The Plant Journal (1999) 18(3), 243–252

Tobacco retinoblastoma-related protein phosphorylated bya distinct cyclin-dependent kinase complex withCdc2/cyclin D in vitro

Hirofumi Nakagami†, Masami Sekine†, Hiroko Murakami

and Atsuhiko Shinmyo*

Graduate School of Biological Sciences, Nara Institute ofScience and Technology (NAIST), Takayama 8916–5,Ikoma, Nara 630–0101, Japan

Summary

The retinoblastoma (Rb) protein was originally identified

as a product of a tumour suppressor gene that plays a

pivotal role in regulating both the cell cycle and differenti-

ation in mammals. The growth-suppressive activity of Rb

is regulated by phosphorylation with cyclin-dependent

kinase (CDK), and inactivation of the Rb function is one of

the critical steps for transition from the G1 to the S phase.

We report here the cloning of a cDNA (NtRb1) from

Nicotiana tabacum which encodes a Rb-related protein,

and show that this gene is expressed in all the organs

examined at the mRNA level. We have demonstrated that

NtRb1 interacts with tobacco cyclin D by using yeast two-

hybrid and in vitro binding assays. In mammals, cyclin D

can assemble with CDK4 and CDK6, but not with Cdc2, to

form active complexes. Surprisingly, tobacco cyclin D and

Cdc2 proteins can form a complex in insect cells, which is

able to phosphorylate tobacco Rb-related protein in vitro.

Using immunoprecipitation with the anti-cyclin D anti-

body, cyclin D can be found in a complex with Cdc2

in suspension-cultured tobacco BY-2 cells. These results

suggest that the cdc2 gene modulates the cell cycle

through the phosphorylation of Rb-related protein by

forming an active complex with cyclin D in plants.

Introduction

Progression through the eukaryotic cell cycle is regulatedby distinct families of cyclin-dependent kinases (CDKs)whose activities are determined by the coordinated bindingof different types of cyclins at each phase of the cell cycle.In mammals, the commitment to enter the cell cycle isgoverned during the G1 phase at a point called the ‘restric-tion (R) point’. The mammalian G1 cyclins consist of

Received 9 November 1998; revised 22 February 1999; accepted 10 March1999.*For correspondence (fax 181 0743 72 5469;e-mail shinmyou@bs.aist-nara.ac.jp).†These two authors contributed equally to this work.

© 1999 Blackwell Science Ltd 243

cyclin D, which associates with CDK4 and CDK6, andcyclin E, which associates with CDK2. Both types of cyclinssequentially phosphorylate the retinoblastoma (Rb) proteinwhich inactivates its growth-suppressive activity (Sherr,1994; Weinberg, 1995). The growth-suppressive functionof Rb is excerted by its binding to a variety of cellularproteins involved in DNA replication and control of the cellcycle (Taya, 1997). The ability of Rb to control the G1/Stransition is mediated largely through its interactions withthe E2F/DP transcription factor family. HypophosphorylatedRb binds to the activation domain of E2F and activelyrepresses its transactivation activity (Hieber et al., 1992;Weintraub et al., 1992). At mid- to late G1, Rb becomeshyperphosphorylated by G1 cyclin-dependent kinases andis released from the promoter-bound E2F, allowing trans-cription of E2F-regulated genes.

Although substantial progress has been made in under-standing the mechanisms that control the cell cycle inyeast and mammals, far less is known about cell cycleregulation in plants. Recently, cDNA clones for Rb-relatedprotein were isolated from maize (Grafi et al., 1996; Xieet al., 1996). Ach et al. (1997a) also reported that maize hastwo genes, RRB1 and RRB2, which encode Rb-relatedproteins. Although the maize Rb-related protein wasshown to be phosphorylated during the course of endo-reduplication in maize endosperm (Grafi and Larkins, 1995),it is not known which cyclin-dependent kinase can phos-phorylate Rb-related protein in plants. The existence ofcyclin D (Dahl et al., 1995; Soni et al., 1995) and Rb-relatedgenes (Ach et al., 1997a; Grafi et al., 1996; Xie et al., 1996)in plants suggests that the mechanisms of G1/S control inplants are more similar to those in mammals than thosein yeast.

Genetic studies with yeasts have revealed a single CDKgene, cdc2 in Schizosaccharomyces pombe and cdc28 inSaccharomyces cerevisiae, required for both the G1/S andG2/M transitions (Nasmyth, 1993; Norbury and Nurse,1992). By contrast, the cell cycle is controlled by a familyof CDKs in mammals (Pines, 1995). Plants contain a numberof cdc2-related genes and several lines of evidence suggestthat different cdc2-related genes are involved in differentphases of the plant cell cycle as in mammals (Burssenset al., 1998). However, which combination of Cdc2-relatedproteins forms active complexes with cyclins has notbeen elucidated (Burssens et al., 1998; Magyar et al., 1993;Magyar et al., 1997).

We have isolated a Rb gene, NtRb1, from tobacco. We

244 Hirofumi Nakagami et al.

Figure 1. Nucleotide sequences and deduced amino acid sequences of the NtRb1 cDNA.The A and B domains of the pocket region are boxed. The N-terminal leucine-rich domain conserved in the Rb family members from plants to mammaliancells is in italic and underlined. Potential CDK-phosphorylation sites (S/TP) are underlined.

have demonstrated that tobacco Cdc2 can form a complexwith tobacco cyclin D in insect cells as well as in suspensioncultured tobacco BY-2 cells, and that the complex expressedin insect cells can phosphorylate tobacco Rb-related proteinin vitro. To our knowledge, this is the first evidence that acomplex of Cdc2 with cyclin D can phosphorylate aRb-related protein.

Results

Isolation of a tobacco cDNA encoding Rb-related protein

A partial cDNA of approximately 2.0 kb encoding aRb-related protein was isolated by screening a culturedtobacco BY-2 cells cDNA library with the cDNA containingalmost a full-length of maize Rb-related cDNA, ZmRb1(Xie et al., 1996). By screening a tobacco SR-1 shoot apexcDNA library with a partial cDNA, several clones wereisolated, and one of the clones (NtRb1) contained anapproximately 3.3 kb insert encoding a 961 amino acidpeptide with a predicted molecular weight of approximately107 kDa (Figure 1). Several in-frame stop codons werefound at the 59 non-translated region of the NtRb1 cDNA,indicating that this clone contains a full-length region.

Analysis of the deduced amino acid sequence of NtRb1

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revealed that it contains the essential domain of homologywith the A and B domains of the pocket region that isconserved among all the mammalian Rb family proteins(Figures 1 and 2). The A domain of NtRb1 protein isapproximately 31% identical and 66% identical in sequenceto the A domains of human Rb protein and maize RRB1protein, respectively. The B domain of NtRb1 protein isapproximately 26% identical and 44% identical in sequenceto the human Rb protein and maize RRB1 protein, respect-ively (Figure 2) (Ach et al., 1997a). The tobacco NtRb1 hasthe shortest B domain region among all the Rb familyproteins (Figure 2). The critical cysteine residue at position706 of human Rb (Kaye et al., 1990) was present at compar-able positions in both the tobacco and maize Rb-relatedproteins (Figure 2). NtRb1 also contains a leucine-richdomain in the N-terminal region which is conserved bothin mammals (Kaye et al., 1990) and in the maize RRB1protein (Figure 2) (Ach et al., 1997a). There are 13 potentialCDK phosphorylation sites in the tobacco NtRb1 which arehighly clustered in the C-terminal region (Figure 1).

NtRb1 is expressed in tobacco plants and culturedtobacco cells

The maize RRB genes are expressed in all the tissues, butthe highest level of expression was seen in the shoot

Rb kinase in plants 245

Figure 2. Sequence alignment of domains A and B, and the N-terminal leucine-rich domain of NtRb1 with mammalian Rb family.Black boxes indicate conserved amino acid residues, and dashes indicate gaps in the sequences. The bars in the A and B domains indicate the α-helix andβ-sheet structures deduced from crystallization analysis (Lee et al., 1998). The arrow in B domain denotes a conserved cysteine residue known to be criticalfor Rb function in mammalian cells (Kaye et al., 1990).

Figure 3. Expression of NtRb1 in various tissues of tobacco plant.Poly(A) RNAs (1 µg) from various tissues of tobacco plant wereelectrophoresed on an agarose–formaldehyde gel, and blotted onto a nylonmembrane. The membrane was probed with the 32P-labelled NtRb1 cDNA.The same membrane was probed with actin cDNA to ensure equal loadingof each sample.

apex (Ach et al., 1997a). To examine NtRb1 expression intobacco, poly(A) RNA from various organs was probedwith a part of NtRb1. In all the organs examined, a transcriptof approximately 3.3 kb was detected. High levels of NtRb1expression were detected in stems, leaves, and roots,which consist predominantly of differentiated cells. Lowlevels of expression were found in flowers and in undiffer-entiated BY-2 cells in suspension culture (Figure 3). Thisresult indicates that NtRb1 is definitely expressed in all theorgans tested.

© Blackwell Science Ltd, The Plant Journal, (1999), 18, 243–252

NtRb1 can bind to tobacco cyclin D

The ability of the mammalian Rb protein family to bind avariety of viral and cellular proteins is conferred exclusivelyby the pocket region (Weinberg, 1991). The conservationof the pocket region in the NtRb1 protein suggests that itcan bind these Rb-binding proteins. To test this possibilitywe used a yeast two-hybrid system to detect binding ofNtRb1 to the tobacco cyclin A, Ntcyc27 (Setiady et al.,1995), and the tobacco cyclin D, designated NtcycD3–1. AcDNA clone representing NtcycD3–1 was isolated from acDNA library of suspension cultured tobacco BY-2 cellsand was classified as the CycD3 class of cyclin D, basedon its high sequence identity to Arabidopsis cyclinδ-3 (Soniet al., 1995) and the CycD3 class of tobacco (Sorrell et al.,1999; Sekine et al., unpublished results).

For two-hybrid assays, a plasmid that contains the partof the NtRb1 encoding amino acids 374–961 of NtRb1 wasconstructed with the Gal4 transactivation domain. Yeaststrain HF7c was transformed with the plasmid togetherwith the plasmids expressing tobacco cyclin D or tobaccocyclin A fused with the Gal4 DNA-binding domains.Although NtRb1 bound to tobacco cyclin D, tobaccocyclin A interacted very weakly with NtRb1 in this assay(Figure 4).

We confirmed the ability of NtRb1 to bind to tobacco

246 Hirofumi Nakagami et al.

Figure 4. Interaction of NtRb1 with NtcycD3–1 in yeast by the two hybridsystem.Plasmid that expresses the NtRb1 protein as a GAL4 DNA-binding domainfusion was transformed pairwise into HF7c yeast cells along with plasmidsthat express the GAL4 activation domain fusions of NtcycD3–1 and tobaccocyclin A, Ntcyc27. The NtRb1 fused with a Gal4 DNA-binding domain wasalso co-transformed into HF7c with pGBT9 vector. These transformantswere streaked on glucose-containing plates with or without histidine.

cyclin D in vitro using experiments with tagged proteins(Figure 5a). NtcycD3–1 was produced in baculovirus-infected insect cells and purified by an anti-FLAG M2affinity gel. SDS–PAGE and staining with the anti-FLAG M2monoclonal antibody showed that the product consistedof a major polypeptide of the size expected for FLAG–NtcycD3–1. Lysates containing equal amounts ofGST–NtRb1 were mixed with lysates of vector-controlinfected cells and with lysates of cells infected with theFLAG–NtcycD3–1 vector. Proteins bound to an anti-FLAGM2 affinity gel were eluted, separated by SDS–PAGE, andanalysed by immunoblotting with the anti-NtRb1 antibody(codons 654–671). NtRb1 immuno-reacted peptides of thesize expected for GST–NtRb1 were only detected in theeluates obtained with lysates containing FLAG–NtcycD3–1(Figure 5a, compare lanes 2–4). A reciprocal binding assayrevealed that protein of the size expected for FLAG–NtcycD3–1 was only detected in lysates containingGST–NtRb1 (data not shown). Taken together, these resultsprovide further support for the physical interaction betweenNtRb1 and a tobacco cyclin D.

Complex of Cdc2/cyclin D can phosphorylate the Rb-related protein in vitro

To determine whether Cdc2 and cyclin D can form a com-plex capable of phosphorylating NtRb1, we co-expresseda His-tagged tobacco Cdc2, cdc2Nt1 (Setiady et al., 1996),together with FLAG–NtcycD3–1 using a baculovirus system.Proteins bound to an anti-FLAG M2 affinity gel were eluted,separated by SDS–PAGE, and analysed by immuno-

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blotting with antisera directed against His tag (Figure 5b,lanes 5–8). Although His–Cdc2Nt1 was equally expressedin insect cells that were or were not expressing FLAG–NtcycD3–1, protein of the size expected for His–Cdc2Nt1was only detected in the eluates obtained with lysatescontaining both His–Cdc2Nt1 and FLAG–NtcycD3–1(Figure 5b, compare lanes 6–8). These results providestrong evidence that the tobacco cyclin D can specificallyinteract with the tobacco Cdc2 in vitro.

We tested whether the tobacco Cdc2/cyclin D complexexhibits Rb kinase activity. Phosphorylation of the GST–NtRb1 was only detected with lysate prepared from insectcells expressing both tobacco Cdc2 and cyclin D, and lysateprepared from insect cells expressing tobacco Cdc2 andcyclin D, and a vector alone had no kinase activity (datanot shown). To exclude the possibility that tobacco Cdc2and/or cyclin D would activate the endogenous CDK/cyclincomplex from insect cells, the tobacco Cdc2/cyclinD complex was purified with the anti-FLAG M2 affinity gelor the TALON metal affinity resin. Phosphorylation of theGST–NtRb1 was only detected with the purified tobaccoCdc2/cyclin D complex in both purification procedures andthe purified Cdc2 and cyclin D alone exhibited no kinaseactivity to the GST–NtRb1 (Figure 5c).

Cyclin D can be found in a complex with Cdc2 in tobaccoBY-2 cells

To confirm that cyclin D binds with Cdc2 in vivo, weimmunoprecipitated extracts from tobacco BY-2 cells withanti-cyclin D, anti-cyclin A, anti-NtRb1 and control anti-bodies. Ntcyc25 (Setiady et al., 1995) was used for thetobacco cyclin A, and Cdc2 was detected by theanti-PSTAIRE antibody which recognizes the conserved‘PSTAIRE’ motif of Cdc2 protein. The eluates from thebeads bound with the anti-NtcycD3–1 antibody, anti-Ntcyc25 antibody, anti-NtRb1 antibody, normal rabbit IgGand anti-PSTAIRE antibody were immunoblotted with theanti-PSTAIRE antibody (Figure 5d). In the eluate from thebeads bound with anti-PSTAIRE antibody, the anti-PSTAIREantibody recognized three polypeptides in which the majorband coincided with a molecular mass of approximately34 kDa and two bands migrated more slowly. In the eluatesfrom the beads bound with both anti-cyclin D and anti-cyclin A antibodies, the anti-PSTAIRE antibody cross-reacted with a polypeptide which has a molecular massof approximately 34 kDa. However, a polypeptide cross-reacted with the anti-PSTAIRE antibody was not detectedin the eluates from the beads bound with anti-NtRb1antibody and normal rabbit IgG used as controls. Thisresult indicated that Cdc2 can be found in a complex withboth cyclin D and cyclin A in tobacco BY-2 cells.

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Figure 5. A complex of Cdc2/cyclin D can phosphorylate Rb-related protein in vitro.(a) NtRb1 can bind to tobacco cyclin D. Lysates of insect Sf9 cells infected with a wild-type baculovirus (Ve) (lanes 1 and 2) and a vector encoding FLAG–NtcycD3–1 (D) (lanes 3 and 4) were incubated with GST–NtRb1 (Rb) for 1 h at 4°C on a rotating stirrer. After incubation, mixtures were bound to an anti-FLAG M2 affinity gel. Elute fractions (lane E) and crude extracts (lane C) were separated on a 10% polyacrylamide gel containing SDS. GST–NtRb1 wasdetected by immunoblotting with anti-NtRb1 antibody (upper column) and FLAG–NtcycD3–1 was detected by immunoblotting with the monoclonal antibodyof anti-FLAG M2 (lower column).(b) Tobacco Cdc2 can form a complex with cyclin D. Lysates of insect Sf9 cells co-infected with baculovirus vectors that express His–Cdc2Nt1 (2) (lanes 1, 2,5 and 6) or His–Cdc2Nt1 and FLAG–NtcycD3–1 (D) (lanes 3, 4, 7 and 8) were purified by an anti-FLAG M2 affinity gel. Elute fractions (lane E) and crudeextracts (lane C) were separated on 10% polyacrylamide gels containing SDS. FLAG–NtcycD3–1 was detected by immunoblotting with the monoclonalantibody of anti-FLAG M2 (lanes 1–4), and His–Cdc2Nt1 was detected by immunoblotting with the antibody for His tag (lanes 5–8). The positions of pre-stained molecular mass protein standards are indicated on the left.(c) Rb kinase activity of Cdc2/cyclin D complex. Purified fractions, prepared from the lysates of insect cells which express the indicated proteins (Ve, vector;2, His–Cdc2Nt1; D, FLAG–NtcycD3–1) with the anti-FLAG M2 affinity gel (F) or the TALON metal affinity resin (T), were incubated with a bacterial GST–NtRb1for 10 min at 30°C in a kinase buffer containing [γ-32P]ATP.(d) Association of NtcycD3–1 with Cdc2 in tobacco BY-2 cells. Total proteins extracted from tobacco BY-2 cells were treated with the indicated immunoaffinitybeads (D, NtcycD3–1; Rb, NtRb1; 25, Ntcyc25; IgG, normal rabbit IgG; P, PSTAIRE). Proteins bound to the beads were eluted and separated on a 10%polyacrylamide gel containing SDS. Cdc2 was detected by immunoblotting with the anti-PSTAIRE antibody. Dots on the right indicate the locations of thethree polypeptides recognized by the anti-PSTAIRE antibody.

Discussion

The first genes encoding plant Rb-related proteins werereported for a monocotyledonous plant, maize (Ach et al.,1997a; Grafi et al., 1996; Xie et al., 1996). We have clonedthe first Rb gene for a dicotyledonous species, tobacco,and show that the Rb-related protein encoded by this genecan bind tobacco cyclin D and is a target for phosphoryla-tion in vitro by a tobacco Cdc2/cyclin D complex.

The conserved A and B pocket domains and the leucine-rich domain of the N-terminal region present in the mam-malian Rb protein family are also present in the maize andtobacco Rb-related proteins, suggesting that the plantand mammalian Rb proteins might have similar functions(Figure 2) (Ach et al., 1997a). Viral oncoproteins have beenidentified that bind the pocket region of Rb family proteins,which disrupts the interaction of these oncoproteins withRb (Chellappan et al., 1992; Zamanian and LaThangue,1992). These oncoproteins bind to Rb via a conservedmotif, LXCXE, which is also found in cyclin D. Therefore,cyclin D can physically interact with Rb via the LXCXEmotif (Dowdy et al., 1993; Kato et al., 1993). We havedemonstrated that the tobacco Rb-related protein binds

© Blackwell Science Ltd, The Plant Journal, (1999), 18, 243–252

a tobacco cyclin D by using a yeast two-hybrid system(Figure 4) and in vitro binding assay (Figure 5). This con-served physical interaction between tobacco Rb-relatedprotein and cyclin D suggests that the pocket region oftobacco Rb-related protein may be involved in binding toa variety of viral and cellular proteins in plants. In fact,several viral proteins and Msi1-like proteins containingWD-40 repeats have been shown to bind maize Rb-relatedproteins (Ach et al., 1997a; Ach et al., 1997b; Grafi et al.,1996; Xie et al., 1996).

Within the B pocket domain, the C706 in human Rb hasbeen shown to be critical for Rb protein function (Kayeet al., 1990). Although the A and B pocket domains arealso conserved in the tobacco NtRb1 protein with theconserved C residue in the B domain, the length of the Bdomain is the shortest among all the identified Rb proteinfamily (Figure 2). The crystal structure of the Rb pocketbound to the human papillomavirus HPV-16 E7 peptidecontaining the LXCXE motif illustrated both that the LXCXEpeptide binds a highly conserved groove on the B pocketdomain and that the A pocket domain is required for stablefolding of the B pocket domain (Lee et al., 1998). The A

248 Hirofumi Nakagami et al.

and B pocket domains each contain the cyclin-fold, a five-helix structural motif which has been found recently in thestructures of cyclins and the basal transcription factor TFIIB(Lee et al., 1998; Noble et al., 1997). However, we havedemonstrated that NtRb1, which does not contain thepart of the cyclin-fold structure in the B pocket domaincorresponding to the α15 to β1 region in human Rb(Figure 2; Lee et al., 1998), can still bind cyclin D (Figures4 and 5). This result suggests that not all of the five-helixstructure motifs in the B pocket domain are essential forthe binding of Rb to proteins containing the LXCXE motif.

The Rb gene is expressed in all the tissues examined inmammals (Bernards et al., 1989). The maize RRB genesare also expressed in all the tissues, but the highest levelof expression was detected in the shoot apex (Ach et al.,1997a). We showed that NtRb1 is expressed at the mRNAlevel in all the organs tested (Figure 3). Although highlevels of NtRb1 expression were detected in stems, leavesand roots, which consist predominantly of differentiatedcells, low levels of expression were found in flowers andin undifferentiated BY-2 cells (Figure 3). It is not knownwhy NtRb1 mRNA levels are relatively high in these organs,but it is possible that NtRb1 could be involved in inducingand/or maintaining a differentiation state in plants.

In plants, a number of cdc2-related genes have beenidentified from several species such as maize (Colasantiet al., 1993), rice (Hashimoto et al., 1992), alfalfa (Magyaret al., 1993, 1997), soybean (Miao et al., 1993), Arabidopsis(Hirayama et al., 1991) and pea (Feiler and Jacobs, 1990).The plant cdc2-related genes are categorized according tothe similarity of the conserved ‘PSTAIRE’ motif (indicatedby the single letter amino acid in the central region of themotif). Some of the plant cdc2-related genes contain analtered PSTAIRE motif, and several show different expres-sion during cell cycle (Burssens et al., 1998; Fobert et al.,1996; Hirt et al., 1993; Magyar et al., 1993; Magyar et al.,1997; Segers et al., 1996). We have previously isolated acDNA clone (cdc2Nt1) that encodes a PSTAIRE-type ofcdc2 gene from tobacco (Setiady et al., 1996). We havedemonstrated that the cdc2Nt1 gene was able to comple-ment at both the G1/S (cdc28–4 and cdc28–13) and G2/M(cdc28–1N) transitions of yeast cdc28 mutants. The cdc2Nt1gene is expressed constitutively throughout the cell cycle(Setiady et al., 1996).

In mammals, cyclin D can associate with CDK4 and CDK6,but not with Cdc2, to form active complexes (Dowdy et al.,1993; Kato et al., 1993). To assay whether tobacco Cdc2 isbound to tobacco cyclin D, we expressed Cdc2Nt1 andNtcycD3–1 in insect cells (Figure 5b). Surprisingly, Cdc2Nt1can bind with NtcycD3–1 in vitro (Figure 5b). Sorrell et al.(1999) recently isolated three tobacco cyclin D clones andfound that two belong to the CycD3 class and the third tothe CycD2 class based on sequence criteria. NtcycD3–1belongs to the CycD3 class but is not identical with their

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clone. This suggests that there are at least three distinctCycD3 cDNAs in tobacco.

We have tested the possibility whether or not Cdc2Nt1/NtcycD3–1 complex exhibits Rb kinase activity. The tobaccoCdc2/cyclin D complex purified from insect Sf9 cells co-infected with baculovirus vectors encoding cyclin D andCdc2 indeed exhibited high level of a protein kinase activitythat phosphorylated a GST–NtRb1 fusion protein in vitro.However, the purified cyclin D and Cdc2 alone did notexhibit Rb kinase activity at all (Figure 5c). This indicatesthat the phosphorylation of tobacco Rb protein by thetobacco Cdc2/cyclin D complex is not due to activation ofendogenous proteins such as CDK and/or cyclins frominsect cells. We also have evidence that lysate of themammalian cyclin D/CDK4 complex produced by a baculo-virus system did not exhibit the phosphorylation of thetobacco Rb protein, while the mammalian cyclin D/CDK4complex can phosphorylate mammalian Rb protein underour assay conditions (data not shown). These results indi-cate that the tobacco Cdc2/cyclin D complex can trulyphosphorylate the tobacco Rb-related protein in vitro.

To confirm that the tobacco cyclin D can bind withCdc2 in vivo, immunoprecipitations were performed withcyclin D, cyclin A, NtRb1 and control antibodies. As shownin Figure 5(d), a polypeptide cross-reacted with the anti-PSTAIRE antibody in the eluates from the beads bound withthe anti-cyclin D and anti-cyclin A antibodies. However, thecross-reacted polypeptide could not be observed in theeluates from the beads bound with the anti-NtRb1 antibodyand normal rabbit IgG. This indicates that Cdc2 can associ-ate physically with cyclin D and cyclin A in tobacco BY-2cells. We found that a polypeptide, cross-reacted with theanti-PSTAIRE antibody, coinciding with a molecular massof approximately 34 kDa was detected in the immuno-precipitate with the anti-cyclin D antibody, while theanti-PSTAIRE antibody cross-reacted mainly with threepolypeptides in tobacco BY-2 cells. It has been reportedthat the anti-PSTAIRE antibody recognizes at least twopolypeptides in plants (Feiler and Jacobs, 1990; Magyaret al., 1993). Two slower-migrating bands were reducedwhen crude extracts were prepared from tobacco BY-2cells without phosphatase inhibitors (data not shown).This suggests that these bands may represent distinctphosphrylation states of the polypeptide with a molecularmass of approximately 34 kDa. However, it remains to beconcluded whether phosphorylation states of Cdc2 proteinaffect the binding with cyclin D. Cdc2Nt1 expressed ininsect cells can be cross-reacted with the anti-PSTAIREantibody (data not shown). We concluded that a singleband cross-reacted with the anti-PSTAIRE antibodyincludes Cdc2Nt1, while we could not rule out the possibil-ity that the band contains other Cdc2-related proteins. Weobserved here that the anti-tobacco cyclin A antibody alsocross-reacted with Cdc2 which can be recognized with the

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anti-PSTAIRE antibody. This observation was supportedby the finding that immunoprecipitation with anti-humancyclin A antibodies allowed the detection of Cdc2-relatedproteins cross-reacted with the anti-PSTAIRE antibody(Magyar et al., 1993). Unfortunately, phosphorylation ofthe tobacco Rb-related protein has not been detected inthe immunoprecipitated proteins with the anti-cyclin Dantibody. It is possible that the tobacco Rb-related proteinmay not be phosphorylated by the Cdc2/cyclin D complexin vivo. Rather than considering this possibility, we believethat the activity of the Cdc2/cyclin D complex in vivo couldnot detected due to a very small amount of immunoprecitit-ated proteins with the anti-cyclin D antibody. Further studywill reveal whether the Cdc2/cyclin D complex can phos-phorylate the tobacco Rb-related protein in vivo.

Our most important finding was that tobacco Cdc2 andcyclin D can form a complex both in vivo and in vitro, andthe complex expressed in vitro phosphorylates Rb-relatedprotein. Further study is required to determine whetherthe partner of the cyclin D is only Cdc2 in plants. Our resultstrongly suggests that the cdc2 gene modulates the cellcycle through the phosphorylation of Rb-related proteinby forming an active complex with cyclin D in plants.Future work should elucidate other combinations of Cdc2-related proteins forming active complexes with varioustypes of cyclins, especially in vivo, and the target proteinsin which the Cdc2/cyclin complexes might participate.

Experimental procedures

Plant materials

Nicotiana tabacum L.cv.SR-1 was grown in a greenhouse. TobaccoBY-2 cells (N.tabacum L.cv. Bright Yellow-2) were cultured at27°C in a modified Linsmaier and Skoog medium as describedpreviously (Setiady et al., 1995).

cDNA library construction and isolation of Rb cDNAs

A tobacco BY-2 cDNA library was constructed with a Uni-ZAP XRkit (Strategene) from RNA prepared from exponentially growingcells in suspension culture. A tobacco SR-1 shoot apex cDNAlibrary was constructed with λ ZipLox kit (Gibco BRL) from RNAprepared from the shoot tip of mature plants. pGAD424Rb1 (kindlyprovided by Crisanto Gutierrez, CSIC-UAM, Spain) which containsZmRb1 (Xie et al., 1996) was digested with EcoRI and BamHI. Theresulting 2.0 kb fragment was gel-purified by a Prep A gene kit(BioRad) and labelled with α-32P-dCTP by random priming (BcaBestlabelling kit, Takara). This probe was used to screen a culturedtobacco BY-2 cell cDNA library as described previously (Setiadyet al., 1995). One positive clone was plaque-purified, and phageDNA was excised in vivo to recover pBluescript SK(–) plasmidaccording to Strategene’s protocol. The tobacco SR-1 shoot apexcDNA library was subsequently screened by a 32P-labelled tobaccocDNA clone. Positive clones were recovered by in vivo excisionaccording to the manufacturer’s manual.

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RNA blot analysis

Total RNA was isolated from various tobacco SR-1 organs aspreviously described (Setiady et al., 1995) and poly(A) RNAs werepurified by Oligotex-dT30 kit (Takara) according to the instructionmanual. Samples of 1 µg were electrophoresed on an 1.0%agarose–formaldehyde gel, blotted on a nylon membrane(Hybond-N1, Amersham), and hybridized with a 32P-labelled2.0 kbp NtRb1 probe (1288–3296 bp in Figure 1) containing the Aand B pocket regions. After autoradiography, the membrane wasstripped and reprobed with a rice actin gene probe (Setiadyet al., 1995).

Yeast two-hybrid assays

The cDNA fragments encoding cyclin D (NtcycD3–1) and cyclin A(Ntcyc27) (Setiady et al., 1995) were subcloned into the two-hybridvector pGBT9, which produced fusion proteins with the Gal4 DNA-binding domain. A fragment encoding amino acids 374–961 ofNtRb1 was fused with the Gal4 transactivation domain to constructa fusion protein in the plasmid pGAD424 as follows. An in-frameBamHI site was introduced upstream of the coding sequence byPCR with the forward primer 59-GGATCCTTGCAATGGCTT-CCCCAGC-39 and the reverse primer 59-GTCGACTAAGACT-CAGGCTGCTCAG T-39 to introduce a SalI site just after the stopcodon of NtRb1. The PCR product was digested with BamHIand SalI and ligated to pGAD424. Yeast transformations wereperformed with S. cerevisiae strain HF7c as described previously(Setiady et al., 1995).

Preparation of GST-NtRb1 fusion proteins

The pGEX-5X-2 plasmid was constructed to express a fusionprotein consisting of amino acids 374–961 encoded by NtRb1fused to glutathione S-transferase (GST). The recombinant plasmidpGAD424 (NtRb1) constructed for yeast two-hybrid assays wasdigested with BamHI and SalI and the insert fragment was sub-cloned into the BamHI and SalI sites of pGEX-5X-2. The recombin-ant plasmid pGEX-5X-2 (NtRb1) was introduced into theEscherichia coli strain BL21 (DE3) pLysS, and the transformantwas grown to an OD600 of 0.4 in LB broth containing ampicillinand chloramphenicol at 37°C. Expression of GST–NtRb1 fusionproteins was induced with 0.25 mM isopropyl-β-D-thiogalacto-pyranoside (IPTG) at 18°C for 16 h. Cells from a 100 ml culturewere lysed by sonication on ice in 5 ml of kinase buffer [50 mM

Tris–HCl at pH 7.5, 10 mM MgCl2, 1 mM EGTA, 1 mM phenylmethyl-sulphonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 10 mM NaF,25 mM β-glycerophosphate, 2 mM sodium orthovanadate] andthen cleared by centrifugation. Glutathion–Sepharose 4B beads(Pharmacia) were washed three times with kinase buffer andsuspended in an equal volume of the same buffer. Washed beads(200 µl) were added to 1 ml bacterial supernatant and incubatedfor 1 h at 4°C on a rotating stirrer. The beads were then washedthree times with kinase buffer and suspended in an equal buffervolume.

Preparation of antibody

Polyclonal antibodies raised against the N-terminal MVELN-NCSNSEENGC peptide of NtRb1, the NLAPNGQIGDIRSPKKVCpeptide corresponding to codons 654–671 of NtRb1, as well asagainst the N-terminal MGIQHNEHNQDQT peptide of NtcycD3–1and the N-terminal MATTQNRRSSVSSA peptide of Ntcyc25 (Seti-

250 Hirofumi Nakagami et al.

ady et al., 1995) were immunized in rabbits by using syntheticpeptides coupled to keyhole limpet haemocyanin through anadditional cysteine residue at their C-termini. Antibodies(MVELNNCSNSEENGC, MGIQHNEHNQDQT and MATTQNR-RSSVSSA) were purified by affinity chromatography in which eachpeptide was coupled to 2-fluoro-1-methyl-pyridinium toluene-4-sulphonate (FMP)-activated cellulofine (Seikagaku Kougyou Co.).

Insect cell culture and baculovirus infection

Spodoptera frugiperda (Sf9) cells were maintained at 27°C inGrace’s insect medium containing 10% fetal bovine serum (FBS)and gentamicin in 100 ml spinner bottles. Virus infection wasperformed in 60 mm diameter dishes. The FLAG tag was fused tothe N-terminal end of the entire coding region of NtcycD3–1 andinserted into the transfer vector pVL1392 (Pharmingen) as follows.To attach a FLAG sequence, an in-frame HindIII site was introducedupstream of the coding sequence by PCR with the forward primer59-AAGCTTATGGGAATACAACACAATGA-39 and the reverse primer59-TCTAGATTAGCGAGGGCTGCCAAC-39 to introduce the XbaI sitejust after the stop codon of NtcycD3–1 and inserted into the HindIIIand XbaI sites of pFLAG-1 (Eastman Kodak). To subclone theFLAG-tagged fragment of NtcycD3–1 into pVL1392, an in-frameBglII site was introduced upstream of the coding sequence ofpFLAG-1 (NtcycD3–1) by PCR with the forward primer 59-AGAT-CTATGGACTACAAGGATGACGATG-39 and the reverse primer asused above for introducing the XbaI site of pFLAG-1 (NtcycD3–1).pVL1392 (FLAG–NtcycD3–1) was co-transfected into Sf9 cells withlinearized BaculoGoldTM DNA (Pharmingen) using a liposome-mediated transfection kit (Gibco BRL).

The cDNA fragment containing the entire coding region ofcdc2Nt1 (Setiady et al., 1996) was inserted into the pFastBac HTaplasmid (Gibco BRL) to construct a pFastBac HTa (His–cdc2Nt1)consisting of a His tag sequence fused to the N-terminal end ofthe Cdc2Nt1. An in-frame BglII site was introduced upstream ofthe coding sequence of cdc2Nt1 by PCR with the forward primer59-AGATCTGGATGGACCAGTATGAAAAAGT-39 and the reverseprimer 59-GTCGACTCACGGAACATACCCAAT-39 to introduce a SalIsite. The resulting fragment was inserted into the BamHI and SalIsites of pFastBac HTa. This plasmid was transformed into E. colistrain DH5aBac (Gibco BRL) for transposition into the bacmid. Therecombinant bacmid was isolated and transfected into Sf9 cellsusing a liposome-mediated transfection kit (Gibco BRL). Recom-binant viruses were assayed for expression of their encodedproteins by immunoblotting. All sequences generated by PCRwere verified by sequencing.

In vitro binding assay

Lysates of E. coli expressing GST–NtRb1 and GST were incubatedwith lysates of insect cells which were or were not expressingFLAG–NtcycD3–1 at 4°C for 1 h on a rotating stirrer. After incuba-tion, the mixtures were purified by the anti-FLAG M2 affinity gel(Eastman Kodak), denatured in gel sample buffer, and separatedon 10% polyacrylamide gels containing SDS. NtRb1 was thendetected by immunoblotting, using the antibody against NtRb1(codons 654–671) with alkaline phosphatase for detection.

Detection of NtcycD3–1/Cdc2Nt1 complex formation

FLAG–NtcycD3–1 and His–Cdc2Nt1 were co-expressed in Sf9 cells,and the lysate was purified by the anti-FLAG M2 affinity gel. Elutefraction was denatured in the gel sample buffer, separated on

© Blackwell Science Ltd, The Plant Journal, (1999), 18, 243–252

10% polyacrylamide gels containing SDS, and then Cdc2Nt1was detected by immunoblotting using the antibody for His tag(Santa Cruz).

In vitro kinase assay

At 72 h post-infection, infected Sf9 cells were lysed, and thenFLAG-NtcycD3–1 and His–Cdc2Nt1 were purified by the anti-FLAGM2 affinity gel and the TALON metal affinity resin (Clontech),respectively. His–Cdc2Nt1/FLAG–NtcycD3–1 complex was purifiedfrom lysates of insect cells expressing both proteins using theanti-FLAG M2 affinity gel or the TALON metal affinity resin. Elutefractions which were prepared in the same way from the lysatesof insect cells infected with a wild-type baculovirus were used asnegative controls. Purified fractions were mixed with a bacterialGST–NtRb1 which were immobilized on glutathione–Sepharose4B beads. Kinase reactions were initiated at 30°C by adding 10 µCiof [γ-32P]ATP (4500 Ci mmol–1, ICN) adjusted with unlabeled ATPto a final concentration of 50 µM. After incubation for 10 min, thebeads were washed twice with cold kinase buffer, and the GST–NtRb1 eluted and resolved on denaturing polyacrylamide gels.The phosphorylated GST–NtRb1 was detected by autoradiography.

Protein extraction and immunoprecipitation

Tobacco BY-2 cells (4 days after subculture) were lysed bysonication on ice in extraction buffer [25 mM Tris–HCl at pH 7.6,75 mM NaCl, 15 mM MgCl2, 15 mM EGTA, 0.1% NP-40, 1 mM

phenylmethylsulphonyl fluoride (PMSF), 10 µg ml–1 leupeptin,50 µg ml–1 N-tosyl-L-phenylalanine chloromethyl ketone (TPCK),5 µg ml–1 pepstatin A, 10 µg ml–1 aprotinin, 5 µg ml–1 antipain,10 µg ml–1 trypsin inhibitor from soybean, 0.1 mM benzamidine,10 mM NaF, 25 mM β-glycerophosphate, 2 mM sodium orthovanad-ate] and then cleared by centrifugation. Protein concentrationswere determined by use of the Protein Assay CBB solution (NacalaiTesque Inc.) with bovine serum albumin as the standard.

The N-terminal anti-NtRb1 antibody, anti-NtcycD3–1 antibody,anti-Ntcyc25 anitibody, anti-PSTAIRE antibody (Santa Cruz) andnormal rabbit IgG (Santa Cruz) were cross-linked to protein ASepharose 4FF beads (Pharmacia) in 0.2 M H3BO3 buffer (pH 9.0)with 20 mM dimethylpimelimidate (DMP). Total protein (500 µg) inextraction buffer was pre-cleared with 10 µl of protein A Sepharose4FF beads for 30 min at 4°C. After centrifugation (15 0003g, 30 min,4°C), supernatants were transferred into the microtubes containing10 µl each of the immunoaffinity beads and incubated for 2 h at4°C. Beads were washed three times with extraction buffer andonce with 150 mM NaCl. The proteins were eluted from thebeads with 10 µl of 0.1 M glycine–HCl (pH 2.5) and immediatelyneutralized with 0.5 µl of 1 M Tris. Eluants were denatured inthe gel sample buffer, separated on 10% polyacrylamide gelscontaining SDS, and then Cdc2 was detected by immunoblottingwith the anti-PSTAIRE antibody.

Acknowledgements

The authors wish to thank Drs Ko Kato, Jun-ya Kato, HiroshiKouchi (National Institute of Agrobiological Resources) and KazuyaYoshida for helpful discussions and suggestions throughout thiswork. We are grateful to Dr Frederik Meins Jr (Friedrich MiescherInstitut) for criticaly reading the manuscript and Dr Dee Wormanfor editing the manuscript. This research was supported by aGrant-in Aid for Scientific Research (Nos 09640773 and 10182217)from the Ministry of Education, Science and Culture, Japan.

Rb kinase in plants 251

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