Cdpks – a Kinase for Every ?Ca2؉ Signal Alice C

trends in plant science Reviews

CDPKs – a kinase for every ?Ca2؉ signal Alice C. Harmon, Michael Gribskov and Jeffrey F. Harper

Numerous stimuli can alter the Ca2؉concentration in the cytoplasm, a factor common to many physiological responses in plant and animal cells. Calcium-binding proteins decode information contained in the temporal and spatial patterns of these Ca2؉ signals and bring about changes in metabolism and gene expression. In addition to calmodulin, a calcium-binding protein found in all eukaryotes, plants contain a large family of calcium-binding regulatory protein kinases. Evidence is accumulating that these protein kinases participate in numerous aspects of plant growth and development.

our types of protein kinases constitute the calcium- (Caenorhabditis elegans). Thus, it is tempting to speculate that dependent protein kinase or calmodulin-like domain protein CDPKs might be present in plants and protozoans only. kinase (CDPK) superfamily. These kinases differ in whether CCaMKs are rarer than CDPKs, and might be expressed in a few F 2ϩ 2ϩ 5 they are regulated by binding Ca (CDPKs), Ca /calmodulin plant tissues only . Like CDPKs, they contain a calcium-binding [calmodulin-dependent protein kinases (CaMKs)], a combination domain6 (Fig. 1), but this domain contains only three EF-hands and of both [calcium and calmodulin-dependent protein kinases is more similar to visinin (another EF-hand protein) than to cal- (CCaMKs)], or neither [CDPK-related protein kinases (CRKs)]. modulin. The autoinhibitory domain contains a binding site for The abundant calcium-stimulated protein kinase activity found calmodulin, and calmodulin stimulates the activity of these kinases. in plant extracts is associated with CDPKs. These enzymes A third type of calcium-regulated protein kinases, the CaMKs, contain three functional domains1–4: catalytic, autoinhibitory and is well characterized from animals and yeast, but only one puta- calcium-binding (Fig. 1). The calcium-binding domain of the tive representative is known in plants7. The plant CaMK is more archetypal CDPK is similar to calmodulin in sequence (~40% similar in sequence to CCaMKs than to animal CaMKs, having an identity) and contains four EF-hand calcium-binding motifs. In identical calmodulin-binding site, but lacking the C-terminal addition to plants, CDPKs are found in protozoans such as para- domain containing EF-hands (Fig. 1). The biochemical properties mecium and Plasmodium falciparum (the causative agent of of this enzyme have not been characterized fully. malaria). Notably, CDPKs are absent from the completed genome The fourth type of protein kinase in the superfamily is the sequence of yeast (Saccharomyces cerevisiae) and of nematode CDPK-related protein kinases (CRKs). They have catalytic domains closely related to those of CDPKs, and their C-terminal domains have some sequence similarity to calmodulin (20% iden- Autoinhibitor tity), but their EF-hands are poorly conserved. Representative members of this group appear to be unresponsive to calcium8–10. It N-terminal Kinase CaM-like is not known how these protein kinases are regulated or what their CDPK physiological roles are. Another type of CRK was reported recently: phosphoenolpyruvate carboxylase kinase has a catalytic CRK domain related to those of CRKs, but no C-terminal domain. This Degenerated protein kinase phosphorylates and regulates phosphoenolpyruvate CCaMK carboxylase in vivo and is regulated at the level of transcription11. Visinin-like In phylogenetic analyses (Fig. 2), the clustering of the plant CaMK CDPKs and CRKs away from the non-plant CaMKs and the Association domain SNF1-like kinases suggests a single common origin for plant Trends in Plant Science CDPK and CRK genes. However, an important evolutionary question remains unresolved. Did different branches of the super- Fig. 1. Domain structure of calcium-dependent protein kinase or family have a common origin or did the fusion of genes encoding calmodulin-like domain protein kinases (CDPKs) and three related a protein kinase and a calcium-binding domain occur more than protein kinases. The N-terminal domain is highly variable in once in evolution? length and sequence. An autoinhibitor is predicted in the region Based on the analysis of the currently available (~70% com- immediately following the kinase domain. A distinguishing feature of CDPKs, CDPK-related protein kinases (CRKs) and cal- plete) genomic sequence from Arabidopsis, we estimate that there cium and calmodulin-dependent protein kinases (CCaMKs) is the will be a total of 40 CDPKs and seven CRKs. An Arabidopsis number of functional EF-hands in a C-terminal regulatory domain: CCaMK sequence has not yet been identified, but southern blot 12 EF-hands that can bind calcium are denoted by black boxes, analysis suggests that there is a single gene . Proliferation of fam- whereas degenerated EF-hands are denoted by gray boxes. In a ily members might be related to expression of some of these genes conventional CDPK the regulatory domain has four EF-hands and in specific tissues, physiological conditions or developmental an overall sequence similarity to calmodulin (CaM). CaMK, stages (reviewed in Ref. 13). Also there might be specialization of calmodulin-dependent protein kinase. cellular roles, which might be related to differences in substrate specificity, subcellular location and calcium sensitivity.

Activity and regulation Regulation of CDPKs by Ca2ϩ CRKs 54Ð56 62 How calcium regulates CDPKs has been 57 CCaMKs 53 58Ð61 the subject of studies with recombinant 67Ð69 Plant and algal 52 ␣ 51 soybean (Glycine max) CDPK and Ara- 63Ð66 CDPKs 70 CaMK (plant) bidopsis CPK1 (Refs 1–4,14). CDPKs are 46,47 48Ð50 − kept in a low basal state of activity by an 40 42 43Ð45 71 Protozoan autoinhibitor located in a junction domain 37Ð39 35,36 72,73 CDPKs that connects the kinase to its C-terminal 31,32 74 33,34 75 calmodulin-like domain. A peptide sequence 30 76 77 from the junction inhibits the activity of 29 78 wild-type enzymes and of a constitutively 27,28 active mutant, in a competitive fashion 25,26 with respect to the peptide substrate, sug- 23,24 gesting that the autoinhibitory sequence 20Ð22 79 19 18 80 functions through a pseudosubstrate mech- 81 3 14Ð17 12,13 SNF1-like anism , analogous to that proposed for a 6Ð10 2Ð4 1 82Ð86 typical CaMK from animals. 11 5 The simplest model for the activation of 87,88 CDPK by Ca2ϩ is provided by analogy to 90Ð92 89 the mechanism for stimulation of animal Trends in Plant Science 93Ð95 CaMKs (non-plant) CaMKs. For CaMKs, calcium promotes a bimolecular binding of calmodulin to a Fig. 2. Unrooted phylogenetic tree showing the relationship between the CDPK superfamily, region immediately downstream of an plant SNF1-like kinases, and the most closely related animal kinases, the calmodulin-dependent autoinhibitory sequence. This binding event protein kinases (CaMKs). Calcium and calmodulin-dependent protein kinases (CcaMKs) and the single plant CaMK (No. 70) share a branch close to the protozoan CDPKs. Plant and proto- somehow disrupts the autoinhibitor and zoan CDPKs form two distinct groups on the tree, with the plant isoforms being found on four results in a ‘release of inhibition’. The dis- branches. The majority of CDPKs from vascular (Nos 1–17, 19–26 and 29–52) and nonvascu- tinction for a CDPK is that this ‘release of lar plants (18, 27 and 28) are interspersed on three branches. The only available CDPK inhibition’ involves intramolecular binding sequence from an alga (No. 53) might represent a first example for a fifth branch of algal with its calmodulin-like domain (Fig. 3). homologs. Few of these CDPKs have been characterized at the biochemical level, so distinc- One line of evidence supporting a close tions cannot be made between the functions of the enzymes on these branches. Sequences of analogy to a CaMK is the observation that plant/algal (1–56) and protozoan (71–78) CDPKs (highlighted in gray) and related kinases in the activity of a truncated CDPK (⌬C), the NCBI nr database were identified by BLAST 2.09 (Ref. 43) – the evolutionary profile method44. Alignments were constructed by ClustalW45, corrected manually, and used to gener- in which the calmodulin-like domain 46 is deleted, can be partially stimulated ate a neighbor-joining tree , which was subjected to 1000 rounds of bootstrap analysis. The tree shown was constructed from sequences of kinase catalytic domains. Each entry contains a two- by either calmodulin or an isolated cal- letter code indicating genus and species, common name (if available) and Accession no. modulin-like domain, with half maximal Entries for CDPKs from Arabidopsis include names (underlined) assigned in Ref. 13 followed 1,2 activation at ~3 ␮M for both activators . by names given in the literature. See http://plantsP.sdsc.edu for additional information on meth- Although this indicates that a CDPK can be ods and sequences. (1) AtCPK20, 3928078; (2) AtCPK1, AK1, 304105; (3) AtCPK2, 1399271; reconstituted as a bimolecular interaction (4) CpCPK1, 1899175: (5) zm3320104; (6) AtCPK26, 4467129; (7) VrCDPK-1, 967125; (8) with calmodulin (i.e. like a CaMK), it is AtCPK6, AtCDPK3, 603473; (9) AtCPK6, 1399275; (10) AtCPK5, 1399273; (11) Zm506413; possible that the natural mechanism of (12) ZmCDPK1, 1632768; (13) ZmCDPK7, 1504052; (14) OsCDPK1, 435466; (15) intramolecular activation (i.e. the whole) is Os6063536; (16) Oscdpk12, 2944385; (17) Oscpk11, 587500; (18) TrCPK1, 2315983; (19) AtCPK12, AtCDPK9, 836946; (20) AtCPK10, AtCDPK1, 604880; (21) AtCPK11, AtCDPK2, distinct from its reconstitution as two sep- ␤ ␣ arate fragments (i.e. the sum of the parts). 604881; (22) AtCPK4, 1399267; (23) GmCDPK , 2501764; (24) GmSK5, GmCDPK , 116054; (25) AtCPK3, AtCDPK6, 2129550; (26) AtCPK3, AtCDPK6alt, 2129553; (27) An important challenge is to understand MpCDPK-a, 4874268; (28) MpCDPK-b, 5162878; (29) Zm639722; (30) AtCPK22, 4115942; the structural basis for activation of CDPK (31) AtCPK27, 5706728; (32) AtCPK31, 5732059; (33) AtCPK15, 2961339; (34) AtCPK21, (and CaMKs), and to determine whether 4115943; (35) AtCPK23, At4115945; (36) AtCPK19, 3367525; (37) Dc1765912; (38) the presence of a tethered calmodulin-like NtCDPK1, 3283996; (39) GmCDPK␥, 2501766; (40) AtCPK9, 1399265; (41) Ib1552214; (42) domain endows CDPKs with unique bio- Mc4336426; (43) OsCPK2, 587498; (44) ZmCDPK2,886821; (45) ZmCDPK9,1330254; (46) chemical and physiological properties. AtCPK13, 1314711; (47) AtCPK14, 1871195; (48) AtCPK8, AtCDPK19, 2129551; (49) One reason for the multiplicity of AtCPK7, 1399277; (50) Fa2665890; (51) AtCPK30, At5882721; (52) AtCPK24, 4589951; CDPKs in a given plant species might be (53) Ce806542; (54) StCPK1,3779218; (55) AtCPK16, 2708745; (56) AtCPK18, 3036811; related to the specialization of different iso- (57) AtCRK3, 3831444; (58) Zm2443388; (59) ZmCRK1, 1313907; (60) ZmMCK1, 1839597; forms with respect to calcium binding and (61) ZmCRK3, 1313909; (62) AtCRK4, AtCP4, 4741927; (63) DcPK421,1103386; (64) AtCRK5, 2154715; (65) AtCRK2, 5020368; (66) AtCRK1, 5020366; (67) LlCCAMK, activation. Dose–response curves (Fig. 4) 860676; (68) NtCCAMK, 4741991; (69) NtCAMK1, 5814023; (70) MdKCCS, 1170626; (71) for three CDPK isoforms from soybean PfCDPK2, 2315243; (72) TgTPK4, 2854042; (73) EtCDPK, 1279425; (74) PfCPK, 422320; show that they are responsive to different (75) PtCPKb, 2271461; (76) PtDPKa, 2271459; (77) TgTPK6, 4325074; (78) TgTPK5, ranges of calcium concentrations15. The 4325072; (79) HvKIN12a, 3341452; (80) At4099088; (81) StPKIN1, 1216280; (82) concentration of calcium required for half- HvBKIN2, 575292; (83) Gm4567091; (84) AtAKIN10, 322596; (85) Cs1743009; (86) ␣ ␤ maximal activity (K0.5) for CDPKs , and NtNPK5, 1076633; (87) CgCMK, 2654181; (88) EmCMKB, 5053101; (89) SpCAMKI, ␥ varies over two orders of magnitude when 3309070; (90) DmCAMKI, 3893099; (91) CeCMK-1, 5672678; (92) RnCAMK1, 3122310; using the synthetic peptide substrate syn- (93) RnCAMKII, 125288; (94) CeCAMKII, 5834390; (95) DmCAMKII, 84904. tide-2. Other CDPKs, such as P. falciparum

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PfCPK1 (Ref. 16), require concentrations of calcium that are + Ca 2+ another order of magnitude higher than those observed for the Basal Activated soybean enzymes. Calcium-binding properties have been experimentally deter- Autoinhibitor mined for only a few CDPKs, and it is difficult to predict from sequence information what the calcium-binding properties of each isoform will be. Some CDPKs appear to have defects in one or KKmore of their EF hands that would affect their calcium-binding properties17. Studies with PfCPK1 (Ref. 16), in which each EF Calmodulin- hand was disabled by the mutation of a critical glutamate residue, Tether like domain showed that for the enzyme to be stimulated by calcium only the Trends in Plant Science first EF-hand must be functional. Therefore, CDPKs with defects in EF hands can still be regulated by calcium. Fig. 3. A ‘release of inhibition’ model for activation of a calcium- Another nuance in the regulation of CDPKs is that their sensi- dependent protein kinase or calmodulin-like domain protein kinase tivity to calcium can be influenced by the type of protein substrate (CDPK) by calcium. The kinase is shown to undergo a confor- (Fig. 4). In the absence of any substrates, CDPK␣ binds Ca2ϩ with mational change in response to calcium that results in an auto- ␮ inhibitory interaction (unbroken line) being displaced (broken line). a Kd of 50 M. However, in the presence of substrates, calcium sensitivity can increase tenfold or more (Fig. 4). These differences in sensitivity to calcium might mean that each isoform of CDPK responds to a specific set of calcium signals, which differ in frequency of oscillation, magnitude and (a) duration depending on the stimulus (reviewed in Refs 18,19). 100 The difficult question of how to test in vivo for differential activities of specific isoforms remains unanswered. 75 αβγPF Regulation by calmodulin 2ϩ 20 2ϩ 50 CCaMK binds both calcium ions and Ca /calmodulin . Ca 0.06 0.4 1.0 15 stimulates autophosphorylation, but not phosphorylation of the in 2ϩ

Activity (%) vitro substrate histone IIAS. By contrast Ca /calmodulin stimu- 25 lates histone IIAS phosphorylation, but inhibits autophosphorylation. Activation is proposed to occur through the binding of 0 Ca2ϩ/calmodulin to a site in the autoinhibitory domain, similar in 0.01 0.10 1.00 10.00 100.00 position and sequence to the intramolecular binding site for the calmodulin-like domain in CDPKs. The isolated autoinhibitory domains of soybean CDPK␣ and (b) Arabidopsis AtCPK1 bind calmodulin2,21, but these enzymes are 100 not greatly stimulated by calmodulin. Because the calmodulin- binding sequence in these CDPKs interacts intramolecularly with 75 the calmodulin-like domain in the holoenzyme, it is probably Syntide-2 unavailable for binding calmodulin. Carrot CRK is also un- affected by the addition of calmodulin (J. Choi, pers. commun.) 50 2+ Histone Ca in spite of the presence of a potential calmodulin-binding site in

Activity or binding its putative autoinhibitory domain. Nevertheless, the question of 25 whether calmodulin might regulate some isoforms is still open, as calcium binding (%) apo-calmodulin can bind to the variable amino-terminal domain 0 of AtCPK1 (Ref. 1). Whether this binding site might have a role in 0.01 0.10 1.00 10.00 100.00 docking the kinase into a protein complex or in modifying a more Free calcium (µM) subtle feature of regulation is not known.

Trends in Plant Science Regulation by myristoylation and lipids Fig. 4. Sensitivity of calcium-dependent protein kinase or The question of regulation by lipids is an important one because calmodulin-like domain protein kinase (CDPK) isoforms to Ca2ϩ. ϩ ϩ this could represent a possible point of crosstalk between signal- (a) Isoform-specific thresholds for Ca2 -activation showing the Ca2 ing pathways, or a means of targeting activity to specific locations dose-response curves for phosphorylation of syntide-2 by soybean ␣ ␤ ␥ by reversible membrane association. CDPKs are found in several CDPKs , and (data from Ref. 15), and phosphorylation of subcellular locations including membranes. The structural basis casein by Plasmodium falciparum CDPK1 (data from Ref. 16). To emphasize differences in calcium sensitivity, the maximal activity for membrane-association is not known because CDPKs have neither predicted membrane spanning regions nor C2 domains, which for each enzyme was set to 100%. The K0.5 for each CDPK is indicated. (b) Substrate-dependent thresholds for Ca2ϩ-activation, are responsible for the interaction of proteins such as protein kinase 2ϩ showing the influence of substrate on the Ca2ϩ sensitivity of C with lipids and Ca . However, it is possible that myristoylation, soybean CDPK␣ (data from Ref. 15). In the absence of any sub- which is known to affect the membrane localization of several pro- ␣ 2ϩ ␮ strates, CDPK binds Ca (broken line) with a Kd of 50 M. In teins, might underlie membrane-association. Many CDPKs have ␮ 13,17 activity assays, the K0.5s were 0.4 and 4.0 M syntide-2 and histone predicted myristoylation sites at their N-termini . A carrot CDPK IIIS, respectively. can be myristoylated during co-expression with appropriate modifying enzymes in E.coli22, and a zucchini (Cucurbita pepo) CDPK

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has been found to be myristoylated in vivo23. Because CDPK together that are involved in two consecutive metabolic steps32. activity is found in the cytosol as well as in membranes, the ques- Roles for 14-3-3s in the activation and targeting of CDPKs need tion arises as to whether their association with membranes might to be explored further. be regulated by a Ca2ϩ-dependent myristoyl switch, in a manner similar to that of recoverin24. Substrates and physiological roles The activity of some CDPK isoforms is stimulated by phos- Insight into the physiological roles of CDPKs has come from pholipids in addition to activation by calcium. Activation of identification of substrates and from experiments using constitu- AtCPK1 (Ref. 21) and carrot (Daucus carota) DcCPK1 (Ref. 10) tively active CDPKs to activate a pathway in the absence of a by phospholipids such as phosphatidylserine and phosphatidyl- calcium signal33. For example, it has been shown that expression inositol is synergistic. However, because these lipids are not con- of a constitutively active version of isoform AtCPK10 [but sidered to be signaling molecules, it is arguable whether they not AtCPK1 or AtCPK11 (numbering as in Ref. 17), or four serve as regulators or as structural components of membrane- protein kinases from another family] led to the expression of a associated CDPKs. One possible scenario is that certain CDPKs stress-, Ca2ϩ- and ABA- responsive reporter gene. This is the first have low activity when located in the cytosol, but are activated stimulus-response pathway shown to be activated by a specific upon translocation to the membrane. isoform. It will be interesting to see what protein(s) in this path- It is possible that some CDPKs are regulated in vivo by signal- way are phosphorylated by this CDPK. ing molecules derived from lipids, but only a few have been Biochemical approaches have identified a variety of CDPK tested. Recombinant DcCPK1 is stimulated by phosphatidic substrates that suggest potential regulatory roles in gene expres- acid10, a component in phospholipase D signaling pathways. It is sion, metabolism and signaling pathway components, traffic of not stimulated by diacylglycerol, which is produced by the cleav- ions and water across membranes, and the dynamics of the age of phosphatidylinositol bisphosphate by phospholipase C and cytoskeleton (Fig. 5). More information on these substrates can be is a regulator of protein kinase C in animals. It will be interesting found at the protein kinase and phosphatase Web site (Box 1), and to see if CDPKs are subject to cross-regulation by components of in a recent review13. Here, we highlight two substrates for which other pathways such as jasmonic acid or brassinosteroids. there is strong experimental support for regulation by CDPK. Sucrose phosphate synthase (SPS) is a key enzyme in the Regulation by phosphorylation sucrose synthesis pathway, and nitrate reductase is the rate-limit- Both native and recombinant CDPKs exhibit intramolecular ing enzyme in the assimilation of nitrogen from nitrate (reviewed autophosphorylation, but the sites of autophosphorylation have in Refs 34,35). Both enzymes are phosphorylated in the dark, not been identified and there is no consensus as to the role of resulting in their inhibition. SPS is directly inhibited by phospho- autophosphorylation. Many CDPK isoforms contain a potential rylation of Ser153, and nitrate reductase is inhibited by a two-step autophosphorylation site (Lys-Gln-Phe-Ser) in their auto- mechanism involving phosphorylation of Ser543 and binding of a inhibitory domains. Because autophosphorylation of CaMKII at a 14-3-3 protein to the phosphorylated site (reviewed in Ref. 28). similar site yields an active enzyme that no longer requires Inhibition of these enzymes in the dark when carbon fixation is Ca2ϩ/calmodulin, it is tempting to speculate that these CDPKs are not occurring diminishes the partitioning of carbon skeletons into potentially activated by a similar mechanism. However, available exported sucrose and amino acids, and conserves glucose and evidence does not support this possibility. Soybean CDPK␣ does fructose for use in other pathways in leaf cells, such as glycolysis not phosphorylate peptides matching the sequence of the auto- or starch synthesis. Evidence that supports this in vivo function inhibitory domain3, and autophosphorylation does not affect the includes co-purification of a CDPK that has been identified as a calcium requirement of either groundnut (Arachis hypogaea)25 or homolog of AtCPK3 (Ref. 26), which phosphorylates both of soybean CDPK␣ (B.C. Yoo and A.C. Harmon, unpublished). In these enzymes at the regulatory sites26,36–38. These observations addition, the activity of a CDPK purified from spinach (Spinacia raise the possibility that a single CDPK can coordinately regulate oleracea) was not altered by incubation in conditions that favor both activities. phosphorylation or by treatment with phosphatase26. In the case of The hypothesis that CDPK down-regulates nitrate reductase CCaMK, autophosphorylation of the lily (Lilium) isoform stimu- and SPS in response to the dark is consistent with the observation lates activity fivefold, but it still requires Ca2ϩ/calmodulin20. that cytoplasmic calcium concentrations are higher at night than However, other reports have shown that autophosphorylation during the day39. However, it should be noted that a calcium-inde- affects the activity of some CDPKs. Autophosphorylation of pendent kinase with properties of SNF1-related kinases also phos- groundnut CDPK is required for its activity, but it occurs at low phorylates Ser153 of SPS and Ser543 of nitrate reductase38,40,41. concentrations of Ca2ϩ and might not have a regulatory role in vivo25. Thus, it is possible that both types of kinase down-regulate By contrast, autophosphorylation of CDPK purified from winged SPS and nitrate reductase in vivo, but in response to a separate bean (Psophocarpus tetragonolobus) is inhibitory27. Thus, clear signaling pathway. evidence showing phosphorylation-dependent activation of a A CDPK might also be involved in activating SPS by phospho- CDPK has not yet emerged. rylation at a site (Ser424) distinct from the inhibitory site42. This phospho-dependent activation occurs in response to hypo-osmotic The 14-3-3 connection stress and presumably increases cytosolic sucrose and thereby The 14-3-3 proteins serve as both regulatory and docking proteins decreases the water potential of the cell to help retain water. These (reviewed in Ref. 28). Several CDPKs bind 14-3-3 proteins, and intriguing results raise the possibility that two different CDPK the activity of at least one is stimulated through binding 14-3-3 pathways that are differentially activated by separate stimuli (Refs 29–31). The 14-3-3s also bind to sites in proteins, such as oppositely regulate SPS activity. nitrate reductase, that have been phosphorylated by CDPK (Ref. 28). Several other proteins including sucrose-phosphate syn- The future thase, trehalose-6-phosphate synthase, glutamine synthetases and In the past ten years we have progressed from identifying the first LIM17 bind 14-3-3 proteins in a phosphorylation-dependent man- member of a new family of calcium-dependent protein kinases to ner30. It has been proposed that 14-3-3s might dock enzymes the understanding that:

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Environmental or developmental signals Box 1. Plant protein kinase and phosphatase Web site

Ca2+ Other pathways This Web site (http://plantsP.sdsc.edu) e.g. calmodulin, focuses on Arabidopsis genes encoding cGMP, G proteins, protein kinases and phosphatases. When CDPKs lipid signals, MAPKs completed, this site will have an annotated database of all families of protein kinases and phosphatases, and will announce the availability of protein kinase and phosphatase mutants as they are identified and Transcription Metabolic Ion pumps Cytoskeletal made available to the research community. factors enzymes and and channels proteins Information on the CPDK family can be G/HBF1? regulators Aquaporins Actin found at http://plantsP.sdsc.edu/cdpk, in- HBP-1a(17)? SPS, NR, SuSy, H+ and Ca2+ depolymerizing cluding fully annotated figures from this GT1BP? HMGR, SAT, pumps, K+ and factor review, expanded information on phylo- EF1-α, Phox anion channels genetic trees and sequence alignments, and a categorized list of literature related to all the CDPKs that have been found in plant and protist species to date. Gene Sugar and Membrane Cell expression amino acid ion traffic architecture metabolism, • Use genetics to identify biological func- signaling tions, as indicated by the phenotypes that result from the disruption of genes or the expression of de-regulated mutants (e.g. calcium independent). Cell growth, development and defense • Use bioinformatics to provide a picture Trends in Plant Science of how CDPK signaling pathways are integrated into the dynamic interactions of Fig. 5. Calcium-dependent protein kinase or calmodulin-like domain protein kinase (CDPK) all signaling pathways in a cell. substrates and physiological roles. A hypothetical model for the roles of CDPKs in plant growth and development and cellular defense responses. CDPK is stimulated by an increase CDPKs represent a potential gold mine of in Ca2ϩ that results from various stimuli. These stimuli might also activate additional signal- opportunity for biotechnology applications. ing pathways and Ca2ϩ might also activate calmodulin-dependent pathways. Activated Given the involvement of calcium signals in CDPK(s) phosphorylate certain substrates, depending on the nature of the stimulus. Other so many aspects of plant biology, including signaling pathways might also be involved in the stimulation of CDPKs or the substrates. biotic and abiotic stress responses, the differ- Phosphorylation of the substrates alters their activities and this contributes to the physiologi- ent CDPK pathways provide many potential cal response. Abbreviations: G/HBF1, soybean G- and H-box binding factor 1; HBP-1a(17), points of intervention to suppress or activate wheat H-box binding factor 1a(17); GT1BP, Arabidopsis GT1-binding protein; SPS, sucrose a specific response. Structure–function stud- phosphate synthase; NR, nitrate reductase; SuSy, sucrose synthase; HMGR, 3-hydroxy-3- ␣ ies on CDPKs have provided an important methylglutaryl-coenzyme A reductase; SAT, serine O-acetyltransferase; EF1- , elongation paradigm by showing how a CDPK can be factor 1-␣; Phox, p67-Phox and p47-Phox are components of NADPH oxidase; aquaporins, water channels including Nodulin 26, a soybean root nodule water and weak anion channel, converted into an active, calcium-indepen- and TIP from tobacco vacuoles. dent kinase to be used as a dominant, posi- tive transgene1. This approach has provided a precedent by establishing a role for one CDPK isoform in selectively activating a • Plants have many CDPK isoforms (Ͼ40 in Arabidopsis), which cold, dark and osmotic stress response pathway in the absence of are predicted to have kinase activities directly activated by cal- other calcium signaling pathways33. cium and potentially modified by cross-talk with other signaling systems. Acknowledgements • CDPKs are multifunctional, with individual isoforms providing We are supported by funding from the National Science Foundation, specific pathways to control transcription, metabolic enzymes, DBI-9975808. membrane transport and cell structure. • Plants also have at least three types of CDPK-related kinases, References with some members regulated by calmodulin and some totally 1 Huang, J.F. et al. (1996) Activation of a Ca2ϩ-dependent protein kinase unresponsive to calcium signals. involves intramolecular binding of a calmodulin-like regulatory domain. A major challenge for the future is to create an integrated picture Biochemistry 35, 13222–13230 of how members of this kinase family are used in plant develop- 2 Yoo, B.C. and Harmon, A.C. (1996) Intramolecular binding contributes to the ment and physiology. To this end, we offer the following basic activation of CDPK, a protein kinase with a calmodulin-like domain. strategy for investigating every isoform in a model plant such as Biochemistry 35, 12029–12037 Arabidopsis. 3 Harmon, A.C. et al. (1994) Pseudosubstrate inhibition of CDPK, a protein • Use biochemistry to define isoform-specific calcium activation kinase with a calmodulin-like domain. Biochemistry 33, 7278–7287 thresholds and substrate specificities. 4 Harper, J.F. et al. (1994) Genetic identification of an autoinhibitor in CDPK, a • Use cell biology tools to delineate subcellular locations. protein kinase with a calmodulin-like domain. Biochemistry 33, 7267–7277

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