- Email: [email protected]
Calmodulin-binding protein kinases in plants
Review
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Calmodulin-binding protein kinases in plants Lei Zhang and Ying-Tang Lu Key Laboratory of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China
Many calmodulin-binding protein kinases have been isolated from plants. Plant calmodulin-binding protein kinases are novel protein kinases that differ from calcium-dependent protein kinases in many important respects. Calmodulin-binding protein kinases are likely to be crucial mediators of responses to diverse endogenous and environmental cues in plants. In this update, we review the structure, regulation, expression and possible functions of plant calmodulin-binding protein kinases. Calcium is a ubiquitous second messenger in plants, playing vital roles during plant growth and development. Intracellular Ca2þ concentrations are modulated in response to various signals, including hormones, light, mechanical disturbances, abiotic stress and pathogen elicitors [1,2]. The information encoded in transient Ca2þ signals is deciphered by various intracellular Ca2þ sensors such as sensor responders and sensor relays that convert the Ca2þ signals into a wide variety of biological responses [3,4]. Calcium-dependent protein kinases (CDPKs) are a class of Ca2þ sensor responders in plants that have been well characterized [5,6]. CDPKs contain four domains: an N-terminal domain of variable length and sequence; a protein kinase catalytic domain; an autoinhibitory junction domain containing a calmodulin (CaM)-binding domain; and a C-terminal CaM-like Ca2þ-binding domain. The activity of CDPKs is regulated by Ca2þ, so they are thought to function in Ca2þ-mediated signal-transduction pathways. However, the precise biological functions of most CDPKs remain elusive [6]. In addition to CDPKs, CDPK-related protein kinases (CRKs) have also been reported to occur in plants. Unlike CDPKs, CRKs do not require Ca2þ for their activities. In addition, biochemical evidence that CRKs bind Ca2þ – CaM is lacking [7,8]. Sensor relays such as CaM and calcineurin-B-like proteins undergo a Ca2þ-induced conformational change and then interact with their target proteins [4,9,10]. In higher plants, the CaM-binding protein kinases (CBKs) are the most unusual of the CaM target proteins. Our intention here is to summarize recent progress in understanding the structure, enzymatic activities and physiological functions of CBKs in plants.
Corresponding author: Ying-Tang Lu ([email protected]).
Structural characteristics CB1 from apple was the first CBK isolated from plant tissue [11], but this was soon followed by the isolation of MCK from maize [12,13], OsCBK from rice [14], AtCBK1 from Arabidopsis [15] and chimeric Ca2þ – CaM-dependent protein kinases (CCaMKs) from lily and tobacco [16,17]. All of these CBKs contain an N-terminal domain of variable length and sequence, a protein kinase catalytic domain, a CaM-binding domain, and a C-terminal domain of variable length and sequence (Fig. 1). However, unlike CDPKs, these CBKs lack well-defined EF hands for Ca2þ binding at their C-termini. CCaMKs are exception, having a C-terminal visinin-like domain consisting of three EF hands, similar to neural visinin-like proteins (Fig. 1). Although CDPKs have CaM-binding domains that can bind Ca2þ – CaM when the C-terminal CaM-like domain is deleted, full-length CDPKs bind only their own intramolecular CaM-like domain rather than free CaM [18]. In this regard, CDPKs differ from CBKs and are not sensors of free cellular CaM. Recently, several new CBKs have been isolated from plants, including NtCaMK1, NtCBK1 and NtCBK2 from tobacco, and AtCBK2 and AtCBK3 from Arabidopsis (Fig. 2) (L. Ma et al., unpublished; W. Hua et al., pers. commun.). Figure 2 shows phylogenetic analyses of CBKs (CDPK in plants and CaMKII in animals). The clustering of these plant CBKs away from the non-plant CaM kinases
CaM-binding domain N-terminal
Kinase domain
Visinin-like domain
CCaMK C-terminal CB1 MCK1 OsCBK TRENDS in Plant Science
Fig. 1. Domain structures of calmodulin (CaM)-binding protein kinases (CBKs) in plants. The N-terminal domain is highly variable in length and sequence. The putative CaM-binding domain is predicted in the region immediately following the kinase domain. A distinguishing feature of CBKs is the C-terminal domain. EF hands that can bind calcium are denoted by black boxes. Abbreviations: CCaMK, a chimeric Ca2þ –CaM-dependent protein kinase; CB1, Ca2þ –CaM-binding protein kinase from apple; MCK1, Ca2þ – CaM-bindng protein kinase from maize; OsCBK, Ca2þ –CaM-binding protein kinase from rice.
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MCK1 MCK2 NtCBK1 NtCaMK1 NtCBK2 DcCRK AtCBK1 AtCBK2 AtCBK3 AtCDPK OsCBK TCCaMK1 TCCaMK2 Lily CCaMK CB1 Rat CaMK II 132.6 120.0
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Fig. 2. Evolutionary relationship between calmodulin (CaM)-binding protein kinases (CBKs) in plants and CaM kinase (CaMK) II in animals. The dendrogram was constructed using CLUSTAL W/DNASTAR 5.02 program (DNASTAR Inc., Madison, WI, USA). GenBank accession numbers are as follows: AtCBK1, AF435449; AtCBK2, AF435447; AtCBK3, AF435448; AtCDPK, 1220099; CB1, 1170626; DcCRK, CAA58750; Lily CCaMK, 860675; MCK1, 1839596; MCK2, AF289237; NtCBK1, AF435450; NtCBK2, AF435452; NtCaMK1, AF550608; OsCBK, AF368282; rat CaMKII, J02942; TCCaMK1, AF087813; TCCaMK2, U70923. The animal gene is indicated in bold.
(CaMKs) suggests a single common origin for plant CBKs and CDPKs. Enzymatic activity of plant CBKs CaM-dependent, CaM-stimulated and CaM-independent protein kinases Many lines of evidence indicate that Ca2þ –CaM-dependent protein phosphorylation occurs in homogenates of plant tissues. However, firm evidence for the presence of CBKs in plants has been established only recently, following the biochemical and molecular characterization of several CBKs. The CCaMKs from lily and tobacco are two examples of CBKs that have been especially well characterized. The C-terminal visinin-like domains of CCaMKs have three Ca2þ-binding EF hands as well as a separate CaM-binding domain (Fig. 1). CCaMKs autophosphorylate in a Ca2þ-dependent manner and phosphorylate substrates in a Ca2þ –CaM-dependent manner, although their autophosphorylation is inhibited by the presence of CaM [17,19,20]. The presence of a visinin-like Ca2þ-binding domain in CCaMKs adds an additional Ca2þ-sensing mechanism, previously unknown in the Ca2þ – CaMmediated signaling cascade in plants [21]. It has also been reported that other plant CBKs show Ca2þ-dependent autophosphorylation and Ca2þ – CaMactivated substrate phosphorylation. For example, ZmCCaMK autophosphorylates in a Ca2þ-dependent manner and substrate phosphorylation in a Ca2þ –CaMactivated manner. In contrast to CCaMKs from lily and tobacco, CaM has no effect on the autophosphorylation activity of ZmCCaMK [22,23]. However, although this maize kinase is named ZmCCaMK, it has not been http://plants.trends.com
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established that its structure is similar to the CCaMKs isolated from lily and tobacco because its gene has not been cloned [22,23]. Recently, an immuno-homolog of ZmCCaMK from pea (PsCCaMK) has been identified, indicating that this type of CBK might be present in other plants [24]. Several other CBKs that differ from CCaMKs in their structural and enzymatic characteristics have recently been identified. These CBKs can be classified into three groups based on the CaM regulation of their enzymatic activities – the CaM-dependent, CaM-stimulated and CaM-independent kinases. NtCaMK1, a Ca2þ – CaMdependent protein kinase from tobacco, is structurally similar to CaMKs from mammal systems, having all 11 subdomains of a kinase catalytic domain and lacking EF hands for Ca2þ-binding (L. Ma et al., unpublished). Biochemical analyses show that NtCaMK1 phosphorylates itself and its substrate only in the presence of Ca2þ –CaM. Once autophosphorylation of the kinase has been initiated, the phosphorylated form of NtCaMK1 displays its activity in a Ca2þ – CaM-independent manner, as is the case for mammalian CaMKII. The biochemical characterizations of NtCaMK1 are similar to those of animal CaMKs, suggesting that some plant CBKs might share both structural and functional homologies with their animal counterparts. CaM-stimulated CBKs have also been characterized in plants. Both AtCBK1 from Arabidopsis and NtCBK2 from tobacco can bind CaM in a Ca2þ-dependent manner. However, the autophosphorylation and substrate phosphorylation activities are shown to be Ca2þ-dependent, and CaM can stimulate their activities up to four- to fivefold [15] (W. Hua et al., pers. commun.). These contrast with the role of CaM in ZmCCaMK, in which CaM has no effect on autophosphorylation activity. AtCBK1 and NtCBK1 are also different from CCaMKs in lily and tobacco, which show an inhibition of autophosphorylation activity in the presence of CaM [17,19]. OsCBK, a novel CaM-binding protein kinase isolated from rice, is classified as a third type of CBK. Although OsCBK binds CaM with high affinity, its autophosphorylation and substrate phosphorylation have been shown to be Ca2þ –CaM independent [14]. The role of CaM binding to OsCBK remains to be determined. Regulation by different CaM isoforms Plants have many CaM isoforms that are subject to different regulation in response to external stimuli, as well as showing differences in their distribution in plant tissues. The existence of multiple divergent CaM isoforms in plants suggests that they might interact with different CaM-binding proteins [25,26]. PCM1 and PCM6 are two CaM isoforms from potato that show differences in their interaction with tobacco CCaMKs. Both PCM1 and PCM6 inhibit Ca2þ-dependent autophosphorylation of NtCCaMK but PCM1 is much more effective than PCM6. However, NtCCaMK requires a higher concentration of PCM1 than PCM6 for substrate phosphorylation [17]. CaM isoforms NtCaM1, NtCaM3 and NtCaM13 have been also used to test their effectiveness in regulating the enzymatic activities of NtCBK2
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(a)
(b)
(g)
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(i)
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Fig. 3. NtCBK1 production patterns in tobacco, the blue color is visualized as a hybridization signal. (a) Flower primordium. (b) Stigma. (c,d) Ovary at different stages. (e) Embryo. (f) Vascular bundle in anther. (g –l) Anthers at different stages: (g) primordium of anther; (h) microspore mother cell; (i) tetrad stage in meiotic division; (j) monokaryotic stage; (k) mature pollen; (l) microspore mother cell (control). (a–d) Scale bar ¼ 400 mm, (e) scale bar ¼ 160 mm, (f– i) scale bar ¼ 80 mm, (j– l) scale bar ¼ 160 mm.
(W. Hua et al., pers. commun.). All of these CaMs stimulated the autophosphorylation and substrate phosphorylation of NtCBK2 but with different efficiencies. Although the activity of NtCBK2 can be amplified up to tenfold by NtCaM3 and NtCaM13, it is enhanced only about twofold by NtCaM1. The effects of different CaM isoforms on the regulation of autophosphorylation and substrate phosphorylation of CBKs suggests that, in addition to the changes in Ca2þ binding that act as a switch, the type and concentration of CaM can also determine the amplification of the Ca2þ signal in Ca2þ – CaM-mediated pathways. Expression and subcellular distribution of CBKs To understand the possible roles of CBKs, their expression profiles have been examined to provide clues to physiological functions during plant growth and development. The spatial and temporal expression patterns of CBKs have been examined using promoter– reporter fusions in transgenic plants and by in situ hybridization with isoform-specific probes. CCaMKs from lily and tobacco are expressed only in anthers [17,27]. Lily CCaMK mRNA and protein are detected in the pollen sac and their distribution is restricted to the pollen mother and tapetal cells [27]. Northern-blot analysis revealed that the genes for both CCaMK1 and CCaMK2 from tobacco were expressed in a http://plants.trends.com
stage-specific manner during anther development. The appearance of mRNA encoding these enzymes coincides with meiosis and becomes undetectable at later stages of anther development. Reverse polymerase-chain-reaction amplification assays using isoform-specific primers showed that mRNAs for both CCaMK1 and CCaMK2 from tobacco are produced in anthers [17]. The stage- and tissue-specific appearance of CCaMKs in anthers suggests that they could play a role in sensing transient changes in free Ca2þ concentration in target cells, thereby controlling developmental events in the anther. Other CBKs such as MCK1, MCK2, NtCBK1, AtCBK1 and OsCBK show similar expression profiles [13 – 15]. NtCBK1 expression (Fig. 3) is highly regulated both temporally and spatially during plant growth and development. The mRNA is abundantly accumulated in the tissue and cells required for rapid growth and metabolic activity, including the growing region of the root, flower primordia, sporogenous cells and tetrads in the anther, as well as in developing embryos. However, NtCBK1 gene expression is undetectable in pollen and anther walls at the mature pollen stage. Physiological functions Ca2þ-regulated protein phosphorylation has been implicated in a range of responses including host– pathogen interactions, cold stress, gravitropism, light-regulated
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gene expression and hypo-osmotic shock. Studies during the past decade have indicated that some of these responses are mediated by CBKs [2,9]. As a major environmental factor, light has been shown to affect almost every phase of plant growth and development, and light-mediated responses have been shown accompanied by changes in cellular Ca2þ and CaM concentrations [28]. ZmCCaMK is downregulated by red light, indicating that this CBK might be involved in the lightdependent signal-transduction pathway. No difference was observed in the production of ZmCCaMK at any intensity of blue light, confirming that the response is specifically mediated at a particular intensity of red light [23]. Many experiments have also led to the conclusion that Ca2þ functions in gravitropic signal-transduction pathways, although the exact mechanism of Ca2þ action has not been established [29]. It has been reported that Ca2þ influences gravitropic signal transduction by interaction with CaM and other Ca2þ-binding proteins, and that MCK1 might be involved in light-regulated root gravitropism because both the CaM-binding activity of MCK1 and light-regulated root gravitropism are inhibited by KN-93 (a drug that can bind to animal CaMKII and inhibit its activity) [12]. Although little information is available about specific functions of CBKs, several protein kinases have been investigated for their roles in regulating plant development and flowering [30]. Recently, experiments have shown that MCK1 plays a role in regulating flowering in tobacco [31]. The overproduction of MCK1 leads to the abortion of flower primordia on the main shoot axis and prolonged the vegetative phase in axillary buds. Flower abortion was first observed in the developing anthers, and the entire flower subsequently senesced. In axillary buds, the prolonged vegetative phase is characterized by atypical elongated, narrow, twisted leaves [31]. A role of CBKs in flowering is also indicated from other experiments with NtCBK1. Overproduction of NtCBK1 resulted in late flowering of tobacco plants (W. Hua et al., pers. commun.). These results suggest a role for CBKs in mediating flowering. Many lines of evidence have indicated the involvement of CBKs in other responses during plant development. The ZmCCaMK homolog from pea (PsCCaMK) has been reported to be involved in stress signaling [24]. The production of NtCaMK1 is stimulated by salt stress: it is greatly enhanced after 30 min with salt-stress treatment, peaks at 70 min and then declines to basal level after 4 h stress treatment. These results suggest that NtCaMK1 might be involved in the salt-stress response of tobacco (L. Ma et al., unpublished). Perspectives During the past decade, significant progress has been made in isolating and identifying CaMs and CBKs from plants. Based on current knowledge, plants contain many unique CBKs with novel regulatory mechanisms that perform plant-specific functions. It is likely that many more novel CBKs will be identified, especially as more plant genome sequences are completed. The challenge now is to elucidate the roles of these CBKs and to http://plants.trends.com
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identify cross-talk among various components of the Ca2þ signaling system. New technologies have emerged to provide facile, rapid methods for identifying the functions of CBKs in plants. Gene-disruption and silencing techniques such as insertional mutagenesis, RNA interference and virus-induced gene silencing can be used to study altered phenotypes [32,33]. Because many CBKs are members of large gene families, there is likely to be functional redundancy or overlap among members of each family. Ascertaining specific functions will thus require the creation of double or triple mutants. Technologies such as reverse genetics using knockout mutants coupled with the analysis of celland tissue-specific production of CBKs using microarrays [34], and non-destructive visualization of protein in live cells using fluorescent reporters should help to provide new insights into the functions of CBKs involved in Ca2þ signaling and deciphering signaling networks. Acknowledgements We thank Russell Jones for his encouragement and the anonymous referees for their helpful comments. Work from our laboratory is supported by grants from National Natural Science Foundation of China and Major State Basic Research Program (G1999011600).
References 1 Rudd, J.J. and Franklin-Tong, V.E. (2001) Unravelling responsespecificity in Ca2þ signaling pathways in plant cells. New Phytol. 151, 7 – 33 2 Reddy, A.S.N. (2001) Calcium: silver bullet in signaling. Plant Sci. 160, 381 – 404 3 Luan, S. et al. (2002) CaMs and CBLs: calcium sensors for specific signal – response coupling in plants. Plant Cell 14 (Suppl.), S389 – S400 4 Sanders, D. et al. (2002) Calcium at the crossroads of signaling. Plant Cell 14 (Suppl.), S401 – S417 5 Harmon, A.C. et al. (2001) The CDPK superfamily of protein kinases. New Phytol. 151, 175 – 183 6 Cheng, S.-H. et al. (2002) Calcium signaling through protein kinases, the Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol. 129, 469– 485 7 Lindzen, E. and Choi, J.H. (1995) A carrot cDNA encoding an atypical protein kinase homologous to plant calcium-dependent protein kinase. Plant Mol. Biol. 28, 785 – 797 8 Furumoto, T. et al. (1996) Plant calcium-dependent protein kinaserelated kinases (CRKs) do not require calcium for their activities. FEBS Lett. 396, 147 – 151 9 Snedden, W.A. and Fromm, H. (2001) Calmodulin as a versatile calcium signal transducer in plants. New Phytol. 151, 35 – 66 10 Reddy, V.S. et al. (2002) Genes encoding calmodulin-binding proteins in the Arabidopsis genome. J. Biol. Chem. 277, 9840– 9852 11 Watillon, B. et al. (1995) Structure of a calmodulin-binding protein kinase gene from apple. Plant Physiol. 108, 847 – 848 12 Lu, Y-T. et al. (1996) Characterization of a calcium/calmodulindependent protein kinase homolog from maize roots showing lightregulated gravitropism. Planta 199, 16– 24 13 Wang, L. et al. (2001) Characterization, physical location and expression of the genes encoding calcium/calmodulin-dependent protein kinases in maize (Zea mays L.). Planta 213, 556– 564 14 Zhang, L. et al. (2002) Molecular and biochemical characterization of a calcium/calmodulin-binding protein kinase from rice. Biochem. J. 368, 145– 157 15 Xie, Q.G. et al. (2002) Characterization of a calmodulin-binding protein kinase from Arabidopsis thaliana. Chin. Sci. Bull. 47, 1650 – 1655 16 Patil, S. et al. (1995) Chimeric plant calcium/calmodulin-dependent protein kinase gene with a neural visinin-like calcium-binding domain. Proc. Natl. Acad. Sci. U. S. A. 92, 4897 – 4901 17 Liu, Z. et al. (1998) Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms. Plant Mol. Biol. 38, 889 – 897
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18 Yoo, B.C. and Harmon, A.C. (1996) Intramolecular binding contributes to the activation of CDPK, a putative kinase with a calmodulin-like domain. Biochemistry 35, 12029 – 12037 19 Takezawa, D. et al. (1996) Dual regulation of a chimeric plant serine/ threonine kinase by calcium and calcium/calmodulin. J. Biol. Chem. 271, 8126 – 8132 20 Sathyanarayanan, P.V. et al. (2001) Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase. J. Biol. Chem. 276, 32940 – 32947 21 Sathyanarayanan, P.V. et al. (2000) Plant chimeric Ca2þ/calmodulindependent protein kinase: role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity. J. Biol. Chem. 275, 30417 – 30422 22 Pandey, S. and Sopory, S.K. (1998) Biochemical evidence of a calmodulin-stimulated calcium-dependent protein kinase in maize. Eur. J. Biochem. 255, 718– 726 23 Pandey, S. and Sopory, S.K. (2001) Zea mays CCaMK: autophosphorylation-dependent substrate phosphorylation and downregulation by red light. J. Exp. Bot. 52, 691 – 700 24 Pandey, S. et al. (2002) A Ca2þ/CaM-dependent kinase from pea is stress regulated and in vitro phosphorylates a protein that binds to AtCaM5 promoter. Eur. J. Biochem. 269, 3193– 3204 25 Zielinski, R.E. (2002) Preparation of recombinant plant calmodulin isoforms. Methods Mol. Biol. 172, 143 – 149
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26 Zielinski, R.E. (2002) Characterization of three new members of the Arabidopsis thaliana calmodulin gene family: conserved and highly diverged members of the gene family functionally complement a yeast calmodulin null. Planta 214, 446 – 455 27 Poovaiah, B.W. et al. (1999) Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers. Planta 209, 161 – 171 28 Mustilli, A.C. and Bowler, C. (1997) Turning into the signals controlling photoregulated gene expression in plants. EMBO J. 16, 5801–5806 29 Konings, H. (1995) Gravitropism of roots: an evaluation of progress during the last three decades. Acta Bot. Neerl. 44, 195– 223 30 Dornelas, M.C. et al. (2000) Arabidopsis thaliana SHAGGY-related protein kinase (ArSK11 and 12) function in perianth and gynoecium development. Plant J. 21, 419– 429 31 Liang, S. et al. (2001) Mediation of flowering by a calmodulindependent protein kinase. Sci. Chin. 44, 506 – 512 32 Waterhouse, P.M. et al. (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. U. S. A. 95, 13959 – 13964 33 Romeis, T. et al. (2001) Calcium-dependent protein kinases play an essential role in plant defense response. EMBO J. 20, 5556 – 5567 34 Schena, M. et al. (1998) Microarrays: biotechnology’s discovery platform for functional genomics. Trends Biotechnol. 16, 301 – 306
2–4 October 2003 3rd International Symposium on Natural Drugs, Naples, Italy Organized by: F. Capasso, A.A. Izzo and N. Mascolo (Naples, Italy) Topics covered: Botanical, chemical and pharmacological aspects of medicinal plants and their applications in gastrointestinal and cardiovascular disturbances, endocrine dysfunctions, immunodeficiences and viral and fungal infections. Contributions to other means will be accepted in a limited number only. Satellite Symposia: Cannabis and cannabinoids; propolis and its components - chemical and pharmacological aspects; green tea and cancer; toxicity of anthraquinone drugs. Venue: Grand Hotel Oriente, Via Diaz 44, 80134 Naples, Italy. Tel: +39 081 5512133 Secretariat: Dr F. Borrelli and Dr N. Milic Department of Experimental Pharmacology, University of Naples Federico II, Via D. Montesano, 49 - 80131 Naples, Italy. Tel: +39 081 678432/436/439 – Fax: +39 081 678403 – E-mail: [email protected] http://plants.trends.com
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Calmodulin-binding protein kinases in plants Lei Zhang and Ying-Tang Lu Key Laboratory of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China
Many calmodulin-binding protein kinases have been isolated from plants. Plant calmodulin-binding protein kinases are novel protein kinases that differ from calcium-dependent protein kinases in many important respects. Calmodulin-binding protein kinases are likely to be crucial mediators of responses to diverse endogenous and environmental cues in plants. In this update, we review the structure, regulation, expression and possible functions of plant calmodulin-binding protein kinases. Calcium is a ubiquitous second messenger in plants, playing vital roles during plant growth and development. Intracellular Ca2þ concentrations are modulated in response to various signals, including hormones, light, mechanical disturbances, abiotic stress and pathogen elicitors [1,2]. The information encoded in transient Ca2þ signals is deciphered by various intracellular Ca2þ sensors such as sensor responders and sensor relays that convert the Ca2þ signals into a wide variety of biological responses [3,4]. Calcium-dependent protein kinases (CDPKs) are a class of Ca2þ sensor responders in plants that have been well characterized [5,6]. CDPKs contain four domains: an N-terminal domain of variable length and sequence; a protein kinase catalytic domain; an autoinhibitory junction domain containing a calmodulin (CaM)-binding domain; and a C-terminal CaM-like Ca2þ-binding domain. The activity of CDPKs is regulated by Ca2þ, so they are thought to function in Ca2þ-mediated signal-transduction pathways. However, the precise biological functions of most CDPKs remain elusive [6]. In addition to CDPKs, CDPK-related protein kinases (CRKs) have also been reported to occur in plants. Unlike CDPKs, CRKs do not require Ca2þ for their activities. In addition, biochemical evidence that CRKs bind Ca2þ – CaM is lacking [7,8]. Sensor relays such as CaM and calcineurin-B-like proteins undergo a Ca2þ-induced conformational change and then interact with their target proteins [4,9,10]. In higher plants, the CaM-binding protein kinases (CBKs) are the most unusual of the CaM target proteins. Our intention here is to summarize recent progress in understanding the structure, enzymatic activities and physiological functions of CBKs in plants.
Corresponding author: Ying-Tang Lu ([email protected]).
Structural characteristics CB1 from apple was the first CBK isolated from plant tissue [11], but this was soon followed by the isolation of MCK from maize [12,13], OsCBK from rice [14], AtCBK1 from Arabidopsis [15] and chimeric Ca2þ – CaM-dependent protein kinases (CCaMKs) from lily and tobacco [16,17]. All of these CBKs contain an N-terminal domain of variable length and sequence, a protein kinase catalytic domain, a CaM-binding domain, and a C-terminal domain of variable length and sequence (Fig. 1). However, unlike CDPKs, these CBKs lack well-defined EF hands for Ca2þ binding at their C-termini. CCaMKs are exception, having a C-terminal visinin-like domain consisting of three EF hands, similar to neural visinin-like proteins (Fig. 1). Although CDPKs have CaM-binding domains that can bind Ca2þ – CaM when the C-terminal CaM-like domain is deleted, full-length CDPKs bind only their own intramolecular CaM-like domain rather than free CaM [18]. In this regard, CDPKs differ from CBKs and are not sensors of free cellular CaM. Recently, several new CBKs have been isolated from plants, including NtCaMK1, NtCBK1 and NtCBK2 from tobacco, and AtCBK2 and AtCBK3 from Arabidopsis (Fig. 2) (L. Ma et al., unpublished; W. Hua et al., pers. commun.). Figure 2 shows phylogenetic analyses of CBKs (CDPK in plants and CaMKII in animals). The clustering of these plant CBKs away from the non-plant CaM kinases
CaM-binding domain N-terminal
Kinase domain
Visinin-like domain
CCaMK C-terminal CB1 MCK1 OsCBK TRENDS in Plant Science
Fig. 1. Domain structures of calmodulin (CaM)-binding protein kinases (CBKs) in plants. The N-terminal domain is highly variable in length and sequence. The putative CaM-binding domain is predicted in the region immediately following the kinase domain. A distinguishing feature of CBKs is the C-terminal domain. EF hands that can bind calcium are denoted by black boxes. Abbreviations: CCaMK, a chimeric Ca2þ –CaM-dependent protein kinase; CB1, Ca2þ –CaM-binding protein kinase from apple; MCK1, Ca2þ – CaM-bindng protein kinase from maize; OsCBK, Ca2þ –CaM-binding protein kinase from rice.
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MCK1 MCK2 NtCBK1 NtCaMK1 NtCBK2 DcCRK AtCBK1 AtCBK2 AtCBK3 AtCDPK OsCBK TCCaMK1 TCCaMK2 Lily CCaMK CB1 Rat CaMK II 132.6 120.0
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Fig. 2. Evolutionary relationship between calmodulin (CaM)-binding protein kinases (CBKs) in plants and CaM kinase (CaMK) II in animals. The dendrogram was constructed using CLUSTAL W/DNASTAR 5.02 program (DNASTAR Inc., Madison, WI, USA). GenBank accession numbers are as follows: AtCBK1, AF435449; AtCBK2, AF435447; AtCBK3, AF435448; AtCDPK, 1220099; CB1, 1170626; DcCRK, CAA58750; Lily CCaMK, 860675; MCK1, 1839596; MCK2, AF289237; NtCBK1, AF435450; NtCBK2, AF435452; NtCaMK1, AF550608; OsCBK, AF368282; rat CaMKII, J02942; TCCaMK1, AF087813; TCCaMK2, U70923. The animal gene is indicated in bold.
(CaMKs) suggests a single common origin for plant CBKs and CDPKs. Enzymatic activity of plant CBKs CaM-dependent, CaM-stimulated and CaM-independent protein kinases Many lines of evidence indicate that Ca2þ –CaM-dependent protein phosphorylation occurs in homogenates of plant tissues. However, firm evidence for the presence of CBKs in plants has been established only recently, following the biochemical and molecular characterization of several CBKs. The CCaMKs from lily and tobacco are two examples of CBKs that have been especially well characterized. The C-terminal visinin-like domains of CCaMKs have three Ca2þ-binding EF hands as well as a separate CaM-binding domain (Fig. 1). CCaMKs autophosphorylate in a Ca2þ-dependent manner and phosphorylate substrates in a Ca2þ –CaM-dependent manner, although their autophosphorylation is inhibited by the presence of CaM [17,19,20]. The presence of a visinin-like Ca2þ-binding domain in CCaMKs adds an additional Ca2þ-sensing mechanism, previously unknown in the Ca2þ – CaMmediated signaling cascade in plants [21]. It has also been reported that other plant CBKs show Ca2þ-dependent autophosphorylation and Ca2þ – CaMactivated substrate phosphorylation. For example, ZmCCaMK autophosphorylates in a Ca2þ-dependent manner and substrate phosphorylation in a Ca2þ –CaMactivated manner. In contrast to CCaMKs from lily and tobacco, CaM has no effect on the autophosphorylation activity of ZmCCaMK [22,23]. However, although this maize kinase is named ZmCCaMK, it has not been http://plants.trends.com
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established that its structure is similar to the CCaMKs isolated from lily and tobacco because its gene has not been cloned [22,23]. Recently, an immuno-homolog of ZmCCaMK from pea (PsCCaMK) has been identified, indicating that this type of CBK might be present in other plants [24]. Several other CBKs that differ from CCaMKs in their structural and enzymatic characteristics have recently been identified. These CBKs can be classified into three groups based on the CaM regulation of their enzymatic activities – the CaM-dependent, CaM-stimulated and CaM-independent kinases. NtCaMK1, a Ca2þ – CaMdependent protein kinase from tobacco, is structurally similar to CaMKs from mammal systems, having all 11 subdomains of a kinase catalytic domain and lacking EF hands for Ca2þ-binding (L. Ma et al., unpublished). Biochemical analyses show that NtCaMK1 phosphorylates itself and its substrate only in the presence of Ca2þ –CaM. Once autophosphorylation of the kinase has been initiated, the phosphorylated form of NtCaMK1 displays its activity in a Ca2þ – CaM-independent manner, as is the case for mammalian CaMKII. The biochemical characterizations of NtCaMK1 are similar to those of animal CaMKs, suggesting that some plant CBKs might share both structural and functional homologies with their animal counterparts. CaM-stimulated CBKs have also been characterized in plants. Both AtCBK1 from Arabidopsis and NtCBK2 from tobacco can bind CaM in a Ca2þ-dependent manner. However, the autophosphorylation and substrate phosphorylation activities are shown to be Ca2þ-dependent, and CaM can stimulate their activities up to four- to fivefold [15] (W. Hua et al., pers. commun.). These contrast with the role of CaM in ZmCCaMK, in which CaM has no effect on autophosphorylation activity. AtCBK1 and NtCBK1 are also different from CCaMKs in lily and tobacco, which show an inhibition of autophosphorylation activity in the presence of CaM [17,19]. OsCBK, a novel CaM-binding protein kinase isolated from rice, is classified as a third type of CBK. Although OsCBK binds CaM with high affinity, its autophosphorylation and substrate phosphorylation have been shown to be Ca2þ –CaM independent [14]. The role of CaM binding to OsCBK remains to be determined. Regulation by different CaM isoforms Plants have many CaM isoforms that are subject to different regulation in response to external stimuli, as well as showing differences in their distribution in plant tissues. The existence of multiple divergent CaM isoforms in plants suggests that they might interact with different CaM-binding proteins [25,26]. PCM1 and PCM6 are two CaM isoforms from potato that show differences in their interaction with tobacco CCaMKs. Both PCM1 and PCM6 inhibit Ca2þ-dependent autophosphorylation of NtCCaMK but PCM1 is much more effective than PCM6. However, NtCCaMK requires a higher concentration of PCM1 than PCM6 for substrate phosphorylation [17]. CaM isoforms NtCaM1, NtCaM3 and NtCaM13 have been also used to test their effectiveness in regulating the enzymatic activities of NtCBK2
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(a)
(b)
(g)
(h)
(c)
(d)
(i)
(j)
(e)
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Fig. 3. NtCBK1 production patterns in tobacco, the blue color is visualized as a hybridization signal. (a) Flower primordium. (b) Stigma. (c,d) Ovary at different stages. (e) Embryo. (f) Vascular bundle in anther. (g –l) Anthers at different stages: (g) primordium of anther; (h) microspore mother cell; (i) tetrad stage in meiotic division; (j) monokaryotic stage; (k) mature pollen; (l) microspore mother cell (control). (a–d) Scale bar ¼ 400 mm, (e) scale bar ¼ 160 mm, (f– i) scale bar ¼ 80 mm, (j– l) scale bar ¼ 160 mm.
(W. Hua et al., pers. commun.). All of these CaMs stimulated the autophosphorylation and substrate phosphorylation of NtCBK2 but with different efficiencies. Although the activity of NtCBK2 can be amplified up to tenfold by NtCaM3 and NtCaM13, it is enhanced only about twofold by NtCaM1. The effects of different CaM isoforms on the regulation of autophosphorylation and substrate phosphorylation of CBKs suggests that, in addition to the changes in Ca2þ binding that act as a switch, the type and concentration of CaM can also determine the amplification of the Ca2þ signal in Ca2þ – CaM-mediated pathways. Expression and subcellular distribution of CBKs To understand the possible roles of CBKs, their expression profiles have been examined to provide clues to physiological functions during plant growth and development. The spatial and temporal expression patterns of CBKs have been examined using promoter– reporter fusions in transgenic plants and by in situ hybridization with isoform-specific probes. CCaMKs from lily and tobacco are expressed only in anthers [17,27]. Lily CCaMK mRNA and protein are detected in the pollen sac and their distribution is restricted to the pollen mother and tapetal cells [27]. Northern-blot analysis revealed that the genes for both CCaMK1 and CCaMK2 from tobacco were expressed in a http://plants.trends.com
stage-specific manner during anther development. The appearance of mRNA encoding these enzymes coincides with meiosis and becomes undetectable at later stages of anther development. Reverse polymerase-chain-reaction amplification assays using isoform-specific primers showed that mRNAs for both CCaMK1 and CCaMK2 from tobacco are produced in anthers [17]. The stage- and tissue-specific appearance of CCaMKs in anthers suggests that they could play a role in sensing transient changes in free Ca2þ concentration in target cells, thereby controlling developmental events in the anther. Other CBKs such as MCK1, MCK2, NtCBK1, AtCBK1 and OsCBK show similar expression profiles [13 – 15]. NtCBK1 expression (Fig. 3) is highly regulated both temporally and spatially during plant growth and development. The mRNA is abundantly accumulated in the tissue and cells required for rapid growth and metabolic activity, including the growing region of the root, flower primordia, sporogenous cells and tetrads in the anther, as well as in developing embryos. However, NtCBK1 gene expression is undetectable in pollen and anther walls at the mature pollen stage. Physiological functions Ca2þ-regulated protein phosphorylation has been implicated in a range of responses including host– pathogen interactions, cold stress, gravitropism, light-regulated
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gene expression and hypo-osmotic shock. Studies during the past decade have indicated that some of these responses are mediated by CBKs [2,9]. As a major environmental factor, light has been shown to affect almost every phase of plant growth and development, and light-mediated responses have been shown accompanied by changes in cellular Ca2þ and CaM concentrations [28]. ZmCCaMK is downregulated by red light, indicating that this CBK might be involved in the lightdependent signal-transduction pathway. No difference was observed in the production of ZmCCaMK at any intensity of blue light, confirming that the response is specifically mediated at a particular intensity of red light [23]. Many experiments have also led to the conclusion that Ca2þ functions in gravitropic signal-transduction pathways, although the exact mechanism of Ca2þ action has not been established [29]. It has been reported that Ca2þ influences gravitropic signal transduction by interaction with CaM and other Ca2þ-binding proteins, and that MCK1 might be involved in light-regulated root gravitropism because both the CaM-binding activity of MCK1 and light-regulated root gravitropism are inhibited by KN-93 (a drug that can bind to animal CaMKII and inhibit its activity) [12]. Although little information is available about specific functions of CBKs, several protein kinases have been investigated for their roles in regulating plant development and flowering [30]. Recently, experiments have shown that MCK1 plays a role in regulating flowering in tobacco [31]. The overproduction of MCK1 leads to the abortion of flower primordia on the main shoot axis and prolonged the vegetative phase in axillary buds. Flower abortion was first observed in the developing anthers, and the entire flower subsequently senesced. In axillary buds, the prolonged vegetative phase is characterized by atypical elongated, narrow, twisted leaves [31]. A role of CBKs in flowering is also indicated from other experiments with NtCBK1. Overproduction of NtCBK1 resulted in late flowering of tobacco plants (W. Hua et al., pers. commun.). These results suggest a role for CBKs in mediating flowering. Many lines of evidence have indicated the involvement of CBKs in other responses during plant development. The ZmCCaMK homolog from pea (PsCCaMK) has been reported to be involved in stress signaling [24]. The production of NtCaMK1 is stimulated by salt stress: it is greatly enhanced after 30 min with salt-stress treatment, peaks at 70 min and then declines to basal level after 4 h stress treatment. These results suggest that NtCaMK1 might be involved in the salt-stress response of tobacco (L. Ma et al., unpublished). Perspectives During the past decade, significant progress has been made in isolating and identifying CaMs and CBKs from plants. Based on current knowledge, plants contain many unique CBKs with novel regulatory mechanisms that perform plant-specific functions. It is likely that many more novel CBKs will be identified, especially as more plant genome sequences are completed. The challenge now is to elucidate the roles of these CBKs and to http://plants.trends.com
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identify cross-talk among various components of the Ca2þ signaling system. New technologies have emerged to provide facile, rapid methods for identifying the functions of CBKs in plants. Gene-disruption and silencing techniques such as insertional mutagenesis, RNA interference and virus-induced gene silencing can be used to study altered phenotypes [32,33]. Because many CBKs are members of large gene families, there is likely to be functional redundancy or overlap among members of each family. Ascertaining specific functions will thus require the creation of double or triple mutants. Technologies such as reverse genetics using knockout mutants coupled with the analysis of celland tissue-specific production of CBKs using microarrays [34], and non-destructive visualization of protein in live cells using fluorescent reporters should help to provide new insights into the functions of CBKs involved in Ca2þ signaling and deciphering signaling networks. Acknowledgements We thank Russell Jones for his encouragement and the anonymous referees for their helpful comments. Work from our laboratory is supported by grants from National Natural Science Foundation of China and Major State Basic Research Program (G1999011600).
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26 Zielinski, R.E. (2002) Characterization of three new members of the Arabidopsis thaliana calmodulin gene family: conserved and highly diverged members of the gene family functionally complement a yeast calmodulin null. Planta 214, 446 – 455 27 Poovaiah, B.W. et al. (1999) Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers. Planta 209, 161 – 171 28 Mustilli, A.C. and Bowler, C. (1997) Turning into the signals controlling photoregulated gene expression in plants. EMBO J. 16, 5801–5806 29 Konings, H. (1995) Gravitropism of roots: an evaluation of progress during the last three decades. Acta Bot. Neerl. 44, 195– 223 30 Dornelas, M.C. et al. (2000) Arabidopsis thaliana SHAGGY-related protein kinase (ArSK11 and 12) function in perianth and gynoecium development. Plant J. 21, 419– 429 31 Liang, S. et al. (2001) Mediation of flowering by a calmodulindependent protein kinase. Sci. Chin. 44, 506 – 512 32 Waterhouse, P.M. et al. (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. U. S. A. 95, 13959 – 13964 33 Romeis, T. et al. (2001) Calcium-dependent protein kinases play an essential role in plant defense response. EMBO J. 20, 5556 – 5567 34 Schena, M. et al. (1998) Microarrays: biotechnology’s discovery platform for functional genomics. Trends Biotechnol. 16, 301 – 306
2–4 October 2003 3rd International Symposium on Natural Drugs, Naples, Italy Organized by: F. Capasso, A.A. Izzo and N. Mascolo (Naples, Italy) Topics covered: Botanical, chemical and pharmacological aspects of medicinal plants and their applications in gastrointestinal and cardiovascular disturbances, endocrine dysfunctions, immunodeficiences and viral and fungal infections. Contributions to other means will be accepted in a limited number only. Satellite Symposia: Cannabis and cannabinoids; propolis and its components - chemical and pharmacological aspects; green tea and cancer; toxicity of anthraquinone drugs. Venue: Grand Hotel Oriente, Via Diaz 44, 80134 Naples, Italy. Tel: +39 081 5512133 Secretariat: Dr F. Borrelli and Dr N. Milic Department of Experimental Pharmacology, University of Naples Federico II, Via D. Montesano, 49 - 80131 Naples, Italy. Tel: +39 081 678432/436/439 – Fax: +39 081 678403 – E-mail: [email protected] http://plants.trends.com