Protein kinase families and signal transduction. Plant Physiology 108, 451–457.[Abstract]
Sopory SK, Munshi M.1998. Protein kinases and phosphatases and their role in cellular signalling in plants. Critical Reviews in Plant Sciences 17, 245–318.
Takezawa D, Ramachandiran S, Paranjape V, Poovaiah BW.1996. Dual regulation of a chimeric plant serine/threonine kinase by calcium and calcium/calmodulin. Journal of Biological Chemistry 271, 8126–8132.
Tretyn A, Wagner G, Felle HH.1991. Signal transduction in Sinapsis alba root hairs: auxins as external messengers. Journal of Plant Physiology 139, 187–193.
Trewavas AJ, Malho R.1997. Signal perception and transduction: the origin of the phenotype. The Plant Cell 9, 1181–1195.[Web of Science][Medline]
Watillon B, Kettemann R, Boxus P, Burney A.1995. Structure of a calmodulin-binding protein kinase gene from apple. Plant Physiology 108, 847–848.[Web of Science][Medline]
Wellmer F, Kircher S, Rugner A, Frohnmeyer H, Schafer E, Harter K.1999. Phosphorylation of parsley bZip transcription factor CPRF2 is regulated by light. Journal of Biological Chemistry 274, 29476–29482.
Wu Y, Hiratsuka K, Neuhaus G, Chua NH.1996. Calcium and cGMP target distinct phytochrome-responsive elements. The Plant Journal 10, 1149–1154.[Web of Science][Medline]
Wu Y, Kuzma J, Marechal E, Graeff R, Lee HC, Foster R, Chua NH.1997. Abscisic acid signalling through cyclic ADP-ribose in plants. Science 278, 2126–30.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  W. Hua, L. Zhang, S. Liang, R. L. Jones, and Y.-T. Lu A Tobacco Calcium/Calmodulin-binding Protein Kinase Functions as a Negative Regulator of Flowering J. Biol. Chem., July 23, 2004; 279(30): 31483 - 31494. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||