Cdc2 Homologs Research Articles

Learning from the Uncontrollable

Research papers are usually revisionist histories, imagined accounts driven by impeccable logic and unbroken experimental success, which in no way reflect the slowness, messiness, and serendipity of real life in the lab. To give a different hopefully more balanced perspective than what is often told, I want to tell you the story behind some of the discoveries that shaped my early career.“In reality, of course, I had stumbled on the control by complete accident.”

Oocyte maturation failure: a syndrome of bad eggs

To show that disruption of meiotic competence results in cell cycle arrest, and the production of immature oocytes that are not capable of fertilization. Through an extensive review of animal studies and clinical case reports, we define the syndrome of oocyte maturation failure as a distinct oocyte disorder, present a classification system based on clinical parameters, and discuss the potential molecular origins for the disease.

A positive signal from the fertilization of the egg cell sets off endosperm proliferation in angiosperm embryogenesis

Double fertilization of the egg cell and the central cell by one sperm cell each produces the diploid embryo and the typically triploid endosperm and is one of the defining characteristics of flowering plants (angiosperms). Endosperm and embryo develop in parallel to form the mature seed, but little is known about the coordination between these two organisms. We characterized a mutation of the Arabidopsis thaliana Cdc2 homolog CDC2A (also called CDKA;1), which has a paternal effect. In cdc2a mutant pollen, only one sperm cell, instead of two, is produced. Mutant pollen is viable but can fertilize only one cell in the embryo sac, allowing for a genetic dissection of the double fertilization process. We observed exclusive fertilization of the egg cell by cdc2a sperm cells. Moreover, we found that unfertilized endosperm developed, suggesting that a previously unrecognized positive signal from the fertilization of the egg cell initiates proliferation of the central cell.

G2/M arrest caused by actin disruption is a manifestation of the cell size checkpoint in fission yeast.

In budding yeast, actin disruption prevents nuclear division. This has been explained as activation of a morphogenesis checkpoint monitoring the integrity of the actin cytoskeleton. The checkpoint operates through inhibitory tyrosine phosphorylation of Cdc28, the budding yeast Cdc2 homolog. Wild-type Schizosaccharomyces pombe cells also arrest before mitosis after actin depolymerization. Oversized cells, however, enter mitosis uninhibited. We carried out a careful analysis of the kinetics of mitotic initiation after actin disruption in undersized and oversized cells. We show that an inability to reach the mitotic size threshold explains the arrest in smaller cells. Among the regulators that control the level of the inhibitory Cdc2-Tyr15 phosphorylation, the Cdc25 protein tyrosine phosphatase is required to link cell size monitoring to mitotic control. This represents a novel function of the Cdc25 phosphatase. Furthermore, we demonstrate that this cell size-monitoring system fulfills the formal criteria of a cell cycle checkpoint.

Purification of GVBD-inducing protein from the ciliate Tetrahymena thermophila.

Germinal-vesicle-breakdown (GVBD) was induced if a 132,000-g supernatant of Tetrahymena thermophila homogenates was injected into Xenopus oocytes. Using this induction of GVBD as a bioassay system, a GVBD-inducing substance was purified from the Tetrahymena by ultra-filtration, liquid chromatography, and electroelution from a band on native-PAGE gel. Proteins eluted from the single band on the native-PAGE gel induced GVBD in the absence of oocyte protein synthesis. This band resolved into two bands on SDS-PAGE: 60 and 112 kDa. The 60 kDa protein was the active fraction inducing GVBD. Immunoprecipitation of the 60 kDa protein prevented the GVBD-inducing activity, supporting the conclusion that the 60 kDa protein is the GVBD-inducing substance. An immunoblot with anti-60 kDa monoclonal antibody and PSTAIR antibody showed that p13suc1-beads could remove cdc2 homologues from T. thermophila supernatant but could not remove the GVBD-inducing activity. The 60-kDa protein appeared at the same time as micronuclear division and disappeared at the beginning of the macronuclear division during synchronous cell division. The cyclic appearance of the 60-kDa protein in the T. thermophila cell cycle suggests that this protein has a cell cycle function.

Purification of germinal-vesicle-breakdown-inducing proteins from Tetrahymena pyriformis

Summary Germinal-vesicle-breakdown (GVBD) of Xenopus laevis oocytes was induced by injecting 132,000 g supernatant of Tetrahymena pyriformis homogenates into oocytes. Using induction of GVBD as a bioassay system, we succeeded in purifying two GVBD-inducing proteins from the 132,000 g supernatant by liquid chromatography with an anion-exchange MonoQ column, ultra-filtration, and electroelution from bands on a native-PAGE gel. A protein of 40 kDa induced GVBD in the absence of protein synthesis of Xenopus oocytes whereas another protein of 56 kDa induced GVBD only in the presence of the oocyte protein synthesis. These proteins did not react with the PSTAIR antibody, which is specific for cdc2 homologues.

Stage-specific activity of the Leishmania major CRK3 kinase and functional rescue of a Schizosaccharomyces pombecdc2 mutant

Cell cycle control by cdc2-related kinases (CRKs) is essential to the regulation of cell proliferation and developmental processes in many organisms. Alternating phases of growth, arrest, and differentiation are characteristics of the infectious cycle of many trypanosomatid parasites, raising the possibility that members of the trypanosomatid CRK gene family participate in the regulation of these essential processes. Here we describe properties of the CRK3 gene from Leishmania major, which encodes a 36 kDa protein kinase showing 60% amino acid sequence identity with human CDK2, including several conserved sites implicated in regulation of kinase activity. CRK3 mRNA was constitutively expressed throughout the parasite life cycle, but histone H1 kinase activity of an epitope tagged CRK3 protein was greater in log-phase than in stationary-phase promastigotes. When integrated into the genome and expressed at the optimal level, CRK3 was able to rescue the growth defect of a Schizosaccharomyces pombe cdc2 mutant ( cdc2-33 ts), indicating that CRK3 is a functional homolog of cdc2. Mutants of CRK3 at several key regulatory residues showed the expected dominant negative effects on the S. pombe mutant. This is the first example of functional expression of a trypanosomatid CRK in yeast, opening the way for further genetic studies within this amenable organism.

The crk3 Gene of Leishmania mexicanaEncodes a Stage-regulated cdc2-related Histone H1 Kinase That Associates with p12cks1

A cdc2-related protein kinase gene, crk3, has been isolated from the parasitic protozoan Leishmania mexicana. Data presented here suggests that crk3 is a good candidate to be the leishmanial cdc2 homologue but that the parasite protein has some characteristics which distinguish it from mammalian cdc2. crk3 is predicted to encode a 35.6-kDa protein with 54% sequence identity with the human cyclin-dependent kinase cdc2 and 78% identity with the Trypanosoma brucei CRK3. The trypanosomatid CRK3 proteins have an unusual, poorly conserved 19-amino acid N-terminal extension not present in human cdc2. crk3 is single copy, and there is 5-fold higher mRNA in the replicative promastigote life-cycle stage than in the non-dividing metacyclic form or mammalian amastigote form. A leishmanial suc-binding cdc2-related kinase (SBCRK) histone H1 kinase, has previously been described which binds the yeast protein, p13(suc1), and that has stage-regulated activity (Mottram J. C., Kinnaird, J., Shiels, B. R., Tait, A., and Barry, J. D. (1993) J. Biol. Chem. 268, 21044-21051). CRK3 from cell extracts of the three life-cycle stages was found to bind p13(suc1) and the leishmanial homologue p12(cks1). CRK3 fused with six histidines at the C terminus was expressed in L. mexicana and shown to have SBCRK histone H1 kinase activity. Depletion of histidine-tagged CRK3 from L. mexicana cell extracts, by Ni-nitrilotriacetic acid agarose selection, reduced histone H1 kinase activity binding to p13(suc1). These data imply that crk3 encodes the kinase subunit of SBCRK. SBCRK and histidine-tagged CRK3 activities were inhibited by the purine analogue olomoucine with an IC50 of 28 and 42 microM, respectively, 5-6-fold higher than human p34(cdc2)/cyclinB.

Regional patterns of c-Fos expression in rat neocortex following performance of audio-visual discrimination

Cell cycle control by cdc2-related kinases (CRKs) is essential to the regulation of cell proliferation and developmental processes in many organisms. Alternating phases of growth, arrest, and differentiation are characteristics of the infectious cycle of many trypanosomatid parasites, raising the possibility that members of the trypanosomatid CRK gene family participate in the regulation of these essential processes. Here we describe properties of the CRK3 gene from Leishmania major, which encodes a 36 kDa protein kinase showing 60% amino acid sequence identity with human CDK2, including several conserved sites implicated in regulation of kinase activity. CRK3 mRNA was constitutively expressed throughout the parasite life cycle, but histone H1 kinase activity of an epitope tagged CRK3 protein was greater in log-phase than in stationary-phase promastigotes. When integrated into the genome and expressed at the optimal level, CRK3 was able to rescue the growth defect of a Schizosaccharomyces pombe cdc2 mutant (cdc2-33ts), indicating that CRK3 is a functional homolog of cdc2. Mutants of CRK3 at several key regulatory residues showed the expected dominant negative effects on the S. pombe mutant. This is the first example of functional expression of a trypanosomatid CRK in yeast, opening the way for further genetic studies within this amenable organism.

Multiple cdc2 (p34 protein kinase) cDNA clones from shoot cDNA library of soybean

Summary A shoot-derived cDNA library of soybean Glycine max (L.) cv. Bragg inoculated with Bradyrhizobium japonicum was constructed for the purpose of assessing expression changes in autoregulated legume plants. To evaluate the representativeness and utility of the library, cDNA clones homologous to yeast cdc2 / CDC28 gene were isolated. Based on restriction and sequence analysis of their 5′ and 3′ regions, two families of genes were found, suggesting that as many as six different cdc2 homologues exist in soybean. These genes may have functional relevance in tissue-specific regulation of cell division. We conclude that the library has utility for nodulation research, especially aspects dealing with shoot-controlled components of autoregulation.

Plant CDC2 is not only targeted to the pre-prophase band, but also co-localizes with the spindle, phragmoplast, and chromosomes

A polyclonal antiserum against the p34 cdc2 homologue of Arabidopsis thaliana, CDC2aAt, was used in parallel with a polyclonal antiserum against the PSTAIRE motif to study the subcellular localization of CDC2 during the cell cycle of isolated root tip cells of Medicago sativa. During interphase, CDC2 was located in the nucleus and in the cytoplasm. The cytoplasmic localization persisted during the complete cell cycle, whereas the nuclear signal disappeared at nuclear envelope breakdown. At the beginning of anaphase, the anti-CDC2aAt antibody transiently co-localized with condensed chromosomes. The chromosomal co-localization disappeared as anaphase continued and remained excluded from the separated chromosomes until cytokinesis, when CDC2 re-located to the newly forming nuclei. We also observed a co-localization of CDC2 with three microtubular structures, the pre-prophase band, the spindle, and the phragmoplast.

Cell cycle phase specificity of putative cyclin-dependent kinase variants in synchronized alfalfa cells.

The eukaryotic cell division cycle is coordinated by cyclin-dependent kinases (CDKs), represented by a single major serine/threonine kinase in yeasts (Cdc2/CDC28) and a family of kinases (CDK1 to CDK8) in human cells. Previously, two cdc2 homologs, cdc2MsA and cdc2MsB, have been identified in alfalfa (Medicago sativa). By isolating cDNAs using a cdc2MsA probe, we demonstrate here that at least four additional cdc2 homologous genes are expressed in the tetraploid alfalfa. Proteins encoded by the new cdc2MsC to cdc2MsF cDNAs share the characteristic functional domains of CDKs with the conserved and plant-specific sequence elements. Transcripts from cdc2MsA, cdc2MsB, cdc2MsC, and cdc2MsE genes are synthesized throughout the cell cycle, whereas the amounts of cdc2MsD and cdc2MsF mRNAs peak during G2-to-M phases. The translation of Cdc2MsA/B, Cdc2MsD, and Cdc2MsF proteins follows the pattern of transcript accumulation. The multiplicity of kinase complexes with cell cycle phase-dependent activities was revealed by in vitro phosphorylation experiments. Proteins bound to p13suc1-Sepharose or immunoprecipitated with Cdc2MsA/B antibodies from cells at G1-to-S and G2-to-M phase boundaries showed elevated kinase activities. the Cdc2MsF antibodies separated a G2-to-M phase-related kinase complex. Detection of histone H1 phosphorylation activities in fractions immunoprecipitated with antimitotic cyclin (CyclinMs2) antibodies from G2-to-M phase cells indicates the complex formation between this cyclin and a kinase partner in alfalfa. The observed fluctuation of transcript levels, amounts, and activities of kinases in different cell cycle phases reflects a multilevel regulatory system during cell cycle progression in plants.

Distinct classes of cdc2-related genes are differentially expressed during the cell division cycle in plants.

cdc2 and several related genes encode the catalytic subunits of cyclin-dependent kinases, which have been implicated in a number of cellular processes, including control of cell division. As a first step in exploring their function in plants, we isolated four cdc2-related genes from Antirrhinum. Two genes, cdc2a and cdc2b, encode proteins that contain a perfectly conserved PSTAIRE motif characteristic of cdc2 homologs, whereas the products of the two remaining genes, cdc2c and cdc2d, appear to represent a new subclass of proteins that have so far only been identified in plants. Transcripts of these novel genes were localized in isolated cells dispersed throughout actively dividing regions of the inflorescence. This localization is consistent with accumulation that is specific to particular phases of the cell cycle. Correlating cell labeling with nuclear condensation and double-labeling experiments using cdc2 and histone H4 as probes indicated that cdc2c transcripts accumulate during S phase as well as during the G2 and M transition, whereas cdc2d expression was specific to the G2 and M phases. All cells labeled with cdc2d also contained cdc2c label, Indicating that expression of cdc2d completely overlapped with that of cdc2c. Transcripts of cdc2a and cdc2b were detected in all cells within actively dividing regions, but at levels that were only slightly higher than those observed in nondividing areas. These transcripts did not appear to accumulate in a cell cycle-specific fashion. The genes cdc2a and cdc2b were able to partially complement a yeast cdc2 mutation, although all four genes appeared to interfere with the sizing mechanism of yeast cells. We propose that plants contain at least two classes of cdc2-related genes that differ in structure, expression, and perhaps function.

Paul Nurse: cycling up the hill

Paul Nurse: cycling up the hill

Identification and characterization of maturation-promoting factor from catfish,Clarias batrachus

Maturation-promoting factor (MPF) extracted from maturing oocytes of catfishes (Clarias batrachus andHeteropneustes fossilis) and carp (Labeo rohita) induces 100% germinal vesicle breakdown (GVBD) when microinjected intoClarias immature unstimulated oocytes. The presence of a similar MPF activity has also been demonstrated in the active fractions collected after superose 12. SDS-PAGE analyses of cytosolic extracts (CE) prepared from immature and mature oocytes revealed the presence of 34- and 46 kDa proteins apart from a few others. Antibody against the PSTAIR sequence of p34(cdc2) recognized 32- and 34 kDa proteins of immature as well as mature oocytes while, 46 kDa protein of mature oocytes was recognized by anti-cyclin B1 antibody. Moreover, labelling of [(35)S]methionine was observed mainly in the region of 46 kDa protein band indicatingde novo synthesis of this particular protein. Anti-cyclin A antibody did not recognize any proteins of immature or mature oocytes. Cyclin B1 was absent in immature oocytes and ovulated eggs. These findings indicate the presence of p34(cdc2) homologs and cyclin B in the MPF of the catfishes and carp oocytes.

Differential accumulation of the transcripts of 22 novel protein kinase genes in Arabidopsis thaliana

22 novel members of the Arabidopsis thaliana protein kinase family (AKs) were identified by using degenerate oligonucleotide primers directed to highly conserved amino acid sequences of the protein kinase (PK) catalytic domain. Of these 22 genes, 16 turned out to carry intron sequences. Homologies of AK sequences were detected to S-locus receptor protein kinases (SRKs) from Brassica spp., to SRK-like PKs from maize and A. thaliana and to several other receptor PKs from A. thaliana. Sequence similarity was also detected to Ca(2+)-dependent PKs (CDPKs) from rape and soybean, to SNF1 and to CDC2 homologues. The genomic organization and the accumulation of the mRNAs from these 22 AK genes were investigated.

The role of cdc2 and other genes in meiosis in Schizosaccharomyces pombe.

The requirement of the cdc2, cdc13 and cdc25 genes for meiosis in Schizosaccharomyces pombe was investigated using three different conditions to induce meiosis. These genes were known to be required for meiosis II. cdc13 and cdc25 are essential for meiosis I. The cdc2 gene, which is required for the initiation of both mitotic S-phase and M-phase, is essential for premeiotic DNA synthesis and meiosis II. The requirement of cdc2 for meiosis I was unclear. This contrasts with Saccharomyces cerevisiae, where CDC28, the homolog of cdc2, is required for meiosis I but not for premeiotic DNA synthesis. Expression of cdc13 and cdc25 was induced after premeiotic DNA synthesis, reaching a sharp peak before the first nuclear division. Expression of cdc22, encoding the large subunit of ribonucleotide reductase, was also induced but the peak was before premeiotic DNA synthesis. The induction of cdc13 and cdc25 was largely dependent on DNA synthesis and the function of the mei4 gene. The mei4 gene itself was also induced in a DNA synthesis-dependent manner. The chain of gene expression activating cdc25 may be important as part of the mechanism that ensures the dependency of nuclear division on DNA replication during meiosis.

Gibberellin promotes histone H1 kinase activity and the expression of cdc2 and cyclin genes during the induction of rapid growth in deepwater rice internodes.

Partial submergence or treatment with either ethylene or gibberellin (GA) promotes rapid internodal growth in deepwater rice (Oryza sativa L.). Earlier work has shown that GA is the immediate hormonal signal for this growth response, which involves induction of the cell cycle at the G2/M phase transition and subsequent enhancement in the rate of DNA synthesis. In all eukaryotes, onset of mitosis is regulated by the p34cdc2/CDC28 protein kinase, whose activity is assayed by in vitro phosphorylation of histone H1. It was found that GA enhanced the activity of p34cdc2/CDC28-like histone H1 kinase in the intercalary meristem of rice internodes. The enzyme activity showed a sharp peak that correlated with a decrease in the population of cells in the G2 phase during the first 4 h of GA treatment but not with changes in DNA synthesis. The level of histone H1 kinase activity increased again when cell division activity in the intercalary meristem is known to be high. The expression of two cdc2 homologs was examined. The mRNA level of one of these, cdc2Os-2, was increased after 1 h of GA treatment, whereas the mRNA level of the other, cdc2Os-1, was not affected. Two cDNAs, cycOs1 and cycOs2, which show high homology to cyclin cDNAs, were cloned from rice. They share 75.1% sequence identity at the amino acid level, and both of them are encoded by mRNAs of 1.6 kb. Expression of the two corresponding cyclin genes was enhanced by GA, and the time course of the induction was compatible with a role for both cyclins in regulating the G2/M phase transition. The cyclins were expressed in the intercalary meristem and the elongation zone of the internode, but the GA-induced increase in transcript levels was restricted to the meristem only. The results support the hypothesis that induction of mitosis by GA is brought about by increased p34cdc2/CDC28 protein kinase activity, which may be the result of transcriptional activation of the cdc2Os-2, cycOs1 and cycOs2 genes.

A cdc2 homologue and closely related processed retropseudogenes from Norway spruce

The p34cdc2 protein kinase is a key component in the regulation of the eukaryotic cell cycle and has been conserved during evolution. We have isolated cDNA clones corresponding to a cdc2 gene (cdc2Pa) from the conifer Norway spruce, Picea abies (L.) Karst. The deduced amino acid sequence is 85-90% identical to p34cdc2 homologues from other plants, contains eleven subdomains characteristic for the protein kinase family, and three sequence motifs specific for the cdc2 protein kinases. A partial genomic clone of cdc2Pa reveals two introns at positions identical to intron positions in Arabidopsis thaliana cdc2a. A Southern blot analysis shows that cdc2Pa is a single-copy gene belonging to a family of about 10 related genes. Partial genomic sequences of six of the genes in this family (86-92% identical to cdc2Pa) show distinct features of processed retropseudogenes. These lack introns and contain deletions, insertions and/or non-silent point mutations. This result is consistent with the hypothesis that processed retropseudogenes in plants may be common among genes expressed in the apical meristem, that is, in cells which have the potential to take part in the formation of reproductive organs. Although cdc2Pa transcripts were abundant in the epicotyl and thus likely in the apical meristem, we observed no strict coupling of expression to cell division in embryos and seedlings.

A single p34cdc2 protein kinase (encoded by nimXcdc2) is required at G1 and G2 in Aspergillus nidulans

We have cloned and sequenced a homolog of cdc2 from Aspergillus nidulans that can complement the Schizosaccharomyces pombe cdc2-33 mutation. The gene was deleted and is required for continued nuclear DNA replication but not for mitochondrial DNA replication. Three different temperature-sensitive alleles were generated by reverse genetics. All of the mutations generate the nim phenotype of A. nidulans. The new gene was designated nimXcdc2 as it is not allelic to any of the other nim genes (nimA to nimW) of A. nidulans. Reciprocal shift experiments place an essential function for nimXcdc2 in G1 and G2. Antipeptide antibodies were generated that detect NIMXcdc2, and antisera were also generated to detect NIMEcyclinB. The two p34cdc2 protein species previously detected in A. nidulans, p34 and p37, both precipitate using NIMXcdc2 C-terminus-specific antibodies but only p34 co-precipitates with NIMEcyclinB. Dephosphorylation of denatured p34 converts it to the p37 form, showing p37 to be the non-phosphorylated form of NIMXcdc2. The phosphorylation of p34 is therefore associated with its interaction with NIMEcyclinB.