JXB Advance Access originally published online on November 1, 2005
Journal of Experimental Botany 2005 56(422):3183-3192; doi:10.1093/jxb/eri315
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RESEARCH PAPER |
Differential expression of genes encoding protein kinase CK2 subunits in the plant cell cycle
1Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad Autónoma de Barcelona, E-08193 Bellaterra (Barcelona), Spain
2Hospital de Tarrasa, Laboratorio de Biología y Genética Molecular, Carretera de Torrebonica s/n, E-08227 Tarrasa (Barcelona), Spain
* To whom correspondence should be addressed. Fax: +34 93 5811264. E-mail: carmen.martinez{at}uab.es
Received 8 June 2005; Accepted 12 September 2005
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Abstract |
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Protein kinase CK2 is a ubiquitous Ser/Thr/Tyr kinase essential for cell viability in eukaryotes. It comprises
catalytic and ß regulatory subunits, which combine to form the classical tetrameric structure, 2ß2. Although CK2 is a component of the network that controls the eukaryotic cell cycle, very little is known about the expression patterns of genes encoding its constituent subunits, especially in plants. A study of the complexity of CK2- and CK2ß-encoding genes in BY-2 cells was undertaken in this work, and cloning of the different members of the gene families was performed. The expression of the individual members of each family in relation to cell proliferation was measured by real time RT-PCR. The data obtained provide an accurate understanding of the transcriptional regulation of CK2 in relation to the cell cycle and cell proliferation.
Key words:
CK2 subunit, CK2ß subunit, plant cell cycle, protein kinase CK2, tobacco BY-2 cells, transcriptional regulation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Protein kinase CK2 is a ubiquitous Ser/Thr/Tyr kinase with multiple substrates (Allende and Allende, 1995
). Studies in yeast and slime mould have demonstrated that CK2 is essential for cell viability (Kikkawa et al., 1992; Glover, 1998). CK2 activity increases with proliferation rate (Carroll and Marshak, 1989; Issinger, 1993; Espunya et al., 1999) and its mis-expression is implicated in cell transformation and cancer (Guerra and Issinger, 1999; Litchfield, 2003). CK2 activity is required at multiple transitions in the cell cycle, including G0/G1, G1/S, and G2/M (Pepperkok et al., 1994; Hanna et al., 1995; Espunya et al., 1999). Furthermore, CK2 is involved in signal transduction related to DNA damage and other stresses (Toczyski et al., 1997; Ghavidel and Schultz, 2001). The enzyme is composed of two different subunits, showing a typical tetrameric structure, 2ß2, where is the catalytic subunit, and ß the regulatory subunit. The regulatory subunit acts by modulating both substrate specificity and response to different effectors of the holoenzyme. However, individual CK2 and CK2ß subunits are associated with other different polypeptides (Boldyreff and Issinger, 1997; Chen et al., 1997; Hériché et al., 1997; Willert et al., 1997), suggesting roles in the cell other than being subunits of the tetrameric CK2.
Although much is known about CK2 in animals and yeast, less is known about CK2 from plants. Molecular cloning has confirmed the existence of catalytic and regulatory subunit homologues in plants (Dobrowolska et al., 1991; Mizoguchi et al., 1993; Collinge and Walker, 1994; Sugano et al., 1998; Peracchia et al., 1999; Riera et al., 2001), revealing that both and ß subunits belong to multigene families. A comparison of the amino acid sequences shows a high degree of conservation among species, supporting the essential role of CK2 in cellular processes. However, important differences exist between CK2 in animals and plants. For example, plant monomeric forms, composed only of catalytic subunits, are relatively common in plant species and coexist with higher molecular weight forms (presumably tetrameric, according to its molecular size) (Dobrowolska et al., 1992; Klimczak and Cashmore, 1994; Espunya and Martínez, 1997). Moreover, CK2ß subunits from plants show a unique structural feature consisting of an N-terminal extension in their polypeptide chain with 
80 additional amino acids (Collinge and Walker, 1994; Martínez et al., 2001).
Previous studies have demonstrated that cyclic changes of CK2 activity occur during the plant cell cycle, reaching maximum values at the G1/S and M phases (Espunya et al., 1999). In those experiments, mRNA and protein levels of both CK2 and CK2ß subunits were also measured by northern and western blots, respectively, and significant changes were not observed, as opposed to the enzymatic activity results. However, only partial cDNAs encoding tobacco CK2 and CK2ß subunits were available, and they were used as probes in northern blots. Consequently, it was not possible to discriminate between the expression of each particular member of the multigenes families. To obviate this, a study of the complexity of CK2- and CK2ß-encoding genes in BY2 cells was undertaken, and cloning of the different members of the gene families was performed. The expression of the individual members of each family was measured by real time RT-PCR. The data obtained provide an accurate understanding of the transcriptional regulation of CK2 in relation to the cell cycle and cell proliferation.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cloning of tobacco CK2 homologues
Partial sequences encoding tobacco CK2
and CK2ß subunits (Espunya et al., 1999) were used as probes for the screening of a BY-2 cDNA library constructed in the vector 
ZAPII (a gift from Dr N Chaubet-Gigot, CNRS, Toulouse, France). Filters were prehybridized at 37 °C overnight in 6x SSC, 0.5% (w/v) SDS, 50% (v/v) formamide, 5x Denhardt's reagent, and 100 µg ml–1 salmon sperm DNA. Hybridization was performed in the same buffer at 37 °C with the 32P-labelled probe (2x107 cpm). The final washes were done at 0.1x SSC, 0.1% SDS, at 50 °C.
Southern blots
Genomic DNA was prepared from BY-2 cells by the CTAB DNA isolation method (Murray and Thompson, 1980). Ten µg of restricted DNA was electrophoresed for 18 h at 20 mV in 0.8% (w/v) agarose gels and blotted to uncharged Nylon membranes (NytranR R, Schleicher & Schuell, Germany). The DNA gel blots were hybridized at 47 °C in 5x SSC, 50% (v/v) formamide, 0.1% N-lauryl sarcosine (w/v), 0.02% (w/v) SDS, and 2% (w/v) blocking agent (Roche Diagnostics, Indianapolis, USA) and subsequently washed in 2x SSC for 10 min at room temperature and in 0.5x SSC and 0.1% (w/v) SDS for 30 min at 57 °C. The probes were synthesized using the DIG-RNA labelling mix (Roche Diagnostics) and the hybridized bands were detected using the Immun-StarTM substrate (Bio-Rad Laboratories Inc, Hercules, USA) and visualized by exposure of film for 3–5 min.
Cells and cell-cycle synchronization
The tobacco BY-2 cell line was cultivated and synchronized as described in Nagata et al. (1992). Synchronization was achieved by a 24 h subculture of stationary phase cells (7-d-old) in a medium containing 3 µg ml–1 aphidicolin (Sigma Aldrich Co, St Louis, Missouri, USA) followed by extensive washes. The mitotic index was determined by staining with 4',6-diamino-2-phenylindole (DAPI) (10 µg ml–1) (Sigma) in the presence of 1% (v/v) Triton X-100, and DNA synthesis by pulse labelling with [3H]-thymidine (Amersham Biosciences, England).
Isolation of total RNA and real-time quantitative RT-PCR
Total RNA was isolated from BY-2 cells as described in Logemann et al. (1987). RNA samples were incubated with RNase-free DNase I (Roche Diagnostics) for 20 min at 37 °C and RNA concentration was determined at A260. Analysis of expression of CK2- and CK2ß-encoding genes was performed by real-time PCR using a LightCycler Instrument (Roche Diagnostics) and SYBR Green I as a fluorescence dye. The cDNA strand was synthesized from DNase I-treated RNA using a random-primed method and SuperScriptTM II RNase H– reverse transcriptase at conditions recommended by the manufacturer (Invitrogen, USA). Primer pairs for each gene were designed to amplify fragments of 150–300 bp. Due to the high homology between the two CK2ß-encoding cDNAs, specific primers for each gene could not be found and the following strategy was used: three different primers were designed, two of them (C3 and C5) amplified the two CK2ß-encoding cDNAs, and the third one (NC5), combined with the C3 primer, was specific for the CK2B1 cDNA. In this way, mRNA levels for CK2B1 were obtained by using the NC5+C3 primers and those for CK2B2 were obtained by subtraction of CK2B1 levels from the product obtained with the C3+C5 primers. All the results were normalized by using tobacco 18S rRNA as an internal standard, because its expression is constitutive during the cell cycle (Okamura et al., 2003; Kim et al., 2003). The PCR-reactions were performed in 20 µl using the FastStart DNA Master SYBR Green I mix (Roche Diagnostic) according to the manufacturer's recommendations. After heat activation of the polymerase at 95 °C, 45 cycles of denaturation (95 °C, 10 s), annealing (60–67 °C, depending on the gene, 10 s) and amplification (72 °C, 10 s) were performed. During each amplification step, the PCR products were quantified. Melting curves for each PCR reaction were determined by measuring the decrease of fluorescence with increasing temperature (from 75 °C to 95 °C). The specificity of the PCR reactions was confirmed by melting curve analysis using software version 3.5 (Roche) as well as by agarose gel electrophoresis of the products. Plasmids containing each of the cDNAs were used in a standard curve assay and the transcripts were normalized as copy number per nanogram of total RNA.
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Results |
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Cloning of CK2
- and CK2ß-encoding cDNAs from tobacco BY-2 cellsThe isolation of partial clones encoding tobacco CK2
and CK2ß homologues was previously described (Espunya et al., 1999). These partial clones have been used as probes to screen a cDNA library generated from exponentially growing BY-2 cells (a gift from Dr N Chaubet-Gigot). With each probe, several positive clones were identified from 106 plaques screened. Analysis of the isolated sequences identified two different cDNAs coding for CK2 homologues (CK2A1 and CK2A2, accession numbers AJ437635 and AJ438264, respectively). The deduced amino acid sequences show that the corresponding proteins are 333 amino acids long, have a molecular mass of 39.4 kDa and share 98.5% sequence identity (Fig. 1A; Table 1A). A clone with more than 90% identity to CK2A2 in its 5'- and 3'-untranslated regions (UTRs) and one change in the deduced amino acid sequence was also isolated (CK2A3, accession number AJ438263). CK2A2 and CK2A3 are ascribed allelic variants of the same protein, since N. tabacum is an amphidiploid genome resulting from the combination of the genomes of Nicotiana sylvestris and Nicotiana tomentosiformis.
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Two different cDNAs coding for tobacco CK2ß homologues (CK2B1 and CK2B2, accession numbers AJ438265 and AJ488194, respectively) have been characterized. The deduced amino acid sequence revealed that CK2B1 is a protein of 275 amino acids with a molecular mass of 30.8 kDa. CK2B2 is a larger polypeptide, 280 amino acids and a molecular mass of 31.7 kDa, due to two small insertions (57HQH and 146GDMFT). The protein products show 86.6% sequence identity (Fig. 1B; Table 1B). In addition, several positive clones that appeared to code for variants of CK2B1 were isolated. The variants differ either in the number of triplets coding for Gly residues of the two Gly repeats present in the coding region of the CK2ß subunits, or differ in the 3'-UTR region (the characteristics of the Gly repeats are detailed below). The differences found in the 3'-UTR of the CK2B1 variants consisted mainly of different polyA attachment sites that resulted in different lengths of the 3'-UTR of the transcripts. One of the clones also presented an internal deletion of 180 nucleotides within the 3'-UTR (data not shown).
Southern analysis of genomic DNA using CK2A2 and CK2B1 sequences as probes is shown in Fig. 2. The patterns obtained with different restriction enzymes are compatible with the existence of only two genes coding for the CK2 subunit and two genes coding for the CK2ß subunit in tobacco BY-2 cells.
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Sequence analysis
The identity values, at the amino acid level, for the tobacco CK2
and CK2ß homologues are compared with CK2 sequences from other species (Table 1A, B). For CK2 polypeptides, identities are >90% among the plant forms and 
50% when compared to those from animals and yeast. The amino acid sequences of CK2A1 and CK2A2 contain every structural determinant previously described for CK2 subunits (data not shown). CK2ß homologues show identity values of 70% within the plant species and <40% when compared with those from animals and yeast. A striking exception is the high homology found between human and Drosophila sequences, particularly in the case of Drosophila CKB1 that shows 87.9% identity with the human sequence. One peculiarity of the plant CK2ß sequences is the presence of an extra N-terminal extension of 80–90 amino acids (Collinge and Walker, 1994; Martínez et al., 2001) not present in other species. This is the main reason for the low identity values obtained when the plant CK2ß sequences were compared with those of others species. This N-terminal region does not share homology to other known proteins. Alignment of the N-terminal regions from different plant sequences reveals different conserved motifs never described. There are two Gly-repeats of variable size (Fig. 1C). The first Gly repeat (residues 6–13 and 6–8 in CK2B1 and CK2B2, respectively) is found in the two tobacco sequences reported here, and in some of the CK2ß subunits from Arabidopsis (CK2B3), Zea mays (CK2B3), and rice (CK2B1 and CK2B3), whereas other isolated clones from different species show just one triplet coding for Gly at that position. The second Gly cluster is only present in tobacco CK2B1 and rice CK2B3 (residues 52–56 and 57–59, respectively, Fig. 1C), and it is not found in any other species. The motif 16DRKRI20 (Motif A, Fig. 1C)) located just downstream of the first Gly cluster described above, is perfectly conserved in all sequences. Moreover, further along is a region rich in Ser and acid residues that fulfil the consensus requirements for CK2 phophorylation (S/T-X-X-D/E); it also contains the conserved Motif B, DSEXSD/EVSG (underlined residue is the most frequently found in that position), with two Ser residues that fulfil the requirements for CK2 phosphorylation. The rest of the polypeptide chain of tobacco CK2ß sequences presents each of the major conserved features previously described in other species, such as a cysteine-rich motif involved in the zinc-finger structure, a potential destruction box, and an acidic region that is involved in the interaction with polyamines (Allende and Allende, 1995; Litchfield, 2003) (data not shown). As with other plant sequences, tobacco CK2ß sequences do not contain the p34cdc2 phosphorylation consensus motif that is present at the C-terminal end of the human CK2ß sequence (Allende and Allende, 1995).
The phylogenetic relationship between the CK2 and CK2ß polypeptides from different species is shown in Fig. 2B. The CK2 homologues from plants group together, the Arabidopsis sequences being the closest relatives to the two tobacco sequences. The CK2ß homologues from plants also group together and the closest relative to the two tobacco sequences is the Arabidopsis CKB3 sequence. Remarkably, CK2B1 from Zea mays appears to have a different evolutionary origin from CK2B2 and CK2B3. Conversely, the tree also reflects the close relationship between the human CK2ß sequence and the Drosophila CK2B1 (87.9% identity). The only protein in the databases that shows homology to the CK2ß subunits is the product of the Stellate locus in Drosophila (Livak, 1990). However, it is surprising that the product of the Stellate locus appears more closely related to S. cerevisiae CK2ß than to Drosophila CK2ß.
Expression pattern of CK2- and CK2ß-encoding genes during the cell cycle
Cyclic changes of CK2 activity occur during the cell cycle in BY-2 cells, reaching maximum values at the G1/S and M phases (Espunya et al., 1999). Until now, the lack of specific probes has prevented a deeper insight into the transcriptional regulation of CK2 genes that code for the two subunits of the enzyme, and ß. Expression analysis by northern blot is hindered by probe specificity when applied to closely related members of a gene family, and is not accurate enough to give quantitative information on mRNA relative levels of each member of the family. An accurate and specific transcript quantification assay based on fluorescent real-time RT-PCR was developed. Specific primers in the 5'-UTR region were designed and tested for each of the two CK2-encoding genes (Table 2). However, primers could not be found to amplify separately each of the two CK2ß-encoding genes, due to the high homology between them. In consequence, CK2B2 mRNA levels were obtained by subtraction of CK2B1 mRNA levels (for which amplification was specific) from the total product of amplification of the two CK2ß-encoding genes (using the common primers C3 and C5). The three primers used to amplify CK2ß cDNAs are located in the coding region (Table 2). Using these molecular tools, the expression of each one of the four CK2 genes during one complete cell division cycle of BY-2 cells synchronized with aphidicolin was quantified. Due to the sensitivity of the method, an accurate measurement of the total RNA levels in each sample is very important. This was accomplished by using 18S rRNA as the internal standard (amplified with specific primers, Table 2), the expression of which is constitutive during the BY-2 cell cycle (Kim et al., 2003; Okamura et al., 2003). Figure 3 shows the mRNA copy number per nanogram of total RNA during one complete cell cycle. CK2A1 and CK2B2 transcripts are dominant, although the four genes show the same pattern of expression, with peaks of transcript levels at M- and at early S-phase. To avoid possible artefacts due to the 24 h incubation with aphidicolin and in order to locate precisely the points of transcriptional activation, mRNA levels of the CK2 genes were quantitatively analysed during the second S-phase of the cell cycle (14–18 h). The quantitative expression of CK2A1 and CK2B2 genes is similar, both in mitosis (4900 and 4700 copies per ng RNA, respectively) and in the S-phase (4450 and 4500 copies per ng RNA, respectively). However, expression of CK2A2 is significantly lower, around 40% of that of CK2A1, both in the M and S-phases, and that of CK2B1 is only 12% of that of CK2B2. Synchronization experiments were performed three times, giving similar results, and only one of them is shown.
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Expression pattern of CK2
- and CK2ß-encoding genes during the BY-2 growth cycleThe growth cycle of BY-2 cells was used to analyse the expression of the CK2
- and CK2ß-encoding genes during the transition from quiescence to the exponential phase of growth. In the stationary phase (day 7 of the growth curve) more than 90% of the BY-2 cells are in G0/G1 (Hemmmerlin and Bach, 1998) and they resume cell division when subcultured in fresh medium, attaining mitotic indices of 5–8% very quickly (Fig. 4A). Samples were collected every 24 h and mRNA levels for each of the four different genes were quantified by real-time RT-PCR using the same conditions as above. Once again, transcripts of CK2A1 and CK2B2 genes were predominant in the growth curve. The mRNA levels increased immediately after transferring the cells to a fresh medium and attained maximal values at day 2. (Fig. 4B). Notably, CK2B2 transcript levels are 3.5-fold those of CK2A1 at day 2, suggesting that CK2ß supply must be in much greater quantities than that of CK2 in the transition from the quiescent to the proliferative state.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Protein kinase CK2 comprises two different subunits, which combine to form an oligomeric structure,
2ß2. In plants, both - and ß-encoding genes belong to multigene families that are more complex than those found in animals. Regulation of CK2 activity is very complex (Allende and Allende, 1995; Litchfield, 2003) and it is also a matter of controversy due to contradictory results depending on the system used for the study. Tobacco BY-2 cells are a suitable system to study cell-cycle-regulated genes in plants because they are highly synchronizable and have a well-characterized growth curve. The present work reports the cloning of different members of CK2- and CK2ß-encoding cDNAs that, according to Southern blot results, belong to multigene families composed of two members. Analysis of the deduced protein sequences reveals that both CK2 and CK2ß subunits contain all the conserved features previously described in their animal counterparts. In addition, the tobacco CK2ß subunits, like other plant homologues, contain an extra N-terminal sequence of around 80 amino acids, not present in animals or yeasts. Alignment of the CK2ß N-terminal region from plants revealed, for the first time, several conserved amino acid stretches that are described in the Results section. From an evolutionary point of view, it is important to note the presence of two hypervariable regions made up of short sequence Gly-repeats with different sizes. In tobacco CK2B1 and CK2B2 genes, the codon composition of these regions is a mixture of pure trinucleotide tracts (GGA) and of mixed tracts (GGA/GGT/GGC) (data not shown, but nucleotide sequences are available in the databases). A high correlation between amino acid repeats and trinucleotide repeats (first case) is consistent with DNA slippage or misalignment during replication, whereas a mixture of codons could be indicative of selection of the homopeptide region (Albà et al., 2001). The sequences of tobacco CK2ß clones suggest a double origin for the Gly-repeats regions: one subset may recently have been originated by slippage, and may therefore be neutral. However, some of the polyglycine segments are likely to have been preserved throughout evolution, giving a significant functional relevance to this region, unique to plant CK2s.
CK2 and CK2ß transcripts accumulate in meristems and other proliferative tissues in plants (Espunya and Martínez, 2003). However, until now nothing was known about the extent of the transcriptional regulation of these genes in relation to cell proliferation. The results reported here clearly show that mRNA levels rise significantly at M-phase and at the onset of S-phase for all four genes, strongly indicating a transcriptional up-regulation of CK2- and CK2ß-encoding genes at particular phases of the cell cycle. Moreover, transcripts corresponding to one CK2-encoding gene (CK2A1) and to one CK2ß-encoding gene (CK2B2) appear to be dominant. CK2A1 transcript levels rises 17-fold in M-phase and 15-fold in the second S-phase, and CK2B2 transcripts increase in a similar way (20-fold in M and 19-fold in S). CK2 mRNA levels also increase during the exponential phase of growth, reaching a maximum at day 2 after subculture and decaying thereafter. Also in this case, a differential expression in quantitative terms of the different members of CK2- and CK2ß-encoding gene families was detected. The most significant increase was obtained for one of the two CK2ß-encoding genes, CK2B2, whose mRNA levels rose 10-fold. The strong activation of one of the two CK2ß-encoding genes suggests an important role of its encoded protein in the transition from quiescence to proliferation. It might reflect the need to supply an excess of the CK2ß subunit to interact with additional partners beside CK2, as part of the requirements to reactivate cell division. In support of this, it must be mentioned that CK2ß interacts with proteins that are functionally related to cell proliferation control (Boldyreff and Issinger, 1997; Chen et al., 1997) and thus it might have some other roles in the cell besides being a subunit of CK2. In exponentially growing cells the subunit is synthesized in excess of the subunit, although a substantial fraction of the newly synthesized protein is rapidly degraded (Lüscher and Litchfield, 1994). Moreover, an asymmetric expression of CK2 subunits was observed in human kidney tumours, i.e. there is an excess of the ß subunit in tumours versus normal tissues when compared to CK2 (Issinger, 1993). The accumulation of CK2ß protein on day 2 of the BY-2 cell growth curve has been demonstrated by this group in previous work (Espunya et al., 1999).
There is a lot of controversy about the regulation of CK2 by growth factors and hormones that stimulate cell proliferation in animal cells. Whereas some authors found a correlation between cell proliferation rates and higher levels of CK2 (Carroll and Marshak, 1989; Bosc et al., 1999), this phenomenon has not been universally observed (Litchfield et al., 1994). In mouse fibroblasts, the two subunits (CK2 and CK2') are differentially regulated in response to serum stimulation, suggesting a specialization of the ' catalytic subunit in relation to the oncogenic process (Orlandini et al., 1998). Although minimal changes in CK2 protein and enzymatic activity have been detected in human cells examined at different stages of the cell cycle (Bosc et al., 1999) other authors report constant CK2 activity values throughout the cycle, but important changes in CK2 intracellular distribution (Wang et al., 2003). The results presented here clearly demonstrate an up-regulation of CK2- and CK2ß-encoding genes upon stimulation of tobacco BY-2 cells to enter cell proliferation, and at particular phases of the cell division cycle. As both CK2 and CK2ß subunits are encoded by multigene families, the use of real-time RT-PCR technique has been crucial to obtain an accurate quantification of the transcripts. The changes in the transcripts levels of CK2- and CK2ß-encoding genes throughout the cell cycle must be, at least partially, responsible for the cyclic changes in CK2 activity previously demonstrated by this group (Espunya et al., 1999). It is worth noting that there is not a strict coincidence between the peak of CK2 activity and that of the transcript levels in S-phase. This might be because: (i) the relevant information at this point can only be obtained from the second S-phase because the aphidicolin blockage is located at the onset of S-phase. As cells go through the cycle, synchronization is being lost, and thus in the second S-phase the synchronization rate is lower than in the M-phase. This can explain the small differences regarding the point where the maximal values for CK2 activity and mRNA levels were obtained, due to the different sensitivity of the two techniques used; or (ii) it is well known that CK2 activity is regulated at several levels, i.e. by interaction with polyamines and/or other proteins, by phosphorylation, by intracellular localization, etc (Litchfield, 2003). So, there is not necessarily a correlation between activity and transcript levels at a particular moment, because the up-regulation of CK2 activity is multifactorial. Furthermore, some of these variables change during the cell cycle, such as the intracellular localization of CK2 (Wang et al., 2003), or the stimulatory effect of polyamines on CK2 activity which is dependent on the cellular compartment (Shore et al., 1997). Nevertheless, it is concluded that it is a transcriptional regulation of CK2 during the cell division cycle, demonstrated by the cyclic accumulations of transcript levels of its constituent's subunits. The cyclic up-regulation of the transcriptional activity of CK2-encoding genes might be necessary to maintain the optimal pool of CK2 subunits that will constitute the oligomeric enzyme, whose enzymatic activity is necessary for cell cycle progression (Espunya et al., 1999).
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Acknowledgements |
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This work was supported by grants from the Dirección General de Enseñanza Superior (BMC2000-0136 and PB96-1167), and the Comissionat per a Universitats i Recerca (2001SGR00198). We thank Dr N Chaubet-Gigot (CNRS, Toulouse, France) for kindly providing the BY-2 cDNA library, and to the Tobacco Science Research Laboratory, Japan Tobacco Inc. for the BY-2 cell suspension. TL-G was the recipient of a fellowship from the Universitat Autónoma de Barcelona. This work was also partially supported by the Acción Integrada HF1997-0238 (DGES).
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