Competitive binding of calmodulin isoforms to calmodulin-bindingproteins: implication for the function of calmodulin isoforms in plants

Sang Hyoung Lee 1, Min Chul Kim 1, Won Do Heo 1, Jong Cheol Kim 1,Woo Sik Chung, Chan Young Park, Hyeong Cheol Park, Yong Hwa Cheong,

Cha Young Kim, Sung-Ho Lee, Kyung Joo Lee, Jeong Dong Bahk, Sang Yeol Lee,Moo Je Cho *

Department of Biochemistry, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701,South Korea

Received 9 March 1999; received in revised form 10 June 1999; accepted 24 June 1999

Abstract

In plants, multiple calmodulin (CaM) isoforms exist in an organism which vary in their primary structures in as much as 32residues out of their 148 amino acids. These CaM isoforms show differences in their expression patterns and/or target enzymeactivation ability. To further understand the biological significance of CaM isoforms, we examined whether CaM isoformsact on specific regulatory targets. In gel overlay assays on various soybean tissue extracts, surprisingly, two soybean CaMisoforms (SCaM-1 and SCaM-4) did not show significant differences in their target binding protein profiles, although theyexhibited minor differences in their relative target binding affinities. In addition, both SCaM isoforms not only effectivelybound five known plant CaMBPs, but also showed competitive binding to these proteins. Finally, immunolocalizationexperiments with the SCaM proteins in sections of various tissues using specific antibodies revealed similar distributionpatterns for the SCaM isoforms except for root tissues, which indicates that the SCaM isoforms are concomitantly expressedin most plant tissues. These results suggest that CaM isoforms may compete for binding to CaMBPs in vivo. This competitivenature of CaM isoforms may allow modulation of Ca2�/CaM signaling pathways by virtue of relative abundance anddifferential target activation potency. ß 1999 Elsevier Science B.V. All rights reserved.

Keywords: Calmodulin isoforms; Competitive binding calmodulin-binding proteins

1. Introduction

Calmodulin (CaM) plays pivotal roles in the trans-duction of various Ca2�-mediated signals in eukary-otic organisms [32,36]. The function of CaM is soessential that, in yeast and Drosophila, CaM is indis-pensable for survival [11,20]. In animals, CaM is in-volved in diverse cellular functions such as cellgrowth and proliferation, muscle contraction, neuraltransmission, and development. More than 30 CaM-dependent enzymes or proteins have been identi¢ed

0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 1 4 9 - 1

Abbreviations: AP, alkaline phosphatase; CaM, calmodulin;CaMBPs, calmodulin-binding proteins; CKII, casein kinase II;CaN, calcineurin; EST, expressed sequence tag; GST, gluta-thione-S-transferase; HRP, horseradish peroxidase; IPTG, L-iso-propyl-galactopyranoside; PBS, phosphate-bu¡ered saline;PMSF, phenylmethylsulfonyl£uoride

* Corresponding author. Fax: +82-591-759-9363;E-mail : mjcho@nongae.gsnu.ac.kr

1 Contributed equally and should be considered co-¢rst au-thors.

BBAPRO 35966 2-8-99

Biochimica et Biophysica Acta 1433 (1999) 56^67

www.elsevier.com/locate/bba

so far, and the number of CaM-binding proteins(CaMBPs) is still rising. Upon sensing rise of intra-cellular Ca2� concentration, CaM binds Ca2� andundergoes conformational changes to expose hydro-phobic pockets that are important for interactionwith targets [23]. Since the molecular basis of Ca2�/CaM-dependent signal transduction that eventuallyevokes cellular responses depends on the Ca2�-de-pendent binding of CaM to target proteins/enzymeswith subsequent modulation of their biological activ-ities, the identi¢cation of CaMBPs is essential tostudy CaM function in plant cells [40].

As compared to animals, relatively little is knownabout the functional importance of CaM in plantcells. CaM is implicated for diverse processes includ-ing thigmomorphogenesis [6], light signal transduc-tion [33], gibberellic acid signaling [16], and plantdefense response against pathogens [19,21]. However,the regulatory target protein(s) of CaM that mediatethese responses are yet unknown. To unequivocallyestablish the role of CaM in plant cells, CaMBP(s)actually linking Ca2�/CaM signals to speci¢c re-sponse should be identi¢ed.

A number of methods have been developed toidentify, isolate, and purify CaMBPs. The a¤nitychromatography method using CaM-agarose is avery powerful technique in that one can purifyCaMBPs from complex crude protein extracts in asingle step [39]. However, this method has its limita-tion due to the possible co-puri¢cation of proteinsthat interact with CaMBPs and its relatively lowsensitivity. The CaM gel overlay methods using125I- or 35S-labeled CaM protein greatly enhancessensitivity and convenience of CaMBP detection.Crude protein extracts are size fractionated bySDS-PAGE and incubated with radio-labeled CaM[17]. The overlay method can also be extended to thescreening of expression cDNAs of CaMBPs [2,14,28].Recently, the use of biotinylated CaM as a non-ra-dioactive alternative was also reported [3,13].

Plants possess many CaM isoforms in contrast toanimals that produce only two CaM isoforms. PlantCaM isoforms are subject to di¡erential transcrip-tional regulation and thus showed di¡erent responsesto external stimuli and di¡erent tissue distributions[35,36]. Recently, two extremely divergent CaM iso-forms were identi¢ed in soybean which have substi-tutions in 32 out of 148 amino acids of highly con-

served plant CaM [25]. Notably, these plant CaMisoforms showed di¡erence in their target enzymeactivation ability [25^27,30]. Then, a question arises,what is the biological importance to plants of havingso many CaM isoforms? Two possibilities can bethought of. One is that plant CaM isoforms mayhave their own speci¢c target enzymes/proteins tomediate CaM isoform-speci¢c functions. Anotherpossibility is that CaM isoforms may selectively ac-tivate or inhibit CaM-dependent enzymes by virtueof their di¡erential target activation ability. Severallines of evidences support the second model. First,although SCaM-4 is unable to activate NAD kinase,it still retains the ability to bind it [26]. Second, hu-man CLP, a divergent CaM-like protein, competi-tively antagonized the activation of myosin lightchain kinase by animal CaM [12]. Finally, in theactivation of animal CaN and NOS in vitro, thesoybean CaM isoforms not only selectively activatedthese enzymes but also competitively inhibited theactivation of these enzymes mediated by anotherCaM isoform [8]. Thus, plant CaM isoforms act onthese enzymes in a reciprocal manner. However, itstill remains an open question whether plantCaMBPs are regulated in the same manner as wehave observed with animal enzymes. Also, it wouldbe worth noting that, if di¡erent CaM isoforms arespeci¢cally localized in di¡erent type(s) of cells and/or expressed in separate tissue regions, the competi-tion among CaM isoforms for binding to CaMBPswill not occur in vivo. One quick and e¡ective meth-od for testing whether plant CaM isoforms have dif-ferent target proteins is the CaM gel overlay tests ofvarious plant protein extracts using each CaM iso-form as a probe.

The study presented here addressed whether di¡er-ent CaM isoforms have di¡erent binding targets orCaM isoforms bind the same subset of proteins. Us-ing 32P-labeled CaM and CaM: :HRP conjugate asprobes, we provide evidence that CaM isoformsshare binding target proteins rather than have spe-ci¢c binding partners. In addition, competitive bind-ing of SCaM isoforms to plant CaMBPs is beingdemonstrated by competition overlay assays. Finally,we show that the highly conserved and the divergentCaM isoforms have similar spatial distributions, notcell-type speci¢c localization. These data suggest thatcompetition will occur among CaM isoforms in bind-

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^67 57

ing to CaMBPs in vivo. The biological importance ofthis competition among CaM isoforms is discussed interms of possible bifurcation/modi¢cation of Ca2�/CaM-dependent signal transduction pathways inplants.

2. Materials and methods

2.1. Puri¢cation of soybean CaM

Soybean CaM isoforms were over-expressed in Es-cherichia coli and puri¢ed as described previously[25,26]. The homogeneity of puri¢ed CaM proteinswas assessed by sodium dodecyl sulfate^polyacryl-amide gel electrophoresis (SDS^PAGE) analysisand was higher than 99%.

2.2. Preparation of membrane proteins from soybeancell suspension cultures

Soybean cell suspension culture, SB-P, was main-tained as described [22]. After collecting cells by mildvacuum ¢ltration on a Whatman #2 ¢lter paper, thecell paste was rapidly frozen under liquid nitrogenand stored at 380³C prior to use. The frozen cells(50 g) were homogenized in 200 ml of ice-cold ex-traction bu¡er (25 mM Tris^HCl (pH 7.5), 0.25 Msucrose, 2.5 mM DTT, 3 mM EDTA, 0.5 mMPMSF) using a Polytron. After stirring for 30 minat 4³C, the homogenate was ¢ltered through twolayers of Miracloth (Calbiochem). Cell debris wasremoved by centrifugation at 12 000Ug for 10 minat 4³C, and the supernatant was centrifuged at105 000Ug for 1 h at 4³C to obtain membrane pro-teins. The crude membrane pellet was resuspended inthe extraction bu¡er containing 20% (w/v) sucroseand subjected to 20^45% (w/v) sucrose density gra-dient ultracentrifugation (95 000Ug, 1 h at 4³C).Fractions were collected at the interfaces between20^30% (w/v), 30^35% (w/v), and 35^45% (w/v) su-crose cushions [37]. The fractionated membrane pro-teins were immediately frozen under liquid nitrogenand stored at 380³C.

2.3. Conjugation of CaM to HRP

Before conjugating to HRP, CaM (1 mg) was ¢rst

incubated in 25 mM Tris^HCl (pH 7.0), 2 mMEGTA, 0.1 M DTT at 55³C for 1.5 h to fully reducea cysteine residue. After dialysis against two changesof 2 l of degassed PBS at 4³C, CaM was concen-trated to a 1 mg/ml concentration using the Centri-con C10 (Amicon). The reduced and dialyzed CaM(385 Wg) was mixed with 1 mg of maleimide-activatedHRP (Pierce) in PBS (0.5 ml ¢nal volume) to give a1:1 molar ratio and incubated at room temperaturefor 1.5 h. Unconjugated CaM was removed by gel¢ltration, and the CaM: :HRP conjugate was storedat 320³C. The conjugation e¤ciency was determinedto be usually higher than 85% by SDS^PAGE anal-ysis.

2.4. Preparation of 35S- and 32P-labeled CaM

35S-labeled CaM was prepared by metabolic label-ing with Translabel (ICN Biomedicals) as described[14]. In the preparation of 32P-labeled CaM, CaMwas phosphorylated by CK II [15,18,24,38]. Thephosphorylation reaction was performed in a 100Wl reaction mixture containing 50 mM Hepes^NaOH (pH 7.6), 10 mM MgCl2, 5 mM EGTA,5 mM DTT, 10 Wg CaM, 0.86 WM poly-L-lysine,5 WCi [Q-32P]ATP (3000 Ci mmol/l), 250 WU CKII(Boehringer Mannheim) at 30³C for 30 min. The re-action was stopped by heating at 85³C for 10 minand then rapidly cooled on ice. Denatured CKII wasremoved by centrifugation at 12 000Ug for 15 min at4³C. The supernatant containing phosphorylatedCaM was carefully transferred and unincorporated[Q-32P]ATP was removed by gel ¢ltration. The 32P-labeled CaM proteins were saved at 320³C. Two-dimensional phosphoamino acid analysis was per-formed according to Boyle et al. [5] to verifythat SCaMs were phosphorylated at Ser and Thrresidues.

2.5. Preparation of protein extracts from soybeantissues

Soybean seeds were germinated and grown onmoist vermiculite at 28³C in the dark. At 3.5 daysafter imbibition, seedlings were cut into several partswith a razor blade, immediately frozen in liquid ni-trogen, and saved at 380³C. For tissues of matureplants (V2 months old), germinated soybean seed-

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^6758

lings were transferred to soil and grown under green-house conditions. Tissue samples (V1 g) wereground to a ¢ne powder using mortar and pestleunder liquid nitrogen. Two volumes of protein ex-traction bu¡er (50 mM Tris^HCl (pH 7.0), 1 mMEDTA, 14 mM L-mercaptoethanol, 1 mM PMSF,0.1% (w/v) polyvinylpolypyrrolidone) was addedand vigorously vortexed for 5 min. Insoluble materialwas removed by centrifugation at 15 000Ug for 20min at 4³C, and the supernatant was saved as thesoluble protein extract and kept at 380³C untiluse. Protein concentration was determined by Pro-tein Assay (BioRad) using BSA as standard.

2.6. CaM gel overlay assays

For the examination of tissue distribution ofCaMBPs, 100 Wg of soluble protein extract was sep-arated on a 5^20% gradient SDS^polyacrylamide gel(140U110U1 mm format). Gel overlay assay wasperformed without transferring the proteins to amembrane support as described by Glenny and Web-er [17], except that we used 32P-labeled CaM (1U105

cpm/ml) instead of [125I]CaM. In CaM: :HRP over-lay assays, proteins were separated on a 10% SDS^polyacrylamide gels and transferred onto an Immo-bilon-MP membrane (Millipore). After washing themembrane in TBS-T (TBS containing 0.1% (v/v)Tween-20), non-speci¢c protein-binding sites wereblocked by incubating the membrane in 7% (w/v)non-fat dry milk/TBS-T for 2 h or overnight. Alloverlay procedures were performed at room temper-ature. The blocked ¢lter was washed three times withTBS-T for a 5-min period and equilibrated in theoverlay bu¡er (50 mM imidazole^HCl (pH 7.5),150 mM NaCl) for 1 h. A second blocking of themembrane was then performed by incubating the¢lter in the overlay bu¡er containing 9% (v/v) gelatinfrom cold water ¢sh skin (Sigma), 0.5% (v/v) Tween-20, and 1 mM CaCl2 for 3.5 h [42]. SCaM: :HRP wasadded to the gelatin-containing bu¡er at a ¢nal con-centration of 0.2 Wg/ml, and the blot was incubatedfor 1 h. Final washing was done in three steps andeach step consisted of ¢ve repeats of a 5-min wash-ing: ¢rst, in TBS-T/50 mM imidazole^HCl (pH 7.5)/1 mM CaCl2 ; second, in 20 mM Tris^HCl (pH 7.5)/0.5% Tween-20/50 mM imidazole^HCl/0.5 M KCl/1mM CaCl2 ; and third, in 20 mM Tris^HCl (pH 7.5)/

0.1% Tween-20/0.5 M KCl/1 mM MgCl2. BoundCaM-HRP was visualized using the enhanced chem-iluminescence (ECL) detection kit (Amersham). Fordetermination of Ca2�-independent binding of CaM,5 mM EGTA substituted for the 1 mM CaCl2 in alloverlay bu¡ers.

2.7. cDNA expression library screening

A lambda ZAPII library constructed frommRNAs of fungal elicitor-treated rice cell suspensionwas used (C.Y. Kim, M.J. Cho, unpublished). Aftergrowing the phages for 3.5 h at 42³C, a nitrocellulosemembrane pre-soaked in 10 mM IPTG was carefullyoverlaid on top of the agar plates, and the incubationwas continued at 37³C for 6^8 h. The membrane wasthen screened for CaMBPs as described in theCaM: :HRP overlay method. Positive plaques werepuri¢ed by two additional rounds of screening, andcDNA inserts were recovered by in vivo excisionwith helper phage, ExAssist (Stratagene).

2.8. Immunolocalization experiment

Hook and root tip regions of soybean seedlings (3-day-old, etiolated) were cut into several 5-mm piecesand immediately ¢xed in the 5% (v/v) formaldehyde,10% (v/v) acetic acid by applying vacuum for 3 h.Fixed tissues were dehydrated in a gradual series ofethanol and embedded in para¤n (Paraplast plus,VWR Scienti¢c). Sections (8 Wm thickness) were pre-pared using a rotary microtome (Carl Zeiss) andmounted onto poly-Lys-coated glass slides. After re-moving the para¤n, non-speci¢c binding sites in thetissue sections were blocked by incubation in 3% (w/v) BSA dissolved in TBS-T. Anti-SCaM-1 or anti-SCaM-4 antibody was applied to the sections at 2.5Wl/ml concentration in TBS-T and incubated over-night at room temperature. Pre-immune serum(1:500 dilution) was used as a negative control. After¢ve washes in TBS-T for 10-min each, the sectionswere incubated for 1 h in TBS-T containing AP-con-jugated secondary antibody (1:500). The bound anti-body was visualized by 4-nitrophenyl phosphate/5-bromo-4-chloro-3-indolylphosphate staining usingan NBP/BCIP stock solution (Boehringer Mann-heim) according to the manufacturer's instructions.After mounting in Permount (Fisher Scienti¢c), the

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^67 59

slides were examined with the Axiophot microscope(Zeiss) using di¡erential interference optics.

3. Results

3.1. Distribution of CaMBPs in various soybeantissues

Soluble protein extracts were prepared from vari-ous parts of soybean plants to examine the distribu-tion of CaMBPs at various developmental stages andtissues. In gel overlay assays of these protein ex-tracts, 32P-labeled SCaM-1 and SCaM-4 were usedon the following reasons. Although CaM: :HRP con-jugates exhibited higher sensitivity than 32P-labeled

CaM as shown in Fig. 1, the former requires theadditional step of protein transfer to a solid supportmembrane for detection. Since we used 5^20% gra-dient gels to see wide size-ranges of protein species, itwas di¤cult to transfer completely all of protein spe-cies to a membrane. However, 32P-labeled CaMoverlay could be done in situ with the proteins re-maining in the gradient gel thus eliminating the pos-sibility of loss of CaMBPs during protein transfer.

The results of representative gel overlay assay areshown in Fig. 2. Unexpectedly, both CaM isoformsexhibited essentially similar binding patterns to var-ious proteins despite the signi¢cant di¡erences intheir primary structure. CaMBPs were detected inall tissue extracts, but CaM isoform-speci¢c bindingproteins were hardly found. Interestingly, CaM iso-forms showed notable di¡erences in their apparentbinding a¤nities to some target proteins. Since theoverlay band intensity depends on the relativeamounts or the potential degradation/modi¢cationof protein species in the crude extract, it is impossibleto compare relative SCaM-binding a¤nities betweenCaMBPs by simple overlay experiments. However, itis possible to compare relative binding a¤nities forCaMBPs among the SCaM isoforms, because theexperiments were performed under identical condi-tions in which only the CaM probes used for eachoverlay were di¡erent. As shown in Fig. 2, severalprotein species showed di¡erence in signal intensitybetween SCaM-1 and SCaM-4 overlay results, evenwhen the autoradiographs displayed nearly identicalband intensities of the two standard CaMBPs (myo-sin and phosphorylase b ; the 200 and 97 kDa proteinbands in STD lane, respectively). For example, in therecognition of CaN used as a positive control (right-most lane in both overlays), SCaM-1 gave a strongersignal than SCaM-4, suggesting a di¡erence in rela-tive CaN-binding a¤nity. In the recognition ofCaMBPs in soybean extracts, the 75 kDa proteinband in elongating tissue is more strongly boundby SCaM-4 than SCaM-1, but the 55 kDa proteinband in stem tissue extract is more evident in SCaM-1 than in SCaM-4 overlay. Although apparentlymore protein bands were detected in SCaM-4 overlayshown in Fig. 2, longer exposure of SCaM-1 overlayrevealed the additional protein bands that had beenreadily seen in the SCaM-4 overlay (data not shown).Therefore, the additional weak protein bands in the

Fig. 1. Speci¢city and sensitivity of CaN detection by [32P]CaMand CaM: :HRP conjugate in gel overlay assay. Variousamounts of CaN along with 10-fold excess amounts of puri¢edGST protein as indicated above lanes were subjected to 10%SDS^PAGE and transferred onto membranes. CaM overlay as-says were performed with 32P-labeled SCaM-1 (A) or SCaM-1: :HRP conjugate (B) to examine speci¢city and sensitivity ofthese CaM probes in recognizing CaMBPs. The relative posi-tions of CaN and GST are indicated by arrows. 32P-gel overlayblot (A) was exposed to ¢lm at 380³C for 12 h with double in-tensifying screens. The CaM: :HRP overlay result (B) was ob-tained after a 3-min exposure to ¢lm.

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^6760

SCaM-4 overlay may be due to their relatively lowerabundance and/or di¡erent binding a¤nities ratherthan their speci¢c binding to SCaM-4. Furthermore,gel overlay assays using 125I-labeled CaM probesgave similar results to further support this conclusion(data not shown). In summary, both SCaM-1 and -4can bind most of soybean CaMBPs with some di¡er-ences in their apparent binding a¤nity, and SCaMisoform-speci¢c binding proteins, if at all present,represent only a minor portion of plant CaMBPs.

3.2. Detection of CaMBPs located in membrane

A number of events occur in biological membranesincluding ion £uxes, photosynthesis, metabolic path-ways, and initiation of cellular signaling. These func-tions are mediated by membrane-bound or -anchored

proteins such as ion channels and pumps, G proteins,and receptors [7]. Interestingly, a number of theseproteins are shown to be regulated by the Ca2�/CaM complex in animal cells. However, in plants,little is known about the presence and identity ofCaMBPs in biological membranes. But the presenceof plant CaM in the plasma membrane strongly sug-gests that there may exist membrane-localizedCaMBPs in plant cells like there are in animals [9].

Crude membrane pellets were prepared from soy-bean cell suspension culture and further fractionatedby sucrose gradient centrifugation to reveal the rela-tive distribution of CaMBPs in various biologicalmembranes [37]. CaM: :HRP conjugates were usedfor detection of three CaM isoform-speci¢c bindingproteins. As shown in Fig. 3, all three SCaM iso-form: :HRP conjugates readily recognized at least

Fig. 2. Distribution of CaMBPs in various tissues of soybean plants. Soluble protein extracts (100 Wg) were separated by 7.5^20% line-ar gradient SDS^PAGE, and CaM overlay assays were performed using 32P-labeled SCaM-1 (A,C) or 32P-labeled SCaM-4 (B,D) ei-ther in the presence of 1 mM CaCl2 (A,B) or 5 mM EGTA (C,D). Tissues of apical and elongating part of hypocotyls, plumule, andcotyledons were isolated from 3-day-old etiolated soybean seedlings. Other tissues were taken from 7-week-old mature soybean plants.CaN indicates the positive control lane containing 30 ng of puri¢ed bovine brain CaN. The autoradiographs shown were obtained onX-ray ¢lms after exposure for 5 days at 380³C with double intensifying screens.

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^67 61

seven protein bands of approximately 35, 47, 76, 80,111, 115, and 140 kDa. However, there was no sig-ni¢cant di¡erence in their relative binding strength tothese protein species. Interestingly, the 35 kDa pro-tein is more evident in the 30^40% sucrose densityfraction, suggesting its possible localization in moredense membranes such as the rough ER and/or plas-ma membrane. In contrast, other protein bands, ex-cept the 47 kDa protein, were enriched in the lightervesicles of the 20^30% sucrose gradient fraction,which are thought to represent the smooth ER andvacuolar membranes. The CaM-binding to all theseseven proteins was Ca2�-dependent, since the inclu-sion of a Ca2� chelator, EGTA, completely abolishedall the binding (data not shown).

3.3. Competitive binding of SCaM isoforms to plantCaMBPs

To further verify the observation that SCaM iso-forms share their binding proteins, we isolated plantCaMBPs by expression library screening with eitherSCaM-1: :HRP or SCaM-4: :HRP conjugate. Fromthe screening, we isolated several soybean or riceplant CaMBPs including a kinesin-like protein [46],CaM-binding heat-shock protein [44], two CaM-binding proteins of unknown function [45,47], andtwo novel CaMBPs highly homologous to genes in

the EST database (GenBank accession numbersF14036 and T04360) [34]. The cDNA inserts of theseisolated CaMBP clones were expressed in E. coli asL-galactosidase fusion proteins and examined forCaM binding by CaM: :HRP gel overlay assays. Asshown in Fig. 4A and D, both SCaM-1 and SCaM-4: :HRP conjugates e¡ectively bound to all theseplant CaMBPs in a Ca2�-dependent manner (seeFig. 4F). These results suggest that SCaM isoformsmight compete with each other for binding plantCaMBPs. To examine whether this competitive bind-ing does occur, competitive CaM overlays were per-formed in the presence of 2-fold or 10-fold excessunlabeled SCaM protein. The inclusion of nativeSCaM-4 competitively abolished the CaMBP-bindingsignals of HRP-labeled SCaM-1, and likewise, theaddition of SCaM-1 weakened the binding signalsof SCaM-4: :HRP. These results clearly show thatthese SCaM isoforms compete with each other forbinding to plant CaMBPs, when both isoforms arepresent. However, the potential of the SCaM iso-forms to diminish the SCaM: :HRP binding signalswas di¡erent. For example, while a 10-fold excessSCaM-4 is required to completely abolish CaNband in a SCaM-1: :HRP overlay, only 2-fold excessSCaM-1 was needed to obtain the same e¡ect inSCaM-4: :HRP overlays, which suggests thatSCaM-1 has a higher relative a¤nity for CaN than

Fig. 3. Detection of CaMBPs in various membrane fractions of soybean suspension cell culture. Crude membrane proteins (Total)were fractionated on a discontinuous Suc gradient ultracentrifugation, and fractions were collected from each gradient interface.Twenty Wg of proteins from each fraction were separated by 10% SDS^PAGE, and the distribution of CaMBPs was examined usingHRP conjugated SCaM-1, SCaM-4, and SCaM-5.

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^6762

SCaM-4. In recognizing plant CaMBPs, SCaM-1 re-duced the SCaM-4: :HRP-binding signals more e¡ec-tively than SCaM-4 did the SCaM-1: :HRP-bindingsignals (see Fig. 4B,E). These results suggest thatSCaM-1 has a higher relative binding a¤nity forthose proteins than SCaM-4.

3.4. Immunolocalization of SCaM isoforms insoybean tissues

CaM is ubiquitous protein mainly found in thecytoplasm, and highly expressed in regions of the

meristems that are active in cell division and growth[10,43]. Were di¡erent CaM isoforms expressed in acell- or tissue-speci¢c manner in plants, the competi-tion for CaMBP-binding among CaM isoformswould not occur in vivo. To test this hypothesis,tissue distributions of SCaM isoforms were com-pared at the protein level using either anti-SCaM-1or anti-SCaM-4 antibody [25]. The anti-SCaM-1antibody recognized SCaM-1 and SCaM-2 withequal sensitivity, but it did not bind to SCaM-4and SCaM-5. On the other hand, anti-SCaM-4 anti-body cross-reacted with SCaM-5, but did not bind to

Fig. 4. Competitive binding of SCaM isoforms to CaMBPs isolated by cDNA expression library screening. CaMBPs expressed as L-galactosidase fusion proteins in E. coli were examined for CaM-binding by CaM: :HRP gel overlay assays. Each lane contains 10 Wgof soluble protein extracts from each of the IPTG-induced E. coli cells harboring cDNA. The control lane contains protein extractsfrom cells containing an empty vector (pBluescript SK3). Upper panels show CaM: :HRP gel overlay assays results performed witheither SCaM-1: :HRP alone (A), or in the presence of a 2-fold (B) or 10-fold (C) excess SCaM-4. The lower left two panels showoverlay assay results with either SCaM-4: :HRP alone (D) or in the presence of a 2-fold excess SCaM-1 (E). The lower rightmost pan-el shows a SCaM-1: :HRP alone assay result in the presence of EGTA (F). For names of CaMBPs, SCBP-1 denotes soybeanCaMBP-1, T-CBPr tobacco CaMBP, M-CBP maize CaMBP, KLP kinesine-like protein, CB-HSP CaM-binding heat-shock protein,and CaN calcineurin. Asterisks indicate protein bands of CaMBPs that correlate with the predicted protein sizes of CaMBPs deducedfrom the cDNA insert sizes. Other smaller bands are degradation products. The closed arrowhead in the SCBP-1 lane points out adimer form of SCBP-1.

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^67 63

SCaM-1 and SCaM-2 [21]. Therefore, the anti-SCaM-1 or anti-SCaM-4 antibody might identify dif-ferences in the spatial distribution of the highly con-served SCaM isoforms and the divergent SCaM iso-forms. Using the two anti-SCaM antibodies,immunolocalization experiments were performed ontissue sections prepared from various parts of soy-

bean seedlings. As shown in Fig. 5, both SCaM iso-form groups showed essentially similar tissue distri-bution patterns in hook cross-sections andlongitudinal sections of apical meristem regions.However, a signi¢cantly more time (s 10-fold) forsignal development was required to detect SCaM-4and -5 signals, which re£ects their lower expression

Fig. 5. Tissue and cellular distribution of SCaM proteins in soybean seedlings. The distribution of highly conserved CaM isoforms(SCaM-1, -2 and -3) and the divergent CaM isoforms (SCaM-4 and -5) was examined by immunolocalization experiments using anti-SCaM-1 and anti-SCaM-4 antibody, respectively. The localization of SCaM proteins in the shoot apical meristem regions (A^C), inthe hook regions (D^F), or in the root-tip regions of 3-day-old etiolated soybean seedlings (I^J) was determined using either anti-SCaM-1 antibody (B,D,G,I) or anti-SCaM-4 antibody (C,E,H,J). G and H are higher magni¢cations of hook immunolocalization re-sults showing localization of SCaM proteins in the vascular bundle region. Negative control experiments using pre-immune serum areshown in A and F, con¢rming the speci¢city of the immunolocalization procedure. Note that anti-SCaM-1 results shown were ob-tained after 1 h color development, whereas anti-SCaM-4 immunolocalization results required a signi¢cantly longer time (V12 h) forcolor development due to the lower abundance of SCaM-4/5 in these tissues. Scale bars = 100 Wm.

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^6764

level in these tissues. In the root tip regions, thehighly conserved SCaM isoforms were most highlyenriched in the root apical meristem, while they weresigni¢cantly lesser in the root cap region (Fig. 5I).However, the divergent SCaM isoforms were hardlydetectable in the root regions (Fig. 5J). These resultsare very much consistent with the previous resultsobtained by Northern blot analyses [25]. The absenceof any signi¢cant signals in the control slides, thatwere incubated with pre-immune serum, supports thespeci¢city of our immunolocalization procedure (Fig.5A,F). In summary, The data suggest that there ex-ists no di¡erence in speci¢c tissue distribution be-tween the highly conserved CaM isoforms and thedivergent SCaM isoforms, which further supportsour hypothesis of the competitive binding of CaMisoforms to plant CaMBPs.

4. Discussion

In this paper, we investigated whether di¡erentCaM isoforms bind to di¡erent set of CaMBPs inorder to elucidate each CaM isoform-speci¢c func-tion in plant cells. To address this question, we ¢rstexamined pro¢les of CaMBPs in tissues that covervarious developmental stages and organs. Surpris-ingly, the divergent CaM isoforms, SCaM-4 and -5,exhibited similar binding patterns to those of highlyconserved CaM isoform, SCaM-1. This is very intri-guing, because it suggests that di¡erent CaM iso-forms share their target proteins in spite of primarystructural diversity. This notion is further supportedby the observation that both SCaM-1 and -4 canbind all of the CaMBPs present in membranes andthe CaMBPs isolated by the cDNA expression li-brary screening. More importantly, SCaM isoformscompete with each other for binding to CaMBPs inthe competitive gel overlay assays.

What is the signi¢cance of these ¢ndings with re-gard to the functioning of CaM isoforms in plantcells? We have previously shown that SCaM-4 hasdi¡erential ability to activate CaM-dependent en-zymes from SCaM-1 [25]. SCaM-1 and SCaM-4 ex-hibited nearly identical e¡ects on the activation ofphosphodiesterase. In contrast, SCaM-4 cannot acti-vated NAD kinase at all mainly due to amino acidsubstitutions in the domain I [26]. Nevertheless,

SCaM-4 still retained the ability to bind NAD kin-ase, which suggests that competitive antagonism mayarise for NAD kinase activation, when both of thetwo CaM isoforms are present together [26]. Indeed,we have observed this competitive antagonismamong CaM isoforms in the activation of CaN andNOS [8]. SCaM isoforms not only selectively acti-vated these enzymes but also competitively inhibitedthe activation of them by another SCaM isoform.This ¢nding suggests the possibility that particularCa2�/CaM signaling pathways can be modulated bythe presence of di¡erent CaM isoforms, which, inturn, may give plant cells the ability for diverse cel-lular responses to Ca2� signals. We have shown herethat SCaM-1 and SCaM-4 bind most of plantCaMBPs in an isoform-independent but competitivemanner rather than involving speci¢city in bindingtargets. In addition, we demonstrated that SCaMisoforms have similar spatial distributions and arenot compartmentalized by tissue speci¢city. Theseobservations strongly raise the possibility that a re-ciprocal regulation mechanism among CaM isoformsdoes occur in vivo within plant cells. Given the func-tional implication of CaM in very diverse plant sig-nal transduction pathways, this reciprocal regulationmay play an important role in the precise tuning ofcellular responses to speci¢c Ca2� signals [31]. Re-cently, we found strong in vivo evidence supportingthe notion for the di¡erential modulation of Ca2�

signaling in plant cells by CaM isoforms. The diver-gent CaM isoforms, SCaM-4 and SCaM-5, are veryrapidly induced by fungal elicitors or pathogens, tolevels comparable to other highly conserved SCaMisoforms such as SCaM-1, -2, and -3 that are rela-tively abundantly expressed in plant cells [21]. Theirinduction is speci¢cally dependent on the Ca2� signalevoked by pathogen. Surprisingly, transgenic plantsconstitutively expressing these SCaM isoforms exhib-ited plant defense responses against pathogens in theabsence of pathogen challenge, which indicates thatthe divergent SCaM isoforms activate plant diseaseresistance signaling cascade(s). Therefore, althoughwe do not know whether the divergent CaM iso-forms act on speci¢c target(s) involved in plant de-fense responses, these observations strongly supportour hypothesis on the role of CaM isoforms modu-lating Ca2�/CaM signaling in plant cells. Also, theinducible expression of SCaM-4 and -5 genes elimi-

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^67 65

nates possible argument for the disability of theseSCaM isoforms acting as competitive antagonists,which are based on the low abundance in healthyplant cells.

Notably, the SCaM isoforms showed di¡erences intheir relative abundance in various tissues and cells.Others and we have shown developmentally regu-lated expression and di¡erential induction of multi-ple CaM isoform genes to various stimuli or stresses[4,6,21,25,35,43]. The data indicate that the biologi-cal availability of a certain CaM isoform in a givenplant cell is continuously changed. In addition, asshown in the overlay assays, CaM isoforms bindCaMBPs with di¡erent relative a¤nities. The di¡er-ence in relative binding a¤nity may allow forCaMBPs to preferentially bind a speci¢c CaM iso-form. However, as relative CaM isoform levels inplant cells changes, a CaM isoform with lower a¤n-ity might able to compete in the binding to CaMBPs.This may provide plant cells with an additional reg-ulatory mechanism for precisely and e¡ectively ¢ne-tuning cellular activities during developmental stagesand changes in environmental stresses.

CaMBPs were detected in all the tissue/organs ex-amined, but di¡erent CaMBPs are present in a spe-ci¢c manner. This implies a possible role of Ca2�/CaM in plant cell growth and di¡erentiation. Similarobservations were made for the cellular distributionof CaMBPs in Vicia fava using 125I-labeled CaM [29].In soybean membranes, there are at least sevenCaMBPs which all showed nearly identical bindingto three SCaM isoforms. At present we do not knowexactly the identity of these CaM-binding membraneproteins. The only known plant CaMBPs localized inthe membranes are Ca2�-ATPase and a putative iontransport, HvCBT1 [1,41]. Further studies, such aspuri¢cation of these proteins, will be necessary tounravel the biological functions of CaM in variousbiological membranes.

Acknowledgements

We are grateful to Richard Park and Gisela Ho-schek for proofreading the manuscript. We alsothank all members of CaM group in the Plant Mo-lecular Biology Laboratory for discussion. S.-H.L.was supported by a post-doctoral fellowship from

the Korean Research Foundation. This work wassupported by a non-directed research fund from Ko-rea Research Foundation to M.J.C. (1996).

References

[1] P. Askerlund, Plant Physiol. 114 (1997) 999^1007.[2] G. Baum, Y. Chen, T. Arazi, H. Takatsuji, H. Fromm,

J. Biol. Chem. 268 (1993) 19610^19617.[3] M.L. Billinsley, K.R. Pennypacker, C.G. Hoover, D.J. Brig-

ati, R.L. Kincaid, Proc. Natl. Acad. Sci. USA 82 (1985)7585^7589.

[4] J.R. Botella, R.N. Arteca, Plant Mol. Biol. 24 (1994) 757^766.

[5] W.J. Boyle, P. van der Geer, T. Hunter, Methods Enzymol.201 (1991) 110^149.

[6] J. Braam, R.W. Davis, Cell 60 (1990) 357^364.[7] A. Broeks, B. Gerrard, R. Allikmet, M. Dean, R.H. Plas-

terk, EMBO J. 15 (1996) 6132^6143.[8] M.J. Cho, P.L. Vaghy, R. Kondo, S.H. Lee, J.P. Davis, R.

Rehl, W.D. Heo, J.D. Johnson, Biochemistry 37 (1998)15593^15597.

[9] M. Collinge, A.J. Trewavas, J. Biol. Chem. 264 (1989) 8865^8872.

[10] M. Dauwalder, S.J. Roux, L. Hardison, Planta 168 (1986)461^470.

[11] T.N. Davis, M.S. Urdea, F.R. Masiarz, J. Thorner, Cell 47(1986) 423^431.

[12] C.F. Edman, S.E. George, A.R. Means, H. Schulman, P.Yaswen, Eur. J. Biochem. 226 (1994) 725^730.

[13] A.P. Fordham-Skelton, F. Safadi, M. Golvkin, A.S.N.Reddy, Plant Mol. Biol. Rep. 12 (1994) 358^366.

[14] H. Fromm, N.-H. Chua, Plant Mol. Biol. Rep. 10 (1992)199^206.

[15] Y. Fukami, T. Nakamura, A. Nakayama, T. Kanehisa,Proc. Natl. Acad. Sci. USA 83 (1986) 4190^4193.

[16] S. Gilroy, Plant Cell 8 (1996) 2193^2209.[17] J.R. Glenny, K. Weber, Methods Enzymol. 102 (1983) 210^

218.[18] C.B. Graves, R.D. Gale, J.P. Laurino, J.M. McDonald,

J. Biol. Chem. 261 (1986) 10429^10438.[19] S.A. Harding, S.-H. Oh, D.M. Roberts, EMBO J. 16 (1997)

1137^1144.[20] R.G. Heiman, R.C. Atkinson, B.F. Andruss, C. Boldug,

G.E. Kovalick, K. Beckingham, Proc. Natl. Acad. Sci.USA 93 (1996) 2420^2425.

[21] W.D. Heo, S.H. Lee, M.C. Kim, J.C. Kim, W.S. Chung,H.J. Chun, K.J. Lee, C.Y. Park, H.C. Park, J.Y. Choi,M.J. Cho, Proc. Natl. Acad. Sci. USA 96 (1999) 766^771.

[22] M.E. Horn, J.M. Widholm, W.N. Ogren, Plant Physiol. 72(1989) 426^429.

[23] P. James, T. Vorherr, E. Carafoli, Trends Biochem. Sci. 20(1995) 38^42.

[24] L.J. Klimczak, M.A. Collinge, D. Farini, G. Giuliano, J.C.Walker, A.R. Cashmore, Plant Cell 7 (1995) 105^115.

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^6766

[25] S.H. Lee, J.C. Kim, M.S. Lee, W.D. Heo, H.Y. Seo, H.W.Yoon, J.C. Hong, S.Y. Lee, J.D. Bahk, I. Hwang, M.J. Cho,J. Biol. Chem. 270 (1995) 21806^21812.

[26] S.H. Lee, H.Y. Seo, J.C. Kim, W.D. Heo, W.S. Chung, K.J.Lee, M.C. Kim, Y.H. Cheong, J.Y. Choi, C.O. Lim, M.J.Cho, J. Biol. Chem. 272 (1997) 9252^9259.

[27] B. Liao, M.C. Gawienowski, R.E. Zielinski, Arch. Biochem.Biophys. 327 (1996) 53^60.

[28] V. Ling, W.A. Snedden, B.J. Shelp, S.M. Assmann, PlantCell 6 (1994) 1135^1143.

[29] C. Ling, S.M. Assmann, Plant Physiol. 100 (1992) 970^980.[30] Z. Liu, M. Xia, B.W. Poovaiah, Plant Mol. Biol. 38 (1998)

889^897.[31] M.R. McAinsh, A.M. Hetherington, Trends Plant Sci. 3

(1998) 32^36.[32] A.R. Means, M.F. van Berkum, I. Bagchi, K.P. Lu, C.D.

Rasmussen, Pharmacol. Ther. 50 (1991) 255^270.[33] G. Neuhaus, C. Bowler, R. Kern, N.-H. Chua, Cell 73

(1993) 937^952.[34] T. Newman, F. de Bruijn, F.P. Green, K. Keegstra, H.

Kende, L. McIntosh, J. Ohlrogge, N. Raikhel, S. Somerville,M. Thomashow, R.E. Tetzel, C. Somerville, Plant Physiol.106 (1994) 1241^1255.

[35] B.W. Poovaiah, A.S.N. Reddy, CRC Crit. Rev. Plant Sci. 12(1993) 185^211.

[36] D.M. Roberts, A.C. Harmon, Annu. Rev. Plant Physiol.Plant Mol. Biol. 43 (1992) 375^414.

[37] D.G. Robinson, G. Hinz, K. Oberbeck, in: N. Harris, K.J.Oparka (Eds.), Plant Cell Biology, A Practical Approach,IRL Press, Oxford, 1994, pp. 245^272.

[38] E. San Jose, A. Benguria, P. Geller, A. Villalobo, J. Biol.Chem. 267 (1992) 15237^15245.

[39] R.K. Sharma, W.A. Taylor, J.H. Wang, Methods Enzymol.102 (1983) 210^218.

[40] W.A. Snedden, H. Fromm, Trends Plant. Sci. 3 (1998) 299^304.

[41] R.C. Schuurink, S.F. Shartzer, A. Fath, R.L. Jones, Proc.Natl. Acad. Sci. USA 95 (1998) 1944^1949.

[42] R.G. Walker, A.J. Hudspeth, Proc. Natl. Acad. Sci. USA 93(1996) 2203^2207.

[43] T. Yang, S. Lev-Yadun, M. Feldman, H. Fromm, PlantMol. Biol. 38 (1998) 109^120.

[44] Y.T. Lu, M.A. Dharmasiri, H.M. Harrington, Plant Physiol.108 (1995) 1197^1202.

[45] Y.T. Lu, H.M. Harrington, Plant Physiol. Biochem. 32(1994) 413^422.

[46] A.S.N. Reddy, S.B. Narasimhulu, F. Safadi, M. Golovkin,Plant J. 10 (1996) 9^21.

[47] A.S.N. Reddy, D. Takezawa, H. Fromm, B.W. Poovaiah,Plant Sci. 94 (1993) 109^117.

BBAPRO 35966 2-8-99

S.H. Lee et al. / Biochimica et Biophysica Acta 1433 (1999) 56^67 67