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Keywords:

  • cell cycle;
  • cyclin-dependent protein kinase;
  • CDK-activating kinase;
  • cyclin H;
  • rice;
  • poplar

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

cDNAs encoding cyclin H homologs were isolated from poplar (Populus tremulax tremuloides) and rice (Oryza sativa) plants, and were designated Pt;cycH;1 and Os;cycH;1, respectively. The deduced amino-acid sequences showed 40–60% similarity to human cyclin H and Schizosaccharomyces pombe Mcs2, with higher similarity in the cyclin box region. While Pt;cycH;1 and Os;cycH;1 were expressed in all tissues examined, the transcripts accumulated abundantly in dividing cells. Expression of Os;cycH;1 was abundant in the S-phase in partially synchronized suspension cells, and was induced by submergence in internodes of deepwater rice. A yeast two-hybrid assay demonstrated that both Pt;CycH;1 and Os;CycH;1 were able to interact with rice R2 kinase, which is structurally and functionally similar to cyclin-dependent kinase (CDK)-activating kinase (CAK) of vertebrates. Moreover, an in vitro pull-down assay showed that Os;CycH;1 specifically bound to R2 but not to other rice CDKs. When R2 was expressed in budding yeast CAK mutant, the suppression activity in terms of temperature-sensitivity was enhanced by co-expression with Os;cycH;1. Furthermore, in vitro kinase assay indicated that the kinase activities of R2 on CDKs and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II were markedly elevated by binding to Os;CycH;1. Our results suggest that cyclin H is a regulatory subunit of CAK, which positively controls CDK- and CTD-kinase activities in plant cells.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

A family of cyclin-dependent protein kinases (CDKs) plays a key role in the progression of the cell cycle. The kinase activity of CDKs is dependent on binding to cyclins. Like vertebrates, plants express several types of CDKs and cyclins (for reviews see Mironov et al. 1999 ; Renaudin et al. 1996 ), thus different sets of CDK/cyclin pairs might regulate the division of plant cells at each stage of the cell cycle. Phosphorylation of CDKs also controls their kinase activities (for review see Morgan, 1995). Full activation of CDKs requires dephosphorylation of inhibitory phosphorylation sites (Thr-14 and Tyr-15 in human CDK2) by dual-specificity phosphatase, Cdc25, and phosphorylation of a threonine residue within the T-loop region (Thr-160 in human CDK2), which blocks the entry of the substrate to the catalytic cleft when the threonine residue is unphosphorylated ( Russo et al. 1996 ). Phosphorylation of this threonine residue is catalyzed by a CDK-activating kinase (CAK) (for reviews see Draetta, 1997; Nigg, 1996). The catalytic subunit of CAK is one of the CDK family termed CDK7/p40MO15 in mammals and Xenopus ( Fesquet et al. 1993 ; Poon et al. 1993 ; Shuttleworth et al. 1990 ; Solomon et al. 1993 ), while its regulatory subunit is named cyclin H ( Fisher & Morgan, 1994; Labbéet al. 1994 ; Mäkeläet al. 1994 ). In the presence of cyclin H, CDK7 activity is significantly stimulated, while CDK7 alone has a low CAK activity ( Fisher & Morgan, 1994), indicating that cyclin H positively regulates the kinase activity of CDK7. In Schizosaccharomyces pombe, Mcs6/Crk1/Mop1, which is closely related to CDK7/p40MO15, associates with the cyclin H homolog Mcs2 ( Buck et al. 1995 ; Damagnez et al. 1995 ).

In addition to cyclin H, MAT1 (which has a RING finger motif) also interacts with CDK7 to stabilize the CDK7–cyclin H complex ( Devault et al. 1995 ; Fisher et al. 1995 ; Tassan et al. 1995a ). The CDK7–cyclin H–MAT1 complex has also been identified in the general transcriptional factor TFIIH which phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II, and is involved in the initiation and elongation of transcription ( Nigg, 1996; Roy et al. 1994 ; Serizawa et al. 1995 ; Seroz et al. 1995 ; Shiekhattar et al. 1995 ). Another cyclin, cyclin C, shows high similarity (45%) to cyclin H in the cyclin box region, where amino-acid sequences are relatively conserved among different cyclin families ( Lew et al. 1991 ). Cyclin C is known to associate with CDK8 ( Tassan et al. 1995b ) and activate the kinase activity on CTD, suggesting that cyclin C is involved in the regulation of transcription ( Rickert et al. 1996 ).

Rice R2, which is structurally similar to vertebrate CAKs ( Hata, 1991), can phosphorylate the threonine residue within the T-loop of human CDK2 and the rice CDK, Cdc2Os1, and also the CTD of Arabidopsis in vitro ( Yamaguchi et al. 1998 ). Biochemical fractionation of rice protein extract suggests that R2 protein is present in at least three distinct protein complexes of 70, 105 and 190 kDa, respectively, among which only the 105 kDa complex has both CDK- and CTD-kinase activities ( Yamaguchi et al. 1998 ). This indicates that R2 does not have kinase activities as a monomer, and another regulatory subunit(s) is required for activation of R2.

Here we report cDNAs encoding cyclin H homologs of poplar and rice plants. The transcripts were detected in almost all tissues, but the expression level was highest in actively dividing cells of rice suspension culture. In vitro pull-down assay showed that rice cyclin H specifically interacts with R2 but not with the other rice CDKs. Moreover, rice cyclin H enhanced CDK- and CTD-kinase activities of R2 in vitro. Our results indicate that cyclin H positively regulates the kinase activities of R2, as in the case of vertebrate-type CAKs.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Isolation of cDNAs encoding cyclin H homologs from poplar and rice plants

One of the EST clones prepared from the cambial region of poplar (Populus tremula L. × tremuloides) ( Sterky et al. 1998 ) was 1204 bp in length, and encoded a predicted protein of 332 amino acids ( Fig. 1a). The amino-acid sequence showed approximately 40% similarity to human cyclin H and S. pombe Mcs2 ( Fig. 1a). In the cyclin box region the poplar sequence showed about 60 and 70% similarity to human cyclin H and S. pombe Mcs2, respectively ( Fig. 1a). Therefore we designated the poplar clone Pt;cycH;1, as proposed by Renaudin et al. (1996) .

image

Figure 1. Comparison of amino-acid sequences of cyclin H homologs.

(a) Amino-acid sequences of cyclin H homologs were aligned to give the highest similarity to each other. Amino acids identical to those of rice Os;CycH;1 are shown by white letters; the region corresponding to the cyclin box is indicated by a shaded box. Human (Hs) and Drosophila (Dro) cyclin H, S. pombe MCS2, S. cerevisiae CCL1 and rice C-type cyclin, Os;CycC;1, are included.

(b) Phylogenetic tree of C-type, H-type and plant cyclins. The poplar and rice cyclins are denoted by boxes. At, Arabidopsis; Nt, tobacco.

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To isolate the rice homolog of cyclin H, Pt;cycH;1 cDNA was used as probe to screen the rice cDNA library. The longest clone contained a putative open-reading frame (ORF) of 330 amino acids which showed approximately 60 and 80% identity to the whole and the cyclin box region of Pt;CycH;1, respectively ( Fig. 1a). Thus the rice clone was named as Os;cycH;1. In the phylogenetic tree, Pt;CycH;1 and Os;CycH;1 were classified into the cluster including animal cyclin H and S. pombe Mcs2, but were distinct from other plant cyclins ( Fig. 1b). Rice cyclin C homolog, which was included in the animal and yeast cyclin C cluster, was relatively close to Pt;CycH;1 and Os;CycH;1 ( Fig. 1b). DNA gel-blot analysis demonstrated that there are at least two genes for cyclin H in the poplar genome, whereas the rice gene is encoded at a single locus in the genome (data not shown).

Expression of Os;cycH;1 is abundant in S-phase and is induced by submergence in deepwater rice

To examine the expression pattern of cyclin H, RNA was isolated from different tissues of poplar and rice plants and was hybridized with each cDNA. As shown in Fig. 2(a), the transcript level of poplar cyclin H was higher in the cambial zone and differentiating xylem, where cells are fated to be xylem, compared with differentiated tissues such as cortex, phloem fibers, phloem and expanding xylem ( Fig. 2a). The 1.6 kb transcript of Os;cycH;1, corresponding to the full-length cDNA, was detected in all rice tissues examined, and the mRNA level was highest in suspension-cultured cells ( Fig. 2b). When the cDNA of rice R2 was used as a probe, the expression pattern was similar to that of Os;cycH;1 ( Fig. 2b), indicating that cyclin H together with R2 is expressed in almost all tissues, but is highly expressed in actively dividing cells.

image

Figure 2. mRNA accumulation of poplar and rice cyclin H in different tissues.

(a) Dot-blot hybridization of poplar cDNAs with the probe of Pt;cycH;1. mRNA isolated from indicated tissues was amplified by RT–PCR, and the cDNA mixture was hybridized with a probe of full-length cDNA of Pt;cycH;1. Polyubiquitin was used as a control. CO, cortex, between cork and the first fiber bundles; PF, phloem fibers; PH, functional phloem; CZ, cambial zone with differentiating phloem; DX, differentiating xylem (cells fated to be xylem); EX, expanding xylem; SW, zone of visible secondary wall formation.

(b) Northern hybridization analysis of Os;cycH;1 and R2. Total RNA (20 μg) isolated from indicated tissues was probed with the ORF of Os;cycH;1 or R2. Ribosomal RNA (rRNA) visualized with ethidium bromide is shown as a control.

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To examine the expression pattern of Os;cycH;1 during the cell cycle, rice suspension-cultured cells were partially synchronized and Northern hybridization was conducted with isolated RNA. When cells were blocked in the late G1 phase with aphidicolin, expression of Os;cycH;1 was low ( Fig. 3a). As soon as cells were released from the block to enter the S phase Os;cycH;1 expression rose and remained elevated between 3 and 30 h after release when cells passed through the S and G2 phases ( Fig. 3a). Overall, a close correlation was observed between changes in the S-phase population and changes in Os;cycH;1 expression ( Fig. 3a).

image

Figure 3. Expression analysis of partially synchronized suspension cells and submerged deepwater rice.

(a) Cyclin H expression in rice synchronized suspension cells. Rice suspension cells were arrested in the G1/S phase by treatment with aphidicolin and then released from the block. Sample was taken at the time of release (0 h) and every 3 h thereafter up to 36 h, and 25 μg of total RNA from each sample was used for Northern blot analysis.

(b) Cyclin H expression in deepwater rice. Total RNA (23 μg) from the meristematic zone of the internode (up to 5 mm above the second node); from the elongation zone (5–15 mm above the second node); and from the differentiation zone (starting 40 mm above the second node) were used for Northern blot analysis.

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Deepwater rice can be induced to grow rapidly in the intercalary meristem at the base of the internode with partial submergence ( Kende et al. 1998 ). In the growing internode, Os;cycH;1 transcripts were most abundant in the intercalary meristem where cell division takes place. Lower transcript levels were present in the elongation zone, and still less Os;cycH;1 mRNA was found in differentiated cells ( Fig. 3b). Upon submergence, transcript abundance increased in the meristem and in the elongation zone. At 6 h, Os;cycH;1 expression was highest and then moderately decayed ( Fig. 3b). In the elongation zone a minor increase in Os;cycH;1 transcript levels was found after 4 h, and further induction of Os;cycH;1 expression was detected after 12 h of submergence. Low levels of Os;cycH;1 mRNA but no significant increase in Os;cycH;1 expression was detected in differentiated tissues upon growth induction ( Fig. 3b).

Interaction of Os;CycH;1 with R2 enhances the suppression activity of R2 on CAK mutation in budding yeast

To examine whether Os;CycH;1 protein can interact with R2, we applied the yeast two-hybrid system. Recombinant proteins of R2 fused to the GAL4-DNA binding domain (DNA-BD) and Os;CycH;1 fused to the GAL4-transactivation domain (AD) was produced in the Saccharomyces cerevisiae Y190 strain, which has HIS3 and LacZ reporter genes under the consensus sequences of the GAL4-binding site ( Flick & Johnston, 1990; Harper et al. 1993 ). Expression of R2 or Os;cycH;1 did not induce the expression of reporter genes, while cells expressing both R2 and Os;CycH;1 fusion proteins could grow on a medium without histidine and expressed the LacZ protein ( Fig. 4a). These results suggest that Os;CycH;1 and R2 are associated with each other in yeast cells. Interestingly, expression of R2 together with Pt;CycH;1 fused to GAL4-AD also induced the expression of reporter gene in Y190 cells (data not shown).

image

Figure 4. Os;CycH;1 interacts with R2 and enhances the suppression activity of R2 on CAK mutation in budding yeast.

(a) Os;CycH;1 interacts with R2 in the yeast two-hybrid system. S. cerevisiae strain Y190 was transformed with R2 and Os;cycH;1 that were cloned into pAS2-1 (pAS2-R2) and pGAD-GL (pGAD-cycH), respectively, or with the empty vector. Transformants were grown on a minimal medium in the presence (+His) or absence (–His) of histidine at 30°C for 3 days. β-Galactosidase activity of the colonies grown on minimal medium was observed by the filter-lift assay (LacZ). As a positive control, pTD1-1 and pVA3-1 were transformed and assayed.

(b) Co-expression of Os;cycH;1 and R2 results in higher suppression of CAK mutation in budding yeast cells. R2 was cloned into the galactose-inducible vector pYES2 (pYES2-R2). The CAK-deficient mutant GF2351 carrying the indicated plasmids was grown on glucose-containing minimal medium (MVD) or on galactose-containing minimal medium (MVGS) at the temperatures indicated for 5 days.

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In budding yeast, Civ1/Cak1 has a CAK activity but not CTD-kinase activity in vivo ( Espinoza et al. 1996 ; Kaldis et al. 1996 ; Thuret et al. 1996 ). We have previously demonstrated that overexpression of R2 was able to complement CAK mutation in the budding yeast strain GF2351, which carries a temperature-sensitive mutation in the civ1/cak1 gene ( Yamaguchi et al. 1998 ). To examine whether Os;CycH;1 regulates the activity of R2, we introduced R2, Os;cycH;1 or both into GF2351 cells. R2 cDNA was cloned into the expression vector pYES2, which contains the galactose-inducible GAL1 promoter ( Yamaguchi et al. 1998 ), and Os;cycH;1 was inserted into the constitutive expression vector pGAD-GL, which contains truncated adh promoter and expresses GAL4 transactivation domain fused to Os;CycH;1. As shown in Fig. 4(b), cells expressing R2 grew at 34°C on galactose-containing minimal medium (MVGS) but not on a glucose-containing minimal medium (MVD). Cells expressing only Os;cycH;1 could not grow at 34°C ( Fig. 4b). In contrast, cells expressing both R2 and Os;cycH;1 grew on the MVGS medium at 36°C, but at this temperature those expressing only R2 were unable to grow ( Fig. 4b). These results indicate that expression of Os;cycH;1 enhances the suppressive activity of R2 on the civ1/cak1 mutation in budding yeast cells.

R2 specifically binds to Os;CycH;1 and Os;CycC;1 in vitro

To confirm the interaction observed in yeast cells, we performed in vitro binding assays. Rice CDKs fused to GST (GST-Cdc2Os1, GST-Cdc2Os2, GST-Cdc2Os3 and GST-R2, respectively), were expressed in Escherichia coli and immobilized on glutathione-Sepharose beads. Os;CycH;1 was transcribed and translated from the cDNA with the rabbit reticulocyte lysate in the presence of [35S]methionine and incubated with CDK beads. After washing, proteins bound to the beads were separated by SDS–PAGE and detected by autoradiography. As shown in Fig. 5(a), Os;CycH;1 was preferentially retained on the GST-R2 resin, while the other CDKs, as well as GST, hardly retained the Os;CycH;1 protein. These results suggest that Os;CycH;1 specifically interacts with R2 but not with the other CDKs.

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Figure 5. Os;CycH;1 specifically binds to R2.

(a) Os;CycH;1 interacts with R2 but not with other rice CDKs. [35S]methionine-labeled Os;CycH;1 was translated in vitro and incubated with GST; GST-Cdc2Os1 (Os1); GST-Cdc2Os2 (Os2); GST-Cdc2Os3 (Os3); and GST-R2 (R2) which were immobilized on glutathione-Sepharose beads. Proteins bound to the beads were separated by SDS–PAGE and autoradiographed. Half volume of the input of the translation product is included as a control (Input).

(b) R2 interacts with C-type and H-type but not with mitotic cyclins. [35S]methionine-labeled Os;CycA1;1 (A1); Os;CycB2;2 (B2); Os;CycC;1 (C); and Os;CycH;1 (H) were translated in vitro and incubated with GST or GST-R2 immobilized on glutathione-Sepharose beads. Proteins bound to the beads were separated by SDS–PAGE and detected by autoradiography.

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In rice plants, four cyclin cDNAs have been reported ( Sauter et al. 1995 ; Umeda et al. 1999 ), and they have been classified into three groups based on similarity of amino-acid sequences: A1-type (Os;cycA1;1); B2-type (Os;cycB2;1 and Os;cycB2;2); and C-type (Os;cycC;1). To examine the exact type of cyclin that binds to R2, we translated cyclin proteins in vitro as described above and incubated with GST or GST-R2 resins. As shown in Fig. 5(b), Os;CycA1;1 and Os;CycB2;2 proteins did not specifically bind to the GST-R2 resin. In contrast, Os;CycH;1 was retained on the GST-R2 resin, while a trace amount of Os;CycH;1 was retained on the GST resin ( Fig. 5b). Interestingly, Os;CycC;1 also bound to the GST-R2 resin with an affinity similar to Os;CycH;1 ( Fig. 5b). These findings indicate that R2 binds not only to cyclin H but also to cyclin C.

Os;CycH;1 enhances the kinase activities of R2 in vitro

We have previously shown that R2 phosphorylates rice Cdc2Os1, human CDK2 and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II of Arabidopsis in vitro ( Yamaguchi et al. 1998 ). Therefore we tested whether Os;CycH;1 enhances the kinase activities of R2 on these substrates. FLAG-tagged Os;CycH;1 (FLAG-CycH) was purified from baculovirus-infected insect cells and incubated with GST-R2 protein, followed by precipitation with glutathione-Sepharose beads. We confirmed that FLAG-CycH was co-precipitated with GST-R2 but not with GST alone ( Fig. 6a). We then subjected the precipitates to kinase assays using GST-fused human CDK2 and rice Cdc2Os1 as substrates ( Fig. 6b). When GST-R2 alone was included in the reaction, CDK2 and Cdc2Os1 were slightly phosphorylated. However, the kinase activity of GST-R2 was significantly stimulated by preincubation with FLAG-CycH. When the threonine residue within the T-loop region of CDK2 (T160) and Cdc2Os1 (T161) was substituted by alanine, they were not phosphorylated by the precipitates of GST-R2 and FLAG-CycH, indicating that this phosphorylation is specific to the threonine residue. Incubation of the glutathione-Sepharose beads with a mixture of GST and FLAG-CycH did not result in phosphorylation of both substrates. We then used the GST-fused Arabidopsis CTD as a substrate. Phosphorylation activity of GST-R2 was elevated in the presence of FLAG-CycH, while GST-R2 alone had only low activity ( Fig. 6c). The slowly migrating band in the precipitates of GST-R2 and FLAG-CycH represents autophosphorylation of GST-R2 ( Fig. 6c; data not shown). These results indicate that Os;CycH;1 positively regulates the kinase activities of R2 on both CDKs and CTD.

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Figure 6. Os;CycH;1 stimulates the kinase activities of R2 on CDKs and CTD.

(a) A mixture of FLAG-tagged Os;CycH;1 and GST (lane 2), or a mixture of FLAG-tagged Os;CycH;1 and GST-R2 (lane 3) was incubated with glutathione-Sepharose beads, and proteins bound to the beads were immunoblotted with anti-FLAG antibody. As a control, FLAG-tagged Os;CycH;1 protein was included (lane 1).

(b) GST-R2 and/or FLAG-tagged Os;CycH;1 (FLAG-CycH) were preincubated and mixed with glutathione-Sepharose beads. As a control, GST was incubated with FLAG-CycH. Proteins bound to the beads were assayed for CDK2- and Cdc2Os1-kinase activities by using GST-fusion proteins as substrate (WT). The substrates with substitutions at the threonine residue within the T-loop to alanine were represented as T160A for CDK2 and T161A for Cdc2Os1, respectively. Bands corresponding to GST-CDK2 and GST-Cdc2Os1 are indicated.

(c) GST-R2 and/or FLAG-tagged Os; CycH; 1 (FLAG-CycH) were preincubated and mixed with glutathione-Sepharose beads. As a control, GST was incubated with FLAG-CycH. Proteins bound to the beads were assayed for CTD-kinase activity using GST-CTD as substrate. Autophosphorylated protein of GST-R2 is also indicated.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant cyclins are classified into either mitotic cyclins (A- and B-type) or D-type cyclins ( Mironov et al. 1999 ; Renaudin et al. 1996 ). In this study we isolated a third class of cyclins from poplar and rice plants. Their deduced amino-acid sequences had high similarity to that of vertebrate cyclin H, especially in the cyclin box region. Poplar and rice cyclin H was expressed in all tissues examined in this study, although their transcripts were abundant in suspension cells and tissues with cell division activity. Moreover, transcripts of Os;cycH;1 were most abundant in the meristematic region of the growing rice internode, but were also found at lower levels in elongating and differentiated cells in deepwater rice. This is in contrast to mitotic cyclin genes whose expression is restricted to dividing cells ( Umeda et al. 1999 ). This supports the notion that cyclin H is involved not only in cell division, but also in transcription, as discussed below.

In the growing internode, Os;cycH;1 transcripts clearly increased at 6 h after submergence and then moderately decreased. Lorbiecke & Sauter, (1998) previously demonstrated that at 4 h after submergence more cells in the meristem began to replicate, and at 6 h the population of S-phase cells was at its peak with a threefold increase over uninduced plants. Between 6 and 18 h submergence, the number of cells in S phase leveled out to approximately twice the number found at 0 h ( Lorbiecke & Sauter, 1998). Therefore the kinetic pattern of Os;cycH;1 expression was similar to the population of S-phase cells in the meristem. Moreover, in partially synchronized suspension cells of Os;cycH;1, transcripts were abundant in S phase. These data suggest that Os;cycH;1 expression was induced when cells entered S phase at an elevated level, and that CAK activity was required for S-phase progression.

Using the yeast two-hybrid system and in vitro pull-down assay, we demonstrated that Os;CycH;1 interacted with R2 but not with the other rice CDKs. Animal cyclin H physically interacts with CDK7/p40MO15 ( Fisher & Morgan, 1994; Mäkeläet al. 1994 ), and S. pombe Mcs2 binds to the CDK7 homolog Mcs6/Crk1/Mop1 in vitro ( Buck et al. 1995 ; Damagnez et al. 1995 ). R2 is closely related to CDK7/p40MO15 of animals and Mcs2/Crk1/Mop1 of fission yeast ( Yamaguchi et al. 1998 ); thus Os;CycH;1 may be a specific partner of the catalytic subunit of CDK-activating kinase in rice plants. We also demonstrated that the kinase activities of R2 in the presence of Os;CycH;1 were much higher than those in the absence of Os;CycH;1, and that overexpression of Os;cycH;1 elevated the suppression activity of R2 in budding yeast CAK mutant. Human cyclin H also stimulated the CAK activity of CDK7/p40MO15in vitro ( Fisher & Morgan, 1994), indicating that R2 is functional in combination with cyclin H in a manner similar to animal CAKs.

In plants, little information is available about which cyclin binds to which CDK to form an active kinase complex. De Veylder et al. (1997 , 1999) showed that two Arabidopsis D-type cyclins, At;CycD1;1 and At;CycD4;1, were capable of interacting with Cdc2aAt and Cdc2bAt in the yeast two-hybrid system. Recently, Nakagami et al. (1999) demonstrated that immunoprecipitates of tobacco extract with anti-Nt;CycD3;1 antibody contained a protein that cross-reacted with the anti-PSTAIRE antibody, and that the Cdc2Nt1–CycD3 complex was able to phosphorylate Rb-related protein in vitro, while Cdc2Nt1 alone did not exhibit phosphorylation activity. However, whether Nt;CycD3;1 specifically activates Cdc2Nt1 in tobacco remains to be determined. Therefore rice cyclin H would be the first plant cyclin whose specific partner has been characterized.

In the present study, we showed that rice R2 protein binds not only to Os;CycH;1, but also to Os;CycC;1. In contrast, human cyclin C could not interact with CDK7/p40MO15 in the yeast two-hybrid system ( Mäkeläet al. 1994 ). Cyclin C is known to interact with CDK8 ( Tassan et al. 1995b ) and activates its phosphorylation activity on CTD ( Rickert et al. 1996 ), although there has been no direct evidence supporting the crucial role of cyclin C. One of the Alfalfa CDKs, cdc2MsE, had an amino-acid sequence closely related to CDK8 ( Magyar et al. 1997 ). Therefore plant C-type cyclins may interact with homologs of both CDK7 and CDK8 to exert a diverse function in cell division and transcription.

We have recently identified an Arabidopsis cDNA, named cak1At, as a suppresser of temperature-sensitive cak mutant of budding yeast ( Umeda et al. 1998 ). However, Cak1At had low similarity to vertebrate-type CAKs and phosphorylated human CDK2, but not Arabidopsis CTD ( Umeda et al. 1998 ). Cak1At forms a protein complex in vivo, but is unable to interact with poplar or rice cyclin H in the yeast two-hybrid system (M. Umeda, S. Matsubayashi and H. Uchimiya, unpublished results), suggesting that the kinase activity of Cak1At is regulated in a different manner from R2. More recently, we have isolated an Arabidopsis cDNA which encodes a putative protein closely related to R2 (M. Umeda, S. Matsubayashi and H. Uchimiya, unpublished results). Therefore plants appear to have at least two CAK homologs with distinct functions. An interesting question is whether Cak1At interacts with a cyclin subunit to form an active CAK complex. Identification of the subunit should facilitate the search for specific substrates to each CAK protein, and allow the identification of molecular mechanisms underlying post-translational regulation of CDKs that may be involved in the activation process of cell division.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Isolation of cDNAs encoding cyclin H homologs

Poplar cDNA (Pt;cycH;1) was identified from EST clones prepared from the cambial region of poplar (Populus tremula L.× tremuloides) ( Sterky et al. 1998 ). For isolation of the rice clone, a cDNA library was constructed from 4-day-old suspension-cultured cells of rice (Oryza sativa L. var. Yamahoushi) as described by Umeda et al. (1994) . The cDNA fragment corresponding to the ORF of Pt;cycH;1 was labeled with fluorescein using a Gene images random prime labeling module kit (Amersham, Arlington Heights, IL, USA), and used as a probe. Plaque hybridization was conducted as described by Hihara et al. (1996) . The isolated cDNAs were subcloned into pBluescript II SK (Stratagene, La Jolla, CA, USA) by the in vivo excision method, followed by determination of the nucleotide sequences.

Genomic Southern hybridization

Genomic DNAs prepared from poplar and rice plants were digested with diverse restriction enzymes, electrophoresed on agarose gels, and transferred to Hybond-N+ membranes (Amersham) according to the manufacturer's instructions. For hybridization, cDNA fragments corresponding to the full length of Pt;cycH;1 and the ORF of Os;cycH;1 were used as probes. Hybridization was conducted as described by Ueda et al. (1996) .

Expression analyses

Extraction of poly(A)+ mRNA from the cambial region of a hybrid aspen stem and subsequent PCR amplification of cDNAs are described by Hertzberg & Olsson 1998) . Approximately 500 ng of cDNAs were dotted onto a Hybond N nylon membrane (Amersham) and hybridized with radiolabeled full-length cDNA of Pt;cycH;1 as probe. Polyubiquitin was used as a control. Total RNA (20 μg) isolated from diverse tissues of rice was electrophoresed on 1.85% formaldehyde/1.2% agarose gels and transferred to a Hybond-N+ membrane.

Seeds of O. sativa cv. Pin Gaew 56 were originally obtained from the International Rice Research Institute, Los Baños, Philippines. Rice plants were grown as described by Sauter (1997). For growth induction, plants were submerged in a 600 l plastic tank filled with tap water as described, leaving approximately 30 cm of the leaf tips above the water surface ( Lorbiecke & Sauter, 1998). At the times indicated, the meristematic tissue was isolated from 0 to 5 mm above the second highest node. Tissue from the elongating zone was harvested between 5 and 15 mm above the second highest node, and differentiated tissue was harvested from the oldest portion of the internode just below the first node. For hybridization, the cDNA fragment corresponding to the ORF of Os;cycH;1 or R2 was used as a probe.

Synchronization of suspension cells and flow cytometric analysis were performed with modifications as described by Sauter (1997). Suspension cells from rice (cv. IR43) were subcultured weekly. Three days after subculture, cells were treated with 20 μg ml−1 aphidicolin to inhibit DNA replication ( Levenson & Hamlin, 1993). After 9 h, another 20 μg ml−1 aphidicolin were added to the cells. After 18 h in aphidicolin, cells were washed three times in culture medium and resuspended in an equal volume of medium. At the times indicated, cells were harvested and either used for protoplast isolation or frozen immediately in liquid nitrogen for RNA isolation.

Yeast two-hybrid assay and complementation test

The ORF of Os;cycH;1 was amplified by PCR with primers 5′-GGCTCGAGGATGGCGGATTTCCGGACC-3′ and 5′-GGCTCGAG-CTAGCTGTCAAGTTGGGC-3′ which included the recognition sequence for XhoI at the amino-terminal and carboxy-terminal ends. After digestion with XhoI, the amplified fragment was ligated to the XhoI site of pBluescript II SK (Stratagene) to produce pBSII–OscycH, and its nucleotide sequence was confirmed.

For the yeast two-hybrid assay, pBSII–R2 ( Yamaguchi et al. 1998 ) and pBSII–OscycH were digested with EcoRI and XhoI, respectively, and their insert fragments were subcloned into the EcoRI site of pAS2-1 (Clontech, Palo Alto, CA, USA) which contains the GAL4 DNA-binding domain (amino acids 1–147) and a selectable marker TRP1, or into the XhoI site of pGAD-GL (Clontech), which contains the GAL4-transactivating domain (amino acids 768–881) and a selectable marker LEU2, respectively. The resultant plasmids pAS2-R2 and pGAD-cycH, and each vector DNA, were introduced into S. cerevisiae strain Y190 ( Harper et al. 1993 ) by the lithium acetate method ( Gietz et al. 1992 ), and the transformants were incubated on minimal medium lacking tryptophan and leucine or lacking tryptophan, leucine and histidine for 3 days. For the His3 assay, 40 m m 3-aminotriazole was added to the minimal medium lacking tryptophan, leucine and histidine. β-galactosidase assay was performed according to the protocol provided by the manufacturer (Clontech). pVA3-1 ( Iwabuchi et al. 1993 ), which contains the murine p53 fused to GAL4 DNA-binding domain, and pTD1-1 ( Li & Fields, 1993), which contains the SV40 large T-antigen cDNA fused to the GAL4 transactivating domain, were used as positive controls.

pYES2-R2 ( Yamaguchi et al. 1998 ), pGAD-cycH and each vector DNA were introduced into the budding yeast CAK-deficient mutant GF2351 (MATa, civ1-4, ura3, leu2, trp1, lyd2, ade2, ade3) ( Thuret et al. 1996 ). Transformants were incubated on MVD or MVGS at 27, 34 or 36°C for 5 days.

Expression and purification of recombinant proteins

Fusion proteins GST-CDK2 (with the K33R mutation), GST-CDK2 (with the T160A mutation) and GST-CTD were expressed and purified as described by Umeda et al. (1998) , and GST-Cdc2Os1, GST-Cdc2Os2, GST-Cdc2Os3, GST-R2, and GST-Cdc2Os1 (with the T161A mutation) were prepared as described previously ( Yamaguchi et al. 1998 ). The plasmid for expression in baculovirus-infected insect cells was constructed as follows. The DNA fragment encoding FLAG was amplified from the pFLAG1 vector (Eastman Kodak, Rochester, NY, USA) by PCR with the primers, which included the XbaI site at the amino-terminal end, and a series of XhoI, EcoRI and XbaI sites at the carboxy-terminal end, respectively. The PCR product was then digested with XbaI and ligated to the XbaI site of pFAST-BAC1 (Gibco BRL) to produce pFAST-BAC-FLAG1. pBSII–OscycH was digested with XhoI, and the insert fragment was ligated to the XhoI site of pFAST-BAC1-FLAG1 to be in-frame with the FLAG sequence. The resultant plasmid, pFAST-BAC-FLAG1-OsCycH, was transformed into E. coli strain DH5aBac (Gibco BRL, Gaithersburg, MD, USA) for transposition into the bacmid. The recombinant bacmid was then isolated and transfected into Sf9 cells by using the liposome-mediated transfection kit (Gibco BRL) according to the protocol recommended by the manufacturer. To purify the FLAG-tagged Os;CycH;1 protein, total protein was extracted from approximately 1.2 × 107 infected Sf9 cells by sonication in reaction buffer (10 m m Tris–HCl pH 8.0, 150 m m NaCl, 0.5 m m Na3VO4, 1 m m NaF, and 10 m mβ-glycerophosphate), and then incubated with 300 μl bed volume of anti-FLAG M2 affinity gel (Sigma, St Louis, MO, USA). After washing the gel with reaction buffer, bound proteins were eluted with 1 ml elution buffer (100 m m glycine–HCl pH 2.5 and 0.1% v/v Triton-X), followed by neutralization with 50 μl 1 m Trisaminomethane.

In vitro binding and kinase assays

Rice cyclin cDNAs subcloned into pBluescript II SK vector were used for in vitro transcription and translation with TNT Coupled Reticulocyte Systems (Promega, Madison, WI, USA) containing [35S]methionine according to the manufacturer's recommended method. Then 10 μg GST or GST-CDK proteins were immobilized with 15 μl of glutathione-Sepharose in 200 μl extraction buffer ( Yamaguchi et al. 1998 ). The affinity resin was incubated with 10 μl translated product in 100 μl extraction buffer for 90 min at 4°C. The resin was then washed five times with extraction buffer, and proteins retained on the resin were separated by SDS–PAGE.

For the kinase assay, 50 ng of GST or GST-R2 was incubated with 15 μl glutathione-Sepharose and 5 μl purified FLAG-Os;CycH;1 in 200 μl extraction buffer for 2 h at 4°C. Then the resin was washed three times with bead buffer (50 m m Tris–HCl pH 7.5, 5 m m NaF, 250 m m NaCl, 0.1% v/v NP-40, 0.1 m m Na3VO4, 5 m m EDTA, 5 m m EGTA) and once with kinase buffer (25 m m Hepes–NaOH pH 7.5, 10 m m magnesium acetate), and assayed for CDK and CTD kinase activities using 0.1 mg ml−1 GST-CDK2, GST-Cdc2Os1 or GST-CTD as substrates, as described by Poon et al. (1993) . To confirm the interaction of GST-R2 and FLAG-Os;CycH;1, 300 μg GST or GST-R2 was mixed with 30 μl FLAG-Os;CycH;1 and 15 μl glutathione-Sepharose for 2 h at 4°C. Immunoblotting was conducted as described previously ( Yamaguchi et al. 1998 ). FLAG-Os;CycH;1 protein was detected by anti-FLAG M2 antibody (Sigma).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We appreciate Dr Carl Mann for providing GF2351 cells. We also thank Dr Masami Sekine and Dr Hirofumi Nakagami for technical advice on the baculovirus expression system. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (Grant No. 10182102) from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project MA2202). The work in the lab of Rishikesh P. Bhalerao was funded by a grant from NFR. M.Y. was supported by a Research Fellowship (No. 08639) from the Japan Society for the Promotion of Science for Young Scientists.

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  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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Footnotes
  1. DDBJ/EMBL/GenBank accession numbers AF092743(Pt;cycH;1) and AB038234(Os;cycH;1).