1 To whom correspondence should be addressed.
Phone: (604)822-3369
Fax: (604)822-2416
e-mail: berger@zoology.ubc.ca

Note: The Paramecium CDK and cyclin genes and their protein products in this paper have been named or renamed according to the newly proposed genetic nomenclature rules for ciliates (Allen et al., 1998). Here, CDK1 was referred to as CDC2PTA, the CDK associated with the initiation of macronuclear DNA synthesis, which was originally isolated and named by Tang et al., (1995).

Introduction

Eukaryotic cell cycle events are controlled by oscillation of the activity of a highly conserved family of protein kinases, the cyclin-dependent kinases (CDKs), originally identified as products of the CDC28 gene of Saccharomyces cerevisiae and the cdc2 gene of Schizosaccharmyces pombe (For reviews see Fisher, 1997; Morgan, 1995). CDKs are small protein kinases that contain little more than the conserved catalytic domains found in all eukaryotic protein kinases (Hanks, et al., 1988); in domain III, they all share a sequence related to the canonical EGVPSTAIREISLLKE motif (‘PSTAIRE’ region). In yeast, two different cell cycle state transitions, ‘Start’ (when the cell becomes committed to a new cell cycle) and point of commitment to division (PCD) are controlled by a single CDK (p34^CDC28 in S. cerevisiae and p34^cdc2 in S. pombe) (Nurse and Bissett, 1981; Beach et al., 1982). In higher eukaryotes, these transitions are controlled by multiple CDKs in association with distinct cyclins. So far, at least 7 different CDKs have been reported to be implicated in the cell cycle regulation in vertebrates (Nigg, 1995)

As an important regulator of the eukaryotic cell cycle, CDK activity is tightly controlled by various post-translational modifications which include protein-protein interactions, reversible phosphorylations, transcriptional control and protein degradation. The monomeric CDK is inactive as a protein kinase, and activation requires the binding of cyclin, a family of unstable proteins whose levels oscillate during the cell cycle (For review see Pine 1995). Cyclins are diverse in sequences, and sharing homology only over a region of about 100 amino acids that has been designated the ‘cyclin box’ (Pine and Hunter, 1989; Minshull et al., 1989). The ‘cyclin box’ region has been demonstrated to be involved in binding a CDK by interacting with its ‘PSTAIRE’ region (Kobayshi et al., 1992; Pine and Hunter, 1990). In addition to cyclin binding, full activation of CDK requires phosphorylation on a conserved threonine residue (Thr160 in human CDK2 and Thr161 in human CDK1) by the CDK-Activating Kinase (CAK) (Kaldis et al., 1996; Thuret et al., 1996). The active CDKs can also be inhibited by phosphorylation by other kinases including Wee1 kinase on Tyr15 and Thr14 within the ATP binding domain (Haese et al., 1995) or by the binding of CDK inhibitor (CKIs) (Toyoshima and Hunter, 1994, Serrano et al., 1993).

It has been well established that CDK-cyclin as the central regulator of the eukaryotic cell cycle is evolutionarily conserved in a wide range of eukaryotes from yeasts to humans, many of them can complement similar function in heterologous systems. In the past few years, this universality has been further extended into some lower eukaryotes (John et al., 1989; Michaelis and Weeks, 1992; Lohia and Samuelson, 1993; Ross-MacDonald et al., 1994). Considering the great diversity of their cell cycle organization (Reviewed by Berger, 1989), it would be extremely interesting to find out how this conserved CDK-cyclin motif is played out in lower eukaryotic cell cycles.

Some unique features of the cell cycle regulation in Paramecium tetraurelia revealed by previous physiological studies make it an interesting subject for cell cycle studies. It is a ciliate, a group of diverse unicellular organisms emerging about 10⁹ years ago and separating phyletically from the eukaryotic line that led to higher eukaryotes including plants and animals before fungi appeared. Like most ciliates, Paramecium has two types of functionally distinct nuclei within the same cytoplasm, one vegetative, polygenomic macronucleus and two germ-line, diploid micronuclei. The timing of key cell cycle events, including DNA synthesis and nuclear division, occur differently in these two types of nuclei, even though they reside in the same cytoplasm, and both are derived grom the zygote nucleus formed at fertilization (Rasmussen and Berger, 1982; Rasmussen et al., 1986; Pasternak 1967; Adl and Berger, 1992). Macronuclei divide by amitosis whereas micronuclei divide by ‘closed’ mitosis with a normal spindle. Previous physiological and genetic studies demonstrated the occurrence of a single major control point, the point of commitment to division (PCD) late in the cell cycle, which is around 90 min before cell division. At this point cells become committed to division in the present cell cycle and the nature of the next cell cycle (meiotic or vegetative) is set, and the duration of the next G1 interval is established (reviewed by Berger, 1988). This differs from the typical situation in most eukaryotes, in which there are separate control points that gate cells into the DNA replication at the end of the G1 period, and into division late in the cell cycle. In addition, due to the relatively imprecise nature of the amitotic division process, cells must have the ability to correct both DNA content and gene copy shortfall in daughter macronuclei. The way the cell copes with this situation is believed to synthesize a fixed amount of DNA in each cycle, with less than a doubling for large macronuclei and more than a doubling for smaller ones (Berger, 1979), suggesting the exact doubling of macronuclear DNA can not be a precondition for the cell division in Paramecium. In contrast, checkpoint controls act throughout the higher eukaryotic cell cycle to ensure that DNA is replicated once and only once per cycle (Hayles et al., 1994). Furthermore, it is also of great interest to understand how Paramecium cells to co-ordinate the cell cycle events occurred so differently in two types of nuclei.

To initiate analysis of the molecular machinery underlying the Paramecium cell cycle regulation, a search for CDK/cyclin homologues has been initiated. In previous studies, a p34^cdc2-like protein kinase, gene, Cdk1, was cloned by utilizing a homology-based approach (Tang et al., 1995). It has been demonstrated to be involved in the initiation of macronuclear DNA synthesis (IDS), supported by its histone H1 kinase activity profile and its macronuclear-specific localization (Tang et al., 1997). Western blot analysis with anti-PSTAIRE antibodies suggested the presence of two classes of p34^cdc2-like protein kinases, the major one corresponds to Cdk1 which does not bind to yeast p13^suc1, an established affinity matrix for many CDKs, whereas the minor form is capable of binding to p13^suc1 (Tang et al., 1994).

In this paper, we report the sequence of the gene encoding another p34^cdc2 homologue, Cdk2. Its relative molecular mass is about 35 kD, which migrates at the same migration rate as that of the minor polypeptide recognized by anti-PSTAIRE antibody [ref]. However, its incapability of binding to p13^suc1 distinguishes it from the p13 binding cdk protein previously reported [ref], suggesting that Cdk2 is a novel CDK in Paramecium.

Materials and methods

Cell culture, cell cycle synchronization and preparation of protein lysate. Paramecium tetraurelia wild type stock 51-S was grown at 27° C in phosphate-buffered Cerophyl medium (Pines, Lawence, KS) supplemented with 5 m g/ml stigmasterol, and inoculated with Klebsiella pneumoniae (Sonneborn, 1970). Synchronized cells were prepared by centrifugal elutriation, as described by Tang et al. (1994). Briefly, 1-1.5x10⁷ cells were loaded onto a Beckman elutriator chamber (30 ml) on a JE.5 rotor (Palo Alto, CA) at 550 g and at a pump rate of 75 ml/min. Following a 100 ml wash with fresh medium, the fractions collected by stepwise increases of the pump speed were pooled until the composite contained around 5% of the starting cell population. This synchronous population was re-inoculated into the fresh medium and maintained at 27° C and harvested at one hour intervals starting 30 min post elutriation. After washing twice with PBS, samples processed for protein lysate preparation.

Paramecium cells were lysed in 4 volumes of lysis buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 50 mM NaF, 5 mM EDTA, 1 mM DTT, 0.1% NP-40, supplemented with a cocktail of protease inhibitors including 50 m g/ml PMSF, 2 m g/ml leupeptin, 4 m g/ml aprotinin, and 1 m g/ml pepstatin A). The lysate was incubated on ice for 15 min, and then centrifuged at 18,400 g for 15 min at 4° C. The supernatant was kept at -20° C. An aliquot was taken for protein quantitation according tousing Bradford (1976). Unless other specified, chemicals were purchased from Sigma (St. Louis, MO.)

Isolation of cDNA and genomic sequences of CDK2. Degenerate oligonucleotide primers were designed based on the two conserved regions of known CDK sequences. The sense primer, based on GEGTYG (domain I of protein kinases), was 5’-cgctcgagGGA/TGAA/GGGA/TACA/TTAT/CGG-3’. The antisense primer, based on DLKPQN (domain VI), was 5’-gcaagcttG/ATTT/CTA/GA/TGGC/TTTA/TAA/GG/ATC-3’. The restriction enzyme sites included to facilitate subsequent cloning were indicated in lowercase letters. PCR was carried out at low annealing temperature of 37° C. A product of ~400 bp was amplified, which was then purified with Qiagen Gel Purification Kit (Qiagen, Santa Clarita, CA) and subcloned into the plasmid pBluescript II KS+/- (Stratagene, Cambridge, UK). The resulting plasmids were subjected to DNA sequencing by the dideoxynucleotide chain termination method (Sambrook et al., 1989).

3’ and 5’ RACE (Rapid Amplification of cDNA Ends) (Frohman et al., 1990) were applied to obtain 3’ and 5’ end sequences of CDK2, respectively. For the 3’ end, first strand cDNA was synthesized from the total RNA by an oligpo-dT₁₇ primer, and then was used as template in the PCR reaction primed with oligo-dT₁₇ and a gene specific primer (5’-ctctagaTAGTTAGATAGAATGCAA-3’) derived from the original PCR fragment. For the 5’ end, first strand cDNA was made from the total RNA by a gene-specific primer (5’-ATAGTTTAAGGCTTGTATCAT-3’), and then tailed with dAs with Terminal d Transferase (TdT, Gibco-BRL, Gaithersburg, MD). The resulting cDNA was amplified with oligo-dT₁₇ primer and another gene-specific primer (5’-ctctagaTAATCTTGATTCATCATA-3’).

The complete genomic DNA sequence of CDK2 was isolated by PCR using primer pairs derived from both ends of the cDNA sequence with genomic DNA as template. CDK2 sequence was confirmed by performing DNA sequencing on both strands.

Genomic Southern blot analysis. Thirty micrograms of genomic DNA was digested with five restriction enzymes, EcoRI, HindIII, XbaI, PstI and BamH1 (Gibco-BRL), subjected to electrophoresis on 0.7% agarose gel and blotted onto Hybond-N⁺ membrane (Amersham, Arlington, IL) using downward transferring technique in 0.4 M NaOH as described by Koetsier et al., (1993). The membrane was hybridized with a DNA probe encompassing the full-length CDK2 or CDK1 sequences and labeled with DIG-11-dUTP (Boehringer-Mannheim, Germany) prepared by PCR and detected as described by Engler-Blum et al. (1993). The membrane was washed at 65° C.

Preparation of anti-Cdk2 antiserum. Polyclonal antiserum was raised against synthetic peptide corresponding to residues 240-255 of Cdk2 with additional cysteine at its amino terminus (CDFKSTFPRWPTPTNPA) to facilitate coupling of the peptide to keyhole limpet hemocyanin (KLH). Two hundred fifty micrograms of peptide in RIBI adjuvant was injected into New Zealand White rabbit in the first injection, followed by 200 m g of boosters every 4 weeks.

Immmunological techniques. Immunoblotting of proteins was carried out, according to Harlow & Lane (1988). Briefly, proteins were resolved on 12.5% SDS-polyacrylamide gels, transferred to Immobilon-P membranes (Millipore, Bedford, MA). After transfer, membranes were first incubated with blocking buffer (5% non-fat milk and 0.1% Tween-20 in PBS) for 1 h, and then with the primary antibody (1:500 dilution) for 1 h. After three washes in blocking buffer, primary antibody was revealed by enhanced chemiluminescence (ECL kit, Amersham) using horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (1:4000 dilution).

For immunoprecipitation, 600 m g of total protein lysate was first pre-cleared with 50 m l of protein A-agarose beads (Gibco-BRL) (50% v/v in lysis buffer) for 30 min at 4° C. The supernatant was then incubated with 2 m l of antiserum or corresponding pre-immune serum on ice for 3 h, followed by 1 h of incubation with 50 m l protein A-agarose beads (50% v/v) with constant rotation at 4° C. Beads were pelleted and washed five times in lysis buffer. For immunocomplexes destined for immunoblotting, 20 m l of 2´ Laemmli sample buffer (Laemmli, 1970) was added the beads, and the sample was heated at 100° C for 5 min prior to electrophoresis. For histone H1 kinase assay, the pellets were further washed twice with 1´ kinase assay mix (50 mM Tris, pH7.5, 10 mM MgCl_2, 1 mM DTT, 40 m M ATP). Then, 10 m l of kinase assay cocktail containing 0.2 mg/ml bovine histone H1 (StressGen, Victoria, BC) and 320 pmol (10 m Ci) [g -³²P] ATP (Amersham) was added and the reactions were incubated at 27° C for 20 min. The reactions were terminated by addition of equal volume of 2´ Laemmli sample buffer. Aliquots were separated on SDS-polyacrylamide gel. Phosphorylated histone H1 was detected by autoradiography and quantitated by liquid scintillation counting of excised histone H1 bands.

p13^suc1 binding assay. Yeast p13^suc1 conjugated to CNBr-activated Sepharose CL4B was purchased from the Upstate Biotechnology, Inc.(Lake placid, NY). The concentration of coupled p13 was 3.7 mg/ml. Just before use, 80 m l of p13^suc1 beads (50% suspension) was washed with 1 ml of bead buffer (50 mM Tris pH7,4, 25 mM NaCl, 5 mM NaF, 5 mM EDTA, 5 mM EGTA, 100 mM Benzamidine, 0.1% NP-40) and resuspended in 400 m l bead buffer. The protein lysate (400 m l) was added to the beads and kept under constant rotation at 4° C for 5 h. After a brief centrifugation, the beads were washed five times with 1 ml of bead buffer. The beads were then resuspended in 40 m l of 2´ Laemmli sample buffer and boiled for 3 min before loading onto the gel.

Expression of GST-Cdk2 fusion protein. A Bamh1-EcoRI cDNA fragment containing residues 212 to 301 of CDK2 was subcloned into a pGEX-2T vector (Pharmacia, Uppsala, Sweden) and transformed into DH5a bacteria. The resulting clones were confirmed by DNA sequencing on both strands. Production of recombinant GST-Cdk2 fusion protein was induced by 0.2 mM IPTG. Protein extraction and purification were carried out as described by Smith and Johnson (1988).

Results

Primary sequence predicts that Cdk2 is a cyclin-dependent protein kinase. Two degenerate oligonucleotide primers representing two conserved regions were designed by comparison of the amino acid sequences of the known p34^cdc2 homologues, , and used in PCR with Paramecium genomic DNA as template. The PCR was carried out at low annealing temperature of 37° C in order to pick up p34^cdc2-related genes. On agarose gel, the PCR product was observed as a ~400 bp band, which was in the size range expected for products derived from p34^cdc2 homologues with this primer pair. After subcloning into pBluescript KS+/- and blue/white selection, 15 positive colonies were obtained. Twelve of them contained the sequences corresponding to one or the other of the two isoforms of the Cdk1 gene identified in previous work (Tang et al., 1995). Three others were novel, identical and, contained EGVPSTAIREISSLKE motif characteristic of the p34^cdc2 family, suggesting they might encode a novel p34^cdc2-like protein in Paramecium.

Full-length cDNA sequence for CDK2 was obtained by 3’ and 5’ RACE anchor PCRs (Frohman et al., 1990). Even though there is no in-frame stop codon (TGA) found upstream of the presumptive initiation ATG, the sequence is likely to represent the complete open reading frame (ORF) of Cdk2 by considering that its size is close to that of Cdk1. Moreover, a single 1.2 kb transcript was observed for CDK2 (data not shown), which is comparable to the size of CDK1 transcript (1.3 kb) (Tang et al., 1995).

The putative ORF is predicted to encode a protein of 301 amino acids with all protein kinase catalytic domains, suggesting Cdk2 is a protein kinase, like Cdk1 (Fig. 1). The ‘EGVPSTAIREISSLKE’ (‘PSTAIRE’ region) in Cdk2 matches perfectly with the canonical sequence in p34^cdc2(CDC28) of yeast whereas one amino acid substitution was observed in Cdk1 (valine to isoleucine) (Tang et al., 1995). No putative poly-adenylation signal near the 3’ end was identified, as is the case for most of the Paramecium genes cloned so far [ref]. Cdk2 is 7 amino acid residues shorter than Cdk1. The predicted relative molecular mass of Cdk2 is 35 kD, which is the same as the minor polyepeptide recognized by anti-PSTAIRE antibody on western blots (Tang et al., 1994).

The Paramecium genetic code is different from the universal genetic code used by most organisms (Caron & Meyer, 1985; Preer et al., 1985; Horowitz & Gorovsky, 1985), in that it uses UAA and UAG as glutamine codons intstead of as stop codons. Within CDK2 ORF, there are 10 UAA coded glutamines and 2 UAG coded glutamines, suggesting a strong preference for UAA (Fig. 1). This is in accordance with previous results (Martindale, 1989).

Comparison of CDK2 genomic and cDNA sequences revealed a single intron of 24 bp located just downstream of the ‘PSTAIRE’ region (Fig. 1). Its position does not correspond to that of either of the two introns in CDK1 (Tang et al., 1995). Small introns are very common in Paramecium genome (Russell et al., 1994). The cdk1 intron has consensus 5’/3’ splice sites GT/AG without apparent branch point consensus sequences.

Phosphorylation and dephosphorylation of specific residues regulates the kinase activity of CDKs(Krek & Nigg, 1991,1992; Norbury et al., 1991). Homologues to the regulatory residues, Thr14, Tyr15 and Thr161 in human CDK1 , Thr20, Tyr21 and Thr167, are conserved in Cdk2 --- (Fig. 1). Therefore, Cdk2 has the potential to be regulated in an analogous fashion to human CDK1.

We compared the amino acid sequences of Cdk2 and Cdk1 with p34^cdc2 homologues from humans (Lee & Nurse, 1987), Schizosaccharomyces pombe (Hindley & Phear, 1984), Saccharomyces cerevisiae (Lorinze & Reed, 1984), Zea may (Colasanti et al., 1991), Dictystelium discoideum (Michaelis & Weeks, 1992), Plasmodium falciparum (Ross-MacDonald et al., 1994), Entamoebae histolytica (Lohia & Samuelson, 1993) and Trichomonas vaginalis (Riley, et al., 1993) (Fig. 2). The highest similarity was found with Zea may p34^cdc2 (61%) and the lowest was found with Trichomonas vaginalis (33%). Compared to Cdk1, Cdk2 generally shares slightly higher similarities with p34^cdc2 homologues from higher eukaryotes such as yeasts, Zea may and humans, but lower similarities with those from lower eukaryotes such as Trichomonas and Plasmodium. Whether it implies that Cdk1 is more ancient than Cdk2 is not clear. [What is known about cdks associated with DNA synthesis in other organisms. It seems to me that that is the proper comparison to make?]

Strikingly, the two Paramecium CDKs share only 48% identity with each other, suggesting that the evolutionary separation of Cdk1 and Cdk2 is ancient and they must have distinct functions in the Paramecium cell cycle regulation. [This underscores the point that I made in the paragraph above. Direct comparisons are only possible between molecules that are true homologues.

Southern blot analysis of Paramecium genomic DNA digested by EcoRI, XbaI, PstI and HindIII, with a DIG-labelled DNA probe derived from the full-length of CDK2 showed two hybridizing bands in each lane under high stringency conditions, suggesting that there are at least two copies of CDK2 gene in the Paramecium genome (Fig. 3A). Similarly, there are at least two bands when the same blot was stripped and reprobed with a CDK1 DNA probe (Fig. 3B). No recognition sites for the above restriction enzymes were found in both sequences. However, the possibility of those less intense bands in each lane corresponding to the micronuclear copy of the gene can not be eliminated. Furthermore, genomic Southern blot analysis results suggests that CDK2 and CDK1 do not reside tandemly in the genome, as their hybridizing patterns are completely different from each other. [Have you thought about pulling out the second CDK2 sequence?]

The protein encoded by CDK2 corresponds to the polypeptide of 35 kD recognized by anti-PSTAIRE antibody. To study the temporal regulation and biological activities of Cdk2 protein, a rabbit antiserum was raised against a peptide corresponding to residues 240-255 of Cdk2, a region not conserved in Cdk1, and was used for all subsequent experiments. Immunoblot analysis of the Paramecium protein lysate showed that this antibody detected a protein of 35 kD, corresponding the minor polypeptide recognized by anti-PSTAIRE antibody (Fig. 4, lanes 1 and 3) and matching the predicted molecular mass of Cdk2. Preincubation of the serum with excess antigenic peptide abolished the immunoreactivity, implying that this antibody was specific for epitopes within the antigenic peptide (data not shown). Moreover, this antibody showed no cross-reaction with Cdk1 (Fig. 4, lane 2 and 3).

To further confirm the specificity of this antibody, we expressed a partial CDK2 sequence (residues 212 to 301) as glutathione-S-transferase (GST) fusion protein in E coli (Fig. 1). Within this region, the only UAA/UAG coded glutamine codons was Q²¹⁶, which was later converted to CAA, the conventional glutamine codon by including a mismatch in one of the primers used for subcloning the fragment into pGEX-2T vector. Moreover, the antigenic peptide used for generating anti-Cdk2 antibody was also located in this region. After purification on glutathione-Sepharose beads, protein products were resolved on Coomassie Brilliant Blue R250 stained SDS-polyacrylamide gel as a major band of predicted molecular mass for fusion protein (36 kD), and a minor band of 26 kD representing GST portion (data not shown). The presence of GST only protein in the expression products might be due to the different codon usage for Paramecium and E coli (Martindale, 1989; Sharp et al., 1988). On an immunoblot of GST-Cdk2 fusion protein and GST only protein, anti-Cdk2 peptide antibody recognized only the GST-Cdk2 protein (36kD), and not GST (26 kD) (Fig. 5A), whereas anti-GST antibody recognized both proteins (Fig. 5B). These results further support that anti-Cdk2 peptide antibody is specific for the protein encoded by CDK2.

p13^suc1 binding property of Cdk2 revealed a novel class of p34^cdc2 homologue in Paramecium. p13^suc1 was initially isolated as a high copy suppressor of certain S. pombe cdc2 mutants (Hayles et al., 1986), and its homologues have been identified in higher eukaryotes (Richardson et al., 1990). It is an essential cell cycle regulator of yeast (Hindley et al., 1987; Moreno et al., 1989), and interacts with p34^cdc2 protein kinases from a number of species. Both positive and negative effects on the cell cycle progression have been attributed to p13^suc1. Even though the exact mechanisms of p13^suc1 action its physiological role remain unclear, the affinity of p13^suc1 for p34^cdc2 homologues has been well exploited, leading to the extensive use of S. pombe p13^suc1 beads to isolate p34^cdc2 kinase activity from a number of species.

Previous results have shown that Cdk1 does not bind to yeast p13^suc1 (Tang et al., 1994). However, the minor polypeptide of 35 kD recognized by anti-PSTAIRE antibody did show capability for binding to p13^suc1 (Fig. 6A). Moreover, histone H1 kinase activity has been detected on p13^suc1 precipitates, peaking at PCD (Tang et al., 1994).

To test whether Cdk2 corresponds to the p35 polypeptide bound to p13^suc1, a lysate from exponentially growing Paramecium cells was incubated with p13^suc1-Sepharose beads. After extensive washing, proteins from both supernatant and beads were resolved on a SDS-polyacrylamide gel and transferred to Immobilon membrane, and then probed with anti-Cdk2 antibody. Surprisingly, we found that Cdk2 retained in the supernatant, just as Cdk1 did, suggesting that it is incapable of p13 binding (Fig. 6B). As a positive control, p34^cdc2 from S. pombe was only observed on the p13 beads, not in supernatant. (Fig. 6C).

Based on this observation, we proposed that the p35 polypeptide band recognized by anti-PSTAIRE antibody might consist of two different classes of p34^cdc2 homologues with very similar molecular masses, one binding to p13^suc1; the other not. The former corresponds to the kinase activity associated with PCD, the latter is Cdk2. To examine this possibility, a p13^suc1 immunodepletion experiment was carried out. As shown in Figure 7 , we observed that only two rounds of incubation with p13^suc1 was sufficient to deplete all p35 associated with p13^suc1 (Fig. 7A:P1, P2, P3), and the third round of incubation brought virtually no 35 kD polypeptide down but a p35 polypeptide could be detected by anti-PSTAIRE antibody in the supernatants in all samples (Fig. 7A: S1, S2, S3). These results support that p35 recognized by anti-PSTAIRE antibody, in fact, contains two classes of polypeptides. The p35 polypeptide in the supernatants corresponded to Cdk2 (Fig. 7C). As a control, Cdk1 could only be detected in the supernatants (Fig. 7B), in agreement with previous results (Tang et al., 1994).

The p13^suc1 immunodepletion results suggested that Cdk2 is a new member of p34^cdc2 kinase family in addition to Cdk1 and the p13^suc1 binding kinase Cdk2 has similar molecular mass of 35 kD to the p34^cdc2 homologue that binds p13^suc1. However, Cdk2 does not bind to p13^suc1, just like Cdk1. For convenience, we named p35 kinase that binds to p13^suc1 Cdk3, and the one identified in this study that does not bid p13^suc1 Cdk2, based on the newly proposed ciliate genetic nomenclature (Allen et al., 1998).

Cdk2 protein level is invariant during the Paramecium vegetative cell cycle. To study Cdk2 protein levels during the cell cycle, exponentially growing Paramecium cells were synchronized by selecting a population of smallest, newly born daughter cells (predominantly in early G1 phase) using a Beckman centrifugal elutriation rotor. Macronuclear DNA synthesis was monitored by feeding cells with [³H]-thymidine-labelled food bacteria. Initiation of DNA synthesis routinely occurred about 2~2.5 hours after elutriation (Berger 1971; Tang et al., 1997). Median time of cell division in elutriated samples ranges from 8.5 to 9.5 h post elutriation. The extent of synchrony obtained by centrifugal elutriation was comparable to that of hand-selected dividing cells from the same culture (Adl & Berger, 1995, Tang et al., 1997). Immediately after the elutriation, cells were re-inoculated into fresh medium and allowed to proceed through one relatively synchronous cell cycle and cells were sampled at one hour intervals starting 30 min after elutriation.

To examine the pattern of Cdk2 protein expression during the vegetative cell cycle, immunoblot analysis with anti-Cdk2 antibody was performed at each time point when samples were collected . Cdk1 was used as an equal loading control (data not shown). As shown in Figure 8, Cdk2 appeared as a single band of 35 kD, and was expressed at roughly comparable levels throughout the vegetative cell cycle, just like Cdk1 (Tang et al, 1997).

We also examined Cdk2 protein expression in starved cells. Athough it was still detectable, Cdk2 protein was present at much lower level in starved cells than in exponentially growing cells (Fig. 9), suggesting that Cdk2 activity is associated with cell proliferation.

Cdk2 histone H1 kinase activity is associated with cytokinesis. Since Cdk2 appears to be a protein kinase based on its primary sequence, we examined kinase activity as a function of cell cycle stage. As Cdk1 has previously been shown to be able to phosphorylate bovine histone H1 in vitro (Tang et al, 1997), histone H1 was chosen as a starting substrate for testing Cdk2 kinase activity.

Anti-Cdk2 antibody was capable of specifically immunoprecipitating a histone H1 kinase activity from the Paramecium lysate. Immunoprecipiates by anti-Cdk2 antibody of lysates from exponentially growing cells showed significant histone H1 kinase activity whereas immunoprecipitates by pre-immune serum did not (data not shown).

To examine Cdk2 kinase activity during the cell cycle, immunoprecipitation was performed with anti-Cdk2 antibodyusing lysates from synchronous . As shown in Figure 10, the protein kinase activity varied periodically throughout the cell cycle, rising to a peak about 9.5 hours after elutriation. This peak corresponds to the point at which more than 70% of cells had undergone cytokinesis (Fig. 10B). The rise of protein kinase activity was sharp and increased to about 10 times that observed during early stages of the cell cycle (Fig.10B). This observation suggested that Cdk2 functionmight be associated with cytokinesis..

Discussion

We used PRC techniques to clone and identify a novel gene, CDK2, whose predicted amino acid sequence bears a high degree of similarity with p34^cdc2 protein kinases from other organisms. Like Cdk1, the Paramecium p34^cdc2 homologue cloned previously (Tang et al., 1995), Cdk2 sequence has all catalytic domains of protein kinases and contains the conserved p34^cdc2 hallmark - ‘PSTAIRE’ region without alteration. Residues equivalent to the regulatory phosphorylation, Thr14, Tyr15 and Thr161 in human CDK1, can be found in the Paramecium sequence at comparable locations, which suggests that regulation of Cdk2 activityin Paramecium may be similar to that in yeast p34^cdc2(CDC28). While Cdk2 displays 33-61% identity with p34^cdc2 homologues from other organisms, it shows only 48% homology with Cdk1, suggesting they may have distinct functions in the cell cycle regulation. As a comparison, human CDK1 and CDK2 share 65% identity at amino acid level (Meyerson et al., 1992). [There are, howeve, other more divergent human CDKs]

Previous results suggested the presence of two classes of p34^cdc2 homologues in Paramecium that differed from each other their p13^suc1 binding property (Tang et al., 1994). By comparing Cdk2 with Cdk1 sequences, we found that Cdk2 had better conservation of putative p13^suc1 binding sites than Cdk1 (Fig. 2). Specifically, among 13 residues involved in p13^suc1 binding (Ducommun et al., 1991, Marcote et al., 1993), ten can be found in Cdk2 whereas only 6 in Cdk1. However, p13^suc1 binding experiment with anti-Cdk2 antibody shows that Cdk2 in fact does not bind to p13^suc1. Therefore, we postulated that the p35 polypeptide recognized by anti-PSTAIRE antibody might contain two different polypeptides with the same migration rate on SDS-PAGE, but with distinct p13^suc1 binding properties. Immunodepletion of p13^suc1 bound polypeptide from lysates confirmed that a portion of p35 polypeptide recognized by anti-PSTAIRE antibody did not possess affinity for p13^suc1, and this might correspond to Cdk2. That anti-Cdk2 antibody only recognized this portion of p35, not that bound to p13^suc1 (Cdk3) further suggests that the unbound form of p35 is Cdk2. Identification of Cdk2 as a new CDK suggests that Paramecium may have more p34^cdc2-related protein kinases than was previously thought. It also strengthens the notion that multiple CDKs are present in Paramecium, and that cell cycle regulation in ciliates may be more complicated than was thought before However, we do not yet know whether these CDKs have overlapping functions or whether each plays distinct function at specific stage of the Paramecium cell cycle.. Existence of multiple CDKs may at least in part explain why CDKs have not been identified as recessive cell cycle mutants in Paramecium thus far. [Careful here. It is not the different types of CDK that would result in the absence of mutants, but the presence of isoforms of the same molecule that would be much more likely to have overlapping function].

As predicted from its primary sequence, Cdk2 immunoprecipitate exhibits histone H1 kinase activity. While Cdk1 kinase activity peaks at the initiation of the macronuclear DNA synthesis (Tang et al.,1997), Cdk2 activity reaches its maximal level at cytokinesis, when more than 70% cells have divided. This further distinguishes it from the Cdk3 kinase activity associated with p13^suc1. The maximum activity level of CDK3 was reached ~90 min before cytokinesis (Tang et al, 1994), which coincides temporally with PCD, the major control point in the Paramecium cell cycle. Thus, the profile of Cdk2 kinase activity suggests that it has a role in cytokinesis.

Since the abundance of Cdk2 protein remained almost constant during the vegetative cell cycle, it is likely that Cdk2 kinase activity, like most CDKs, is regulated by post-translational modifications, given that both cyclin binding domains and phosphorylation sites are conserved within the sequence. Consistent with this, two classes mitotic cyclin homologues have been identified in Paramecium (Zhang and Berger, submitted). Results from co-immunoprecipitation experiment indicated that Cdk2 formed a complex with one of the cyclins (cyc1), and that histone H1 kinase activity associated with that cyclin also reached maximal level at cytokinesis.

However, there is no direct evidence available so far for the involvement of any CDKs in the cell cycle regulation in Paramecium. Part of reason is due to the difficulty associated with the expression the full-length sequence in heterologous systems, such as yeast, because of its non-universal genetic codon. Furthermore, gene knockout techniques are still not mature enough both theoretically and practically in Paramecium to allow us to examine the functions of these CDKs in vivo. Nevertheless, further studies on the subcellular localization of Cdk2 and regulatory mechanisms such as phosphorylation by anti-phosphotyrosine antibodies may shed more light on the roles of CDKs in the Paramecium cell cycle control and its coordination..

Taken together, our results demonstrate a family of three p34^cdc2-related protein kinases in Paramecium tetraurelia: Cdk1, Cdk2 and Cdk3. Given that their kinase activities reach maximal levels at different points within the cell cycle, and that the proteins exhibit different affinity for p13^suc1 beads, it is likely that multiple p34^cdc2 protein kinases have evolved to regulate different cell cycle events early in the evolutionary history of ciliates.. This view is further supported by the identification of two anti-PSTAIRE reactive proteins from another ciliate, Tetrahymena thermophila, with different affinities for p13^suc1 (Tang, et al, 1994; Roth et al., 1991). One of CDK genes of Tetrahymena has been cloned (Zhang and Berger, unpublished).

Acknowledgments

This research was completed under support of NSERC Canada to JDB. We are grateful to Drs. Liren Tang and Sarb Nil for helpful discussion, technical supports. Anti-GST polyclonal antibody was kindly provided by Dr. Greg Mullen.

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66. Figure legends

Fig. 1. Nucleotide sequence of CDK2, and the predicted amino acid sequence of the gene product, Cdk2. The conserved ‘PSTAIRE’ region is double underlined. Putative phosphorylation sites, Thr20, Tyr21 and Thr164 in Cdk2, equivalent to Thr14, Tyr15 and Thr161 of human CDK1, respectively, are indicated by bold italics. A 24-nucleotide intron in the sequence is in lower cases. Q* is glutamine acid encoded by TAA or TAG. "*" indicates the stop codon TGA. The sequence used for designing the antigenic peptide is underlined. The region that was expressed as GST fusion protein is in parentheses ‘[ ]’. The GenBank accession number of this sequence is U95166.

Fig. 2. Comparison of the amino acid sequence of Cdk2 and cdk1 with p34^cdc2 homologues from Tichomonas vaginalis (Tv), Entamoeba histolytica (Eh), Plasmodium falciparum (Pf), Dictyodtelium discoideum (Dd), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Zea mays (Zm) and Homo sapiens (Hs). Dashes indicate identity with cdk2, and gaps have been introduced where necessary to maximize the alignment. The ‘PSTAIRE’ region is double underlined. The regulatory phosphorylation sites corresponding to Thr14, Tyr15 and Thr161 of human CDK1 are underlined. ‘^’ indicates the residues involved in p13^suc1 binding in human CDK1.

Fig. 3. Genomic Southern blot analysis. Paramecium genomic DNA was digested with EcoRI (lane 1), XbaI (lane 2), PstI (lane 3), HindIII (lane 4), and probed with the DIG-labelled CDK2 probe (A). The membrane was then stripped and re-probed with the DIG-CDK1 probe (B). Arrowheads indicate some faint bands in the blot. The 1-kb DNA marker from Gibco-BRL are indicated on the left.

Fig. 4. Detection of p34^cdc2 homologues in Paramecium lysate. Lysate prepared from unsynchronized cells was analyzed by immunoblotting with anti-PSTAIRE antibody (lane 1), anti-GST-Cdk1 fusion protein antibody (lane2, Tang et al., 1997) and anti-Cdk2 peptide antibody (lane 3). Molecular marker from Bio-Rad is indicated.

Fig. 5. Specificity of anti-Cdk2 peptide antibody. Lane 1, purified GST-Cdk2 protein. Lane 2, purified GST only protein. The immunoblot was first probed with anti-Cdk2 peptide antibody (A). Then, it was stripped and re-probed with anti-GST antibody (B).

Fig. 6. p13^suc1 affinity of the Paramecium CDKs. Panels A and B show protein lysates from Paramecium; Panel C shows protein lysates from S. pombe. W: whole lysate; P: bound fraction eluted from p13^suc1 beads; S: supernatants (unbound fraction) . The blots were probed with antibodies indicated.

Fig. 7. p13^suc1 immunodepletion experiment showing Cdk2 is a novel p34^cdc2 homologue. Aliquots of resulting bound portion (P1, P2, P3) and supernatant (unbound, S1, S2, S3) after each round of p13^suc1 incubation were immunoblotted with anti-PSTAIRE antibody (A), anti-Cdk1 (B), and anti-Cdk2 (C). W: whole lysate.

Fig. 8. Expression of Cdk2 protein during the vegetative cell cycle. Paramecium lysates were prepared from synchronized samples by centrifugal elutriation at 1 h intervals, starting from 0.5 hr to 11.5 hr after elutriation. Immunoblotting of equal amount of lysate protein was performed with anti-Cdk2 antibody. PI was lysate from asynchronized sample probed with preimmune serum as a control.

Fig. 9. Cdk2 protein level in exponentially-growing and starved cells. Immunoblotting of equal amount of proteins from exponentially-growing cells (lane 1) and starved cells (lane 2) was performed with anti-Cdk2 antibody.

Fig. 10. Cdk2 exhibits protein kinase activity towards bovine histone H1 which fluctuates in a cell-cycle dependent manner. Small early G1 cells were selected by centrifugal elutriation and re-inoculated into fresh medium, and aliquots were taken for determination of cumulative percentage of cell division (B). The equal amount of protein lysates from synchronized samples were immunoprecipitated with anti-Cdk2 antibody. The resulting immunocomplexes were subjected to histone H1 kinase assay, and aliquots of reaction were run on a SDS-PAGE (A). Quantitation of the kinase activity was performed by scintillation counting of the excised phosphorylated histone H1 bands (B). PI shows the background activity in the immunocomplexes by pre-immune serum. This figure presents composite results derived from several batches of synchronized samples due to the limited amount of proteins available from each elutriation.