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First published online July 25, 2002; 10.1105/tpc.002550 American Society of Plant Biologists Phosphorylation of Retinoblastoma-Related Protein by the Cyclin D/Cyclin-Dependent Kinase Complex Is Activated at the G1/S-Phase Transition in TobaccoGraduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0101, Japan 2 To whom correspondence should be addressed. E-mail sekine{at}bs.aist-nara.ac.jp; fax 81-743-72-5469
In mammals, D-type cyclin-associated kinases mainly regulate the G1/S transition by phosphorylating the retinoblastoma (Rb) protein. We previously demonstrated that in tobacco, cyclin D (Nicta; CycD3;3) is complexed with the PSTAIRE-containing cyclin-dependent kinase (CDKA) from tobacco. Here, we show that Nicta; CycD3;3–associated kinases phosphorylate both the tobacco Rb-related protein (NtRb1) and histone H1. Although NtRb1 kinase activity was detected only during the middle G1- to early S-phase, histone H1 kinase activity was observed as two peaks in G1- to S-phase and G2/M- to M-phase. Importantly, we show that the proportion of cells in the G1-phase was reduced in transgenic Bright Yellow-2 cells overexpressing Nicta; CycD3;3-GFP. Mutational analyses revealed that phosphorylation of Thr-191 in Nicta; CycD3;3 possibly is required for both full kinase activity and localization predominantly to the nucleus. These data suggest that Nicta; CycD3;3 acts as a rate-limiting regulator in the G1/S transition by forming active complexes with CDKA or its related kinases to phosphorylate Rb-related protein and potentially plays a novel role during G2/M and mitosis.
Cell division plays a crucial role in the growth and development of multicellular organisms (Meyerowitz, 1997  ; Polymenis and Schmidt, 1999). Plants have unique properties with respect to cell growth, plasticity of body plan, and organization and development, which suggests that they also may have different controls in place to regulate cell division. Plant cells potentially have the continuous proliferative capacity that is found postembryonically in shoot and root apical meristems. Additionally, plants are able to change their fate in response to environmental signals throughout the life cycle (Deng, 1994; Francis, 1998). However, the external factors that control cell division, and ultimately plant growth, are unknown.
In mammals, changes in the cell cycle machinery play an important role in controlling growth in response to extracellular signals (Sherr, 1994
Elucidation of the mechanisms that govern the G1/S transition in plants is essential to understanding plant growth and development. The recent identification of plant homologs of Rb-related protein (Grafi et al., 1996
The Arabidopsis cyclin D, Arath; CycD3;1, is induced by the plant hormone cytokinin and a carbon source (Suc), whereas Arath; CycD2;1 is induced by Suc alone (Soni et al., 1995
Although cyclin D activity is critical for cell cycle control, development, and growth in plants, very little is known about cyclin D activity on Rb-related protein, which is the most likely candidate to play a significant role in the control of the G1/S-phase transition. In mammals, the G1/S transition is controlled by CDK4 and CDK6 associated with cyclin D and CDK2 associated with cyclin E, and the G2/M transition is controlled by cdc2 (CDK1) associated with cyclin A and cyclin B (Pines, 1995
Plants contain multiple CDKs, including those of the PSTAIRE type, known as cdc2a or CDKA (Doonan and Fobert, 1997 In this study, we demonstrate that Nicta; CycD3;3–associated kinases phosphorylate NtRb1 during the middle G1- to early S-phase and that the phosphorylation of histone H1 occurs at two discrete points within the cell cycle: at the G1/S and G2/M boundaries. Acceleration of the G1-phase progression was observed in tobacco Bright Yellow-2 (BY-2) cells overexpressing Nicta; CycD3;3-GFP. Mutational analyses revealed that Thr-191 in Nicta; CycD3;3 is likely to be involved in both full kinase activity in complex with CDKA and its localization to the nucleus.
The Nicta; CycD3;3/Nicta; CDKA;3 Complex Phosphorylates Both Histone H1 and NtRb1 Using a baculovirus expression system, we demonstrated previously that Nicta; CycD3;3 forms an active complex with Nicta; CDKA;3, a member of CDKA (cdc2a) group that exhibits kinase activity against the tobacco Rb-related protein NtRb1 (Nakagami et al., 1999 ). Because we found that the C-terminal region of NtRb1 (amino acids 823 to 961) was phosphorylated equally by the Nicta; CycD3;3/Nicta; CDKA;3 complex in vitro as the region used previously (amino acids 374 to 961) (Nakagami et al., 1999), we opted to continue our experiments using the C-terminal region as a substrate. In this study, we also evaluated the kinase activity of the Nicta; CycD3;3/Nicta; CDKA;3 complex on histone H1 in vitro.
His-tagged Nicta;CDKA;3 was coexpressed in insect cells with FLAG-tagged Nicta; CycD3;3 using a baculovirus expression system (Figure 1A)
. A human cyclin D1/CDK4 complex, used as a control, phosphorylated human Rb but not histone H1 (Figure 1A), consistent with a previous result describing histone H1 as a poor substrate (Kato et al., 1993
To verify that the Nicta; CycD3;3/Nicta; CDKA;3 complex is responsible for the kinase activity seen in lysates, the complex was purified with an anti-FLAG M2 affinity gel or TALON metal affinity resin. Although proteins purified from cells transfected with Nicta; CycD3;3, Nicta; CDKA;3, or vector alone did not exhibit kinase activity, proteins purified from insect cells expressing both Nicta; CycD3;3 and Nicta; CDKA;3 exhibited kinase activity against histone H1 and NtRb1 (Figure 1B). The presence of a weak signal in Nicta; CDKA;3 alone is most likely the result of complex formation between Nicta; CDKA;3 and endogenous cyclins derived from insect cells, resulting in some weak kinase activity.
Thr-191 of Nicta; CycD3;3 Is Required for Kinase Activity in Vitro
To determine whether these residues also are important for the function of plant D-type cyclins, mutations were introduced into the putative phosphorylation sites in Nicta; CycD3;3. We replaced Thr-191 of Nicta; CycD3;3, which corresponds to Thr-156 of human cyclin D1, with the nonphosphorylatable amino acid Ala (T191A). Thr-191 was further mutated to Ser (T191S) to provide a different phosphorylatable residue. Ser-296 and Ser-300 in Nicta; CycD3;3, which correspond to Thr-286 of human cyclin D1, were replaced with Ala (S296,300A). The triple mutant (T191A,S296,300A), in which all three phosphorylatable sites are mutated, also was constructed. We evaluated whether these mutants form active complexes with Nicta; CDKA;3. His-tagged wild-type and mutant Nicta; CycD3;3 were coexpressed with FLAG-tagged Nicta; CDKA;3 in insect cells (Figure 3A) . Equivalent levels of wild-type and mutant Nicta; CycD3;3 expression were detected in insect cells by immunoblotting with a His tag–specific antibody (data not shown). Although lysates containing Nicta; CDKA;3 and either wild-type or mutant Nicta; CycD3;3 (T191S or S296,300A) exhibited kinase activity against histone H1 and NtRb1, lysates containing Nicta; CDKA;3 with Nicta; CycD3;3 (T191A) or Nicta; CycD3;3 (T191A,S296, 300A) had no kinase activity. The Nicta; CycD3;3 (T191A) mutant retained the ability to bind Nicta; CDKA;3, as shown by an in vitro binding assay (Figure 3B).
Nicta; CycD3;3 (T191A) Localizes Predominantly to the Cytoplasm The subcellular localization of wild-type and mutant Nicta; CycD3;3 proteins fused to green fluorescent protein (GFP) was examined in exponentially growing tobacco BY-2 cells and compared with that in BY-2 cells expressing GFP alone. Expression of the GFP-fused proteins was analyzed by immunoblotting with a GFP-specific antibody, and expression of polypeptides corresponding to the expected size was determined for each of the proteins (data not shown). Fluorescence microscopy of the BY-2 cells expressing GFP alone showed that GFP fluorescence was observed in both the nucleus and the cytoplasm (Figure 4) . By contrast, green fluorescence exhibited by Nicta; CycD3;3-GFP and Nicta; CycD3;3 (S296,300A)-GFP was found primarily in the nucleus, whereas that exhibited by Nicta; CycD3;3 (T191A)-GFP and Nicta; CycD3;3 (T191A,S296,300A)-GFP was localized predominantly in the cytoplasm. These results suggest that phosphorylation of Thr-191 is required for the nuclear localization of Nicta; CycD3;3.
Thr-191 of Nicta; CycD3;3 Is Required for Full Kinase Activity in Immunoprecipitates from Cell Extracts GFP-fused Nicta; CycD3;3 proteins were immunoprecipitated from exponentially growing cells with a GFP-specific antibody and immunoblotted subsequently with an anti-PSTAIRE antibody recognizing the conserved PSTAIRE motif of CDKA. The anti-PSTAIRE antibody cross-reacted with a polypeptide of  34 kD in immunoprecipitates from the BY-2 cells expressing GFP-fused Nicta; CycD3;3 proteins (Figure 5)
. However, no band was detected with an anti-PSTAIRE antibody in the case of wild-type BY-2 cells and BY-2 cells expressing GFP alone. This finding suggests that GFP-fused wild-type and mutant Nicta; CycD3;3 proteins bind to CDKA in tobacco cells.
Phosphorylation of histone H1 was detected in immunoprecipitates prepared from BY-2 cells expressing Nicta; CycD3;3-GFP and Nicta; CycD3;3 (S296,300A)-GFP, whereas immunoprecipitates from BY-2 cells expressing either Nicta; CycD3;3 (T191A)-GFP or Nicta; CycD3;3 (T191A, S296,300A)-GFP demonstrated only weak kinase activity (Figure 5). Phosphorylation of NtRb1 also was reduced significantly in immunoprecipitates from BY-2 cells expressing Nicta; CycD3;3 (T191A)-GFP compared with cells expressing Nicta; CycD3;3-GFP. These results suggest that phosphorylation of Thr-191 in Nicta; CycD3;3 is required for full kinase activity in immunoprecipitates from cell extracts.
Nicta; CycD3;3-Associated Kinases Phosphorylate Rb-Related Protein during the Middle G1- to Early S-Phase
Nicta; CycD3;3–associated kinases were obtained by immunoprecipitation with an antibody raised against the 13 N-terminal amino acids of Nicta; CycD3;3 (Nakagami et al., 1999 ). Immunoprecipitates obtained with the Nicta; CycD3;3– specific antibody exhibited kinase activity against both histone H1 and NtRb1. Phosphorylation of histone H1 occurred in two peaks at G1- to S-phase and G2/M- to M-phase, whereas NtRb1 kinase activity was present only during G1- to S-phase (Figure 6C). The immunoprecipitated kinase activity resulted from Nicta; CycD3;3–associated kinases, because preimmune serum immunoprecipitated only background kinase activity and no phosphorylated bands were detected in the assay without the addition of the substrates (data not shown).
Because the yeast p13SUC1 protein binds various plant CDKA-related kinases (De Veylder et al., 1997
There is growing evidence that Rb also has effects during S-phase and mitosis in mammals (Lukas et al., 1999 Although Nicta; CycD3;3–associated kinases phosphorylated histone H1 during S-phase to the next (G1) phase, their kinase activities were reduced twice at 2 to 4 h and 12 h. However, NtRb1 was phosphorylated only at 14 h, corresponding to the middle to late G1-phase (Figure 6D), which is consistent with the result obtained by synchronization of BY-2 cells treated with aphidicolin and propyzamide (Figure 6C). p13SUC1-bound kinases also phosphorylated histone H1 during all phases tested, and their kinase activities were reduced at 2 to 4 h and 12 to 14 h, whereas NtRb1 was phosphorylated only at 0 to 2 h, corresponding to middle to late S-phase (Figure 6D). Thus, Nicta; CycD3;3–associated kinases phosphorylate NtRb1 during a very short period in middle G1-phase to early S-phase, and p13SUC1-bound kinases exhibit kinase activity on NtRb1 during late G1-phase to late S-phase.
Overexpression of Nicta; CycD3;3 Reduces the Proportion of Cells in G1-Phase
The cell lines that overexpress Nicta; CycD3;3 (T191A)-GFP, named T191A, had no discernible cell cycle effects on growing cells compared with the control cells that express GFP alone, named GFP (Figure 7, Table 1). By contrast, overexpression of Nicta; CycD3;3-GFP, named CycD3;3, and Nicta; CycD3;3 (S296,300A)-GFP, named S296,300A, both of which form the active complexes with CDKA, reduced the proportion of cells in G1-phase and increased the proportion of S-phase cells (Figure 7, Table 1). Furthermore, the doubling times of CycD3;3 and S296,300A were reduced relative to that of GFP. For the S296,300A mutant, this was particularly marked, corresponding to a 25% reduction in doubling time. These results suggest that Nicta; CycD3;3–associated kinases promote the cell cycle progression from G1- to S-phase and that Nicta; CycD3;3 regulates the G1/S transition in tobacco cells.
We also measured the cell number for each cell line for 8 days after subculturing under identical conditions in culture medium supplemented with the antibiotic kanamycin (Figure 8) . S296,300A showed faster growth and reached higher cell density than CycD3;3. T191A grew more slowly than both S296,300A and CycD3;3 during the first 4 days, but ultimately it reached the highest cell density. We did not observe a clear difference in cell size between these transgenic cell lines and wild-type BY-2 cells, so overexpression of Nicta; CycD3;3 and its mutants appears to have no effect on cell size.
In plants, D-type cyclins respond to signals such as cytokinin and Suc at the G1/S boundary (Riou-Khamlichi et al., 1999 , 2000). Therefore, cellular mechanisms that sense extracellular signals must be integrated with the cell cycle control machinery during the G1/S transition in plants. Although the existence of cyclin D– and Rb-related genes suggests that the G1/S control mechanisms in plants may be similar to those in mammals, this is by no means confirmed (Gutierrez, 1998; de Jager and Murray, 1999; Mironov et al., 1999). In particular, the phosphorylation of Rb by plant cyclin D complexes prepared from cell extracts has not been demonstrated. In this study, we have demonstrated that, in tobacco, Nicta; CycD3;3–associated kinases phosphorylate Rb-related protein during middle G1-phase to early S-phase.
Mammalian cyclin D associates with CDK4 and CDK6, which contain the sequence P(I/L)ST(V/I)RE, to form active complexes that selectively phosphorylate Rb protein (Sherr, 1994
Arabidopsis CycD2 and CycD3 proteins have been shown to interact with the PSTAIRE-containing CDKA in vivo and to phosphorylate histone H1 (Cockcroft et al., 2000
Rb contains several functional domains. Domains A and B are highly conserved from human to plants, and they form a "pocket" domain that binds many cellular factors, which is critical to the function of Rb (Weinberg, 1995 These findings suggest that phosphorylation within the plant A/B pocket region may be allowed by its conformational change caused primarily by phosphorylation in the C-terminal region. We used the C-terminal region alone as a substrate, and it remains to be determined whether the timing and pattern of phosphorylation of intact Rb-related protein are different from those of the C-terminal region alone. In addition, it will be interesting to determine the relative contributions of each of 13 putative phosphorylation sites in tobacco Rb-related protein to the release of important regulators from the pocket, which may stimulate the commitment to progress through G1- into S-phase.
In agreement with the findings that Nicta; CycD3;1 and Nicta; CycD2;1 transcripts accumulate during G2/M- to M-phase (Sorrell et al., 1999
Because the fission yeast protein p13SUC1 binds largely to CDKA-related proteins containing a conserved PSTAIRE motif (De Veylder et al., 1997
One possible explanation for this finding is that the weak activity of the Nicta; CycD3;3–associated kinases bound to p13SUC1 would not be detected during middle to late G1-phase against this background. Alternatively, other complexes than CycD3;3/CDKA also might contribute to phosphorylate NtRb1 at the G1/S transition. There are at least two CycD3 genes and one CycD2 gene in tobacco (Sorrell et al., 1999
By contrast, two peaks of histone H1 kinase activity were observed at the G1/S and G2/M boundaries. This result is consistent with reports that alfalfa histone H1 kinase activity was increased at both G1- to S-phase and G2/M- to M-phase, suggesting that CDKA forms different complexes in the G1/S- and G2/M-phases (Magyar et al., 1993
p13SUC1-bound proteins recovered from synchronized cells in various cell cycle phases contained equivalent levels of CDKA, as determined by immunoblotting with an anti-PSTAIRE antibody (data not shown). This finding suggests that the activities of CDKA-associated kinases may be regulated by several different cyclins and, more importantly, by additional regulatory factors, such as CDK-activating kinase and CDK inhibitors (Umeda et al., 1998
Presuming that NtRb1 is a preferred target of the Nicta; CycD3;3–associated kinases, it is likely that the kinase complexes undergo transport into the nucleus. Fluorescence microscopy of transgenic cells revealed the localization of Nicta; CycD3;3-GFP to the nucleus, whereas Nicta; CycD3;3 (T191A)-GFP remained predominantly in the cytoplasm (Figure 4). Human cyclin D1 accumulates in the nucleus during G1-phase, disappearing subsequently from nuclei during DNA synthesis (Diehl and Sherr, 1997 After overexpression of Nicta; CycD3;3-GFP, the proportion of G1-phase cells decreased in growing cells (Figure 7). In addition, overexpression of Nicta; CycD3;3 (S296,300A)-GFP resulted in a reduction in the lengths of G1- and G2/M-phases and significantly reduced the doubling time (Table 1), suggesting that Nicta; CycD3;3–associated kinase activity not only promotes the G1/S transition but also plays a potential role during G2/M- and M-phase. In contrast, overexpression of Nicta; CycD3;3 (T191A)-GFP had no discernible cell cycle effects; rather, it prolonged the doubling time. It should be noted that Nicta; CycD3;3 (T191A)–associated kinase activity is very weak, compared with that of both wild-type and mutant Nicta; CycD3;3 (S296,300A) in immunoprecipitates from cell extracts (Figure 5). In plants, Nicta; CycD3;3/CDKA complexes exhibit kinase activity against Rb-related protein during middle G1-phase to early S-phase, and phosphorylation of histone H1 is increased at both G1- to S-phase and G2/M- to M-phase. This is in sharp contrast to findings in mammals, in which cyclin D–associated kinases mainly regulate the G1/S transition through the phosphorylation of Rb proteins. Although it remains to be determined whether the phosphorylation of Rb-related protein stimulates the G1/S transition in plants, our data provide further evidence that Rb-related protein plays a potentially significant role in plant cell cycle control.
Plant Material and Cell Cycle Synchronization Tobacco cells (Nicotiana tabacum cv Bright Yellow-2 [BY-2]) were cultured in a modified Linsmaier and Skoog medium as described previously (Nakagami et al., 1999 ). Cells were synchronized using 5 µM aphidicolin (Wako Pure Chemical, Osaka, Japan) and 3 µM propyzamide (Wako Pure Chemical) essentially as described by Nagata et al. (1992).
Cell Cycle Analysis of Tobacco BY-2 Cells
Construction of Nicta; CycD3;3 Mutants
Construction of Binary Plasmids and Transformation of Tobacco BY-2 Cells
Preparation of the GST-NtRb1 Fusion Protein
Insect Cell Culture and Baculovirus Infection A His tag was fused to the N terminus of wild-type and mutant Nicta; CycD3;3. Sequences encoding wild-type and mutant Nicta; CycD3;3 were amplified with the following primers: forward, 5'-GGATCCATGGGAATACAACACAATGAG-3'; reverse, 5'-GTCGAC-TTAGCGAGGGCTGCCAACA-3'. The DNA fragments generated by PCR were inserted into the BamHI and SalI sites of pFastBac HTb (Gibco BRL). Recombinant bacmid, intermediate plasmid, was isolated and transfected into Sf9 cells using a liposome-mediated transfection kit (Gibco BRL).
In Vitro Kinase Assay
Detection of Complex Formation
Protein Extraction and Immunoprecipitation
Proteins were immunoprecipitated from 300-µg protein extracts using 4 µg of Nicta; CycD3;3–specific antibody (Nakagami et al., 1999
Kinase assays were performed on proteins immobilized on protein A–Sepharose beads or p13SUC1-agarose beads. The reaction was initiated by adding 10 µL of kinase buffer containing 2.5 µg of histone H1 (Gibco BRL) or GST-NtRb1 and 370 kBq of After denaturation of beads in gel sample buffer, proteins were separated on 10% SDS–polyacrylamide gels, and cyclin-dependent kinase was detected by immunoblotting with an anti-PSTAIRE antibody (Santa Cruz Biotechnology).
Fluorescence Microscopy
Accession Numbers
The authors thank Ko Kato, Jun-ya Kato, Hiroshi Kouchi, and Kazuya Yoshida for their helpful discussions and suggestions throughout this work. We are grateful to James Murray, Laszlo Bögre, and David Sorrell for their critical reading of the manuscript. We also are grateful to Hirofumi Harashima for substantial assistance with the figures. This research was supported by a Grant-in-Aid for Scientific Research (Grant 12037213) from the Ministry of Education, Science, and Culture, Japan.
Online version contains Web-only data.  Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002550.
1 Current address: Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Austria. Received February 21, 2002; accepted April 9, 2002.
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Abstract
INTRODUCTION
-32P-ATP.
inhibitor aphidicolin and the anti-tubulin drug propyzamide (Planchaisa et al., 2000
-glycerophosphate, and 2 mM sodium orthovanadate) and then cleared by centrifugation. Kinase reactions were initiated by adding the lysates to the kinase buffer containing 2.5 µg of histone H1 (Gibco BRL) or GST-NtRb1 and 370 kBq of 
