Characterization of the in vitro reconstituted cyclin A or B1-dependent cdk2 and cdc2 kinase activities

Human cyclins A and B1 were assembled with the cdk2 or cdc2 protein to reconstitute their respective kinase activities in vitro. Both cyclins complemented either cdk2 or cdc2, yielding kinase activities that supported the phosphorylation of histone H1. Activation of cdk2-catalyzed H1 kinase activity by cyclin A required a 10-min preincubation of the two components, whereas cdc2 kinase supported phosphate incorporation without a detectable time lag upon the addition of cyclin B1, suggesting a slower association rate of cdk2 with cyclin A compared with cdc2 and cyclin B1. Both cdk2 and cyclin A, as well as cdc2 and cyclin B1, formed stable complexes in the absence of ATP and substrate that could be isolated after glycerol gradient centrifugation. Incubation of the isolated complexes with ATP and histone H1 supported the phosphorylation of the substrate. Cyclin A-activated cdk2 or cdc2 phosphorylated p107, a pRB-related cellular protein, 10 times more effectively than the cyclin B1-complexed kinases. This was most likely due to a direct association of cyclin A with p107 (Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. (1992) Science 255, 85-87; Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D., and Harlow, E. (1992) Science 255, 87-90). The reconstituted cdc2-cyclin B1 complex incorporated 4-5-fold more phosphate into the p34 subunit of the three-subunit (p70, p34, and p14) human single-stranded DNA-binding protein (also called RP-A), a DNA replication and DNA repair factor, than cdc2-cyclin A. No detectable phosphorylation of the p34 protein was observed with cdk2 complexed with either cyclin B1 or A. These data indicate that both cyclins as well as the catalytic subunits are important factors in controlling the rate of phosphorylation of a given substrate. The cyclin-activated cdc2 family kinases may target their cellular substrates through cyclin-mediated protein-protein interactions.     

Molecular evolution of cdc2 pseudogenes in spruce (Picea)

The p34cdc2 protein and other cyclin-dependent protein kinases (CDK) are important regulators of eukaryotic cell cycle progression. We have previously cloned a functional cdc2 gene from Picea abies and found it to be part of a family of related sequences, largely consisting of pseudogenes. We now report on the isolation of partial cdc2 pseudogenes from Picea engelmannii and Picea sitchensis, as well as partial functional cdc2 sequences from P. engelmannii, P. sitchensis and Pinus contorta. A high level of conservation between species was detected for these sequences. Phylogenetic analyses of pseudogene and functional cdc2 sequences, as well as the presence of shared insertions or deletions, support the division of most of the cdc2 pseudogenes into two subfamilies. New cdc2 pseudogenes appear to have been formed in Picea at a much higher rate than they have been obliterated by neutral mutations. The pattern of nucleotide changes in the cdc2 pseudogenes, as compared to a presumed ancestral functional cdc2 gene, was similar to that previously found in mammalian pseudogenes, with a strong bias for the transitions C to T and G to A, and the transversions C to A and G to T.     

Two functional soybean genes encoding p34cdc2 protein kinases are regulated by different plant developmental pathways

We have isolated two cDNA clones (cdc2-S5 and cdc2-S6) encoding p34cdc2 protein kinases, homologs of yeast cdc2/CDC28 genes, from a soybean nodule cDNA library. The two sequences share 90% sequence homology in the coding regions. The 5' and 3' noncoding regions are distinct from each other, however, indicating that at least two genes encode p34cdc2 protein kinases in soybean. Both sequences can rescue the cdc28 mutation in Saccharomyces cerevisiae but rescue it with different efficiency. Genomic Southern analysis showed the existence of two copies for each of these genes, which are not closely linked and are nonallelic. The relative expression level of the two soybean p34cdc2 genes varies in different tissues. Expression of cdc2-S5 is higher in roots and root nodules, whereas cdc2-S6 is more actively expressed in aerial tissues, indicating that regulation of these two p34cdc2 genes is coupled with plant developmental pathways. Expression of cdc2-S5 is, furthermore, enhanced after Rhizobium infection, whereas cdc2-S6 fails to respond, suggesting that cdc2-S5 plays a role in nodule initiation and organogenesis. This latter gene preferentially responds to auxin (alpha-naphthaleneacetic acid) treatment, indicating that phytohormones may be involved in the control of cell division mediated by Rhizobium infection. Thus, different p34cdc2 protein kinases may control cell division in different tissues in a multicellular organism and respond to different signals--e.g., phytohormones.     

Introduction of cyclin B induces activation of the maturation-promoting factor and breakdown of germinal vesicle in growing zebrafish oocytes unresponsive to the maturation-inducing hormone

When treated with 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), a natural maturation-inducing hormone in fishes, fully grown zebrafish oocytes are induced to mature via the activation of the maturation-promoting factor (MPF), which consists of cdc2 (a catalytic subunit) and cyclin B (a regulatory subunit). In contrast, 17alpha,20beta-DP is unable to induce growing (previtellogenic and vitellogenic) oocytes to mature. To know the reason growing oocytes fail to mature upon 17alpha,20beta-DP treatment, we investigated changes in the components of machinery responsible for MPF activation during zebrafish oogenesis. Immunoblotting experiments using monoclonal antibodies against cdc2, cyclin B, and cdk7 (an activator of cdc2) have revealed that the concentrations of cdc2 and cdk7 are almost constant during oogenesis. Cyclin B was present in mature oocytes but absent in growing and fully grown immature oocytes. These results, which are identical to those in goldfish, strongly suggest that cyclin B is synthesized from stored (masked) mRNA after 17alpha,20beta-DP stimulation and that its binding to the preexisting cdc2 allows cdk7 to activate MPF. Microinjection of cyclin B protein induced MPF activation and germinal vesicle breakdown in growing oocytes, as well as in fully grown oocytes, indicating that cdk7 present in growing oocytes is already active. Northern blot analysis revealed the presence of cyclin B mRNA in both previtellogenic and fully grown oocytes. These results indicate that, as in fully grown oocytes, growing oocytes are already equipped with the catalytic subunit of MPF (cdc2) and its activator (cdk7) and that the appearance of the regulatory subunit of MPF (cyclin B) is sufficient for initiating maturation. Therefore, the unresponsiveness of growing oocytes to 17alpha,20beta-DP is attributable to a deficiency in the processes leading to cyclin B synthesis, which include 17alpha,20beta-DP reception on the oocyte surface, subsequent signal transduction pathways, and unmasking the stored cyclin B mRNA.     

A cdc2-like kinase associated with commitment to division in Paramecium tetraurelia

Cell division in higher eukaryotes is mainly controlled by p34cdc2 or related kinases and by other components of these kinase complexes. We present evidence that cdc2-like kinases also occur in Paramecium. Two polypeptides reacted with an antibody directed against the perfectly conserved PSTAIR region found in cdc2 kinases in other eukaryotes. Only the less abundant peptide bound to p13suc1 from Schizosaccharomyces pombe. Using centrifugal elutriation to select cells on the basis of size, we isolated highly synchronous Paramecium G1 cells. With this procedure, we demonstrated that the p13suc1-associated cdc2-like histone H1 kinase was activated before cell division at the point of commitment to division in Paramecium. Further, we show that Paramecium cdc2-like proteins occurred principally as monomers and that these monomers were active as histone H1 kinases in vitro.     

cdc2 kinase-mediated phosphorylation of splicing factor SF2/ASF

SR proteins are a family of splicing factors which are important components of spliceosomes. Recent studies suggested that phosphorylation of SR protein might be a key event for the regulation of pre-mRNA splicing and is prevalent in metaphase cells. To investigate the role of cdc2 kinase in cell cycle-dependent phosphorylation of SR protein, we examined its phosphorylation of SF2/ASF, a representative SR protein. SF2/ASF was phosphorylated both by recombinant cdc2 kinase, a cdc2-cyclin B complex, and by cdc2 kinase immunoprecipitated from G2/M phase HeLa cells. In vitro phosphorylation and phosphopeptide mapping of several mutant proteins revealed that cdc2 kinase specifically phosphorylates the RS domain of SF2/ASF with serines 227, 238 and presumably 199 as major phosphorylation sites. These findings suggest the possibility that cdc2 kinase takes part in the cell cycle-dependent phosphorylation of SR protein which regulates the function of spliceosomes.     

Biogenic decomposition of bone collagens

In diapausing eggs of the silkworm Bombyx mori, embryonic cells are arrested at G2 phase. The ability to undertake cell division is resumed in the course of diapause termination caused by such a treatment as acclimation to 5 degrees C. As an initial trial to investigate the relationship between diapause and embryonic cell cycling, we have cloned and sequenced two Bombyx cDNAs encoding two distinct cdc2-related Ser/Thr protein kinases. One (Bm cdc2) encoded a 37.0 kDa protein which had all of the domains characteristic of other Cdc2 kinase. The other (Bcdrk) encoded a 45.1 kDa protein that was most similar to Drosophila and human cdc2-related protein kinases (Dcdrk protein and PISSRLE kinase). Northern blot analysis was carried out to examine levels of Bm cdc2 and Bcdrk mRNA during embryogenesis of non-diapause eggs. The result demonstrated that the mRNA level of Bm cdc2 appeared to correspond to the activity of nuclear/cellular division in non-diapause eggs, and that the developmental profile in the level of Bcdrk mRNA was somewhat different from that of Bm cdc2 mRNA.     

The prognostic significance of p34cdc2 and cyclin D1 protein expression in prostate adenocarcinoma

BACKGROUND: Cyclin-dependent kinases (CDK) and cyclins constitute the subunits of the maturation-promoting factor that controls the process of cell division. High levels of these proteins have been reported in human malignancies of the stomach, colon, breast, and lung, and have been implicated in aberrant cell division and dysregulated tumor growth. METHODS: p34cdc2 CDK and cyclin D1 (D1) protein expression were evaluated in 140 radical prostatectomy specimens harboring adenocarcinoma (PAC), using the respective monoclonal antibodies on archival tissue sections. In each case, slides stained with hematoxylin and eosin were examined for evaluation of Gleason's grade and pathologic stage. The DNA content of the tumors was determined by the Feulgen method with the CAS200 Image Analyzer (Cell Analysis Systems, Lombard, IL). Nuclear immunoreactivity for the two proteins was semiquantitatively scored, and results were correlated with Gleason's grade, stage, ploidy, metastatic status, and disease recurrence after radical prostatectomy. RESULTS: p34cdc2 was expressed in 84 of 140 PACs (60%) and correlated with high Gleason's grade (P = 0.0001), advanced pathologic stage (P = 0.01), nondiploid DNA content (P = 0.0001), and metastases (P = 0.04). On multivariate analysis using the Cox proportional hazards model, p34cdc2 immunoreactivity (P = 0.0001) and high Gleason's grade (P = 0.01) each independently predicted disease recurrence. When tumors were of low Gleason's grade and lacked p34cdc2 expression, 4 of 39 PACs (10%) recurred, as compared with 18 of 47 (38%) that recurred when tumors were of high Gleason's grade and expressed p34cdc2 protein. D1 was positive in 31 of 140 PACs (22%) and showed a trend (P = 0.07) of high Gleason's grade, but it did not reach statistical significance with any of the prognostic variables. In the majority of PACs expressing both p34cdc2 and D1 proteins, the adjacent benign prostate acini showed focal, scattered nuclear positivity of the basal and secretory epithelial cells. CONCLUSIONS: p34cdc2 is expressed in a majority of PACs and correlates with high Gleason's grade, advanced pathologic stage, nondiploid DNA content, and metastases. On multivariate analyses high Gleason's grade and p34cdc2 immunoreactivity predict disease recurrence independently of the pathologic stage. Thus, p34cdc2 appears to play a critical role in the evolution, proliferation, and spread of PACs and may be of prognostic value when applied to initial prostate tissue samples taken by needle biopsy.     

The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues

Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin-dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity. The purified kinase was found to phosphorylate not only cdc2, but also cdk2 and a divergent cdc2-like protein from Caenorhabditis, in chimeric complexes including both mitotic and G1/S cyclins. Extensive microsequencing of p40 did not reveal any convincing homology with any known protein. In contrast, p37 is the starfish homologue of the M015 gene product, a kinase previously cloned by homology probing from a Xenopus cDNA library. As expected, immunodepletion of the MO15 protein depleted Xenopus egg extracts of CAK (cdk-activating kinase) activity, which was recovered in immunoprecipitates. Taken together, the above results demonstrate that MO15 is a gene conserved throughout evolution (at least from echinoderms to vertebrates) that encodes the catalytic subunit of a protein kinase that activates cdc2-cdks complexes through phosphorylation of Thr161 (or its homologues).     

Control of cell division in eucaryotes

In eucaryotes, M-phase promoting factor (MPF) triggers meiosis in germ cells and mitosis in somatic cells. MPF is composed of two proteins of which one is homologous with the protein kinase encoded by gene cdc2 of Schizosaccharomyces pombe (p34cdc2) and the other is a cyclin whose concentration oscillates during the cell cycle. Inactivation of p34cdc2 (MPF) requires cyclin degradation, which occurs during the metaphase-anaphase transition of the M-phase. Cyclin degradation is not only associated with cell cycle progression, but is also required for this event. At the G2/M transition, p34cdc2 protein kinase is activated and catalyzes phosphorylation of numerous key proteins, thus enabling cell changes to occur. p34cdc2 undergoes multiple-site phosphorylation in a cell cycle-dependent manner. At onset of mitosis, the protein phosphatase cdc25 catalyzes dephosphorylation of the p34cdc2 kinase at the threonine 14 and tyrosine 15 sites. This event may be the rate-limiting step controlling onset of mitosis in cells of vertebrates. A second protein kinase, encoded by the proto-oncogene c-mos, acts as a cytostatic factor preventing cyclin degradation and keeping unfertilized eggs from progressing beyond the second meiotic metaphase.     

5-substituted 2-bromoindolo[3,2-b]quinoxalines. A class of potential antitumor agents with cdc25 phosphatase inhibitory properties

Several derivatives of 5-substituted 2-bromoindolo[3,2-b]quinoxaline were synthesized and characterized. The synthesized compounds were evaluated for their antitumor activity using the National Cancer Institute-in vitro-disease oriented antitumor screen and two biochemical mechanism-based screens (cdc2 kinase and cdc25 phosphatase). Compound 19 showed broad spectrum antitumor activity with full panel (MG-MID) GI50. TGI, and LC50 of 14.2, 31.6- and 66.2 microM, respectively. In addition it inhibited cdc2 kinase and cdc25 phosphatase with IC50's of 70 and 25 microM, respectively. Thus, compound 19 represents a model for compounds with potential antitumor activity and cdc25 phosphatase inhibitory properties.     

cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization

A protein kinase that phosphorylates a specific KSP sequence [K(S/T)PXK], which is abundant in high molecular weight neurofilament (NF) proteins, was identified and isolated from rat spinal cord. Characterization of this enzyme activity revealed a close relationship with p34cdc2 kinase with respect to its molecular mass (32.5 kDa by SDS/PAGE) and substrate specificities. It could phosphorylate a synthetic peptide analog of the simian virus 40 large tumor antigen, reportedly a specific substrate for p34cdc2 kinase. Histone (H1) and peptide analogs of the KSP sequence present in the C-terminal end of rat and mouse neurofilament proteins were phosphorylated. This kinase did not phosphorylate alpha-casein and peptide substrates of other known second messenger-dependent or -independent kinases. Dephosphorylated rat NF protein NF-H was strongly phosphorylated by the purified enzyme; NF proteins NF-M and native NF-H, but not NF-L, were slightly phosphorylated. Studies on synthetic peptide analogs of KSP repeats with substitution of specific residues, known to be present in the C-terminal regions of NF-H, revealed a consensus sequence of X(S/T)PXK, characteristic of the p34cdc2 kinase substrate. On Western blots, the enzyme was immunoreactive with antibody against the C-terminal end of cdc2 kinase (mouse) and neuronal cdc2-like kinase from rat but not with an antibody against the conserved PSTAIRE region of the p34cdc2 kinase. The antibody against the C-terminal end of cdc2 kinase could immunoprecipitate (immunodeplete) the purified kinase activity. Since the adult nervous system is composed primarily of postmitotic cells, the present observations indicate a nonmitotic role for this cdc2-like kinase activity. The effective phosphorylation of NF-H by this kinase suggests a function in axonal structure.     

Effects of phosphate on in vitro 2-cell block of AKR/N mouse embryos based on changes in cdc2 kinase activity and phosphorylation states

This study demonstrated the effects of phosphate on the 2-cell block of AKR/N mouse embryos at the molecular level and focused on changes in the kinase activity and the phosphorylation state of cdc2, which is shown to regulate the cell division cycle. Removal of phosphate from the culture medium dramatically increased developmental rates to the 4-cell (91.8%) and blastocyst (42.6%) stages compared with those of embryos cultured in 1.17 mM phosphate (3.3% and 0%, respectively). The rate of development to the 4-cell stage was significantly inhibited by 0.001 mM phosphate (p < 0.05), and no morula formation was observed at 1.0 mM. The patterns of cdc2 kinase activity during the first cell cycle in AKR/N embryos were similar to those of control MCH embryos, showing the highest activity at M phase and low activity during the interphase. The phosphorylated form of cdc2 increased during the interphase, indicating that the synthesis of cyclin B and accumulation of inactive pre-maturation-promoting factor (pre-MPF) as well as abrupt dephosphorylation of cdc2 at the first cleavage correlated with the activation of cdc2 kinase. When phosphate was absent, the activation pattern of cdc2 kinase during the second cell cycle in AKR/N embryos was similar to that in the first cell cycle. On the other hand, no dephosphorylation of cdc2 was observed and the kinase activity remained at a low level until 56 h after insemination in the presence of phosphate, although an increase in phosphorylated cdc2 was observed as in the phosphate-free group. Treatment of AKR/N embryos arrested at the 2-cell stage with okadaic acid resulted in the dephosphorylation and activation of cdc2, confirming the presence of a sufficient amount of pre-MPF. These results show that phosphate has a deteriorative effect on the in vitro development of AKR/N embryos and suggest that this effect was not on the synthesis of cyclin B but on the dephosphorylation of phosphorylated cdc2.     

Articular cartilage lesions of the knee

We have evaluated the role of various protein kinases on the induction of the gadd (growth arrest and DNA damage inducible) genes, using a panel of protein kinase inhibitors. Our data indicate that three different stress response pathways mediating gadd gene induction are most likely regulated by different protein kinases or combinations of protein kinases. The protein kinase inhibitor staurosporine and the temperature sensitive (ts) p34cdc2 mutant reduced induction by the alkylating agent methylmethane sulfonate (MMS) of the rodent gadd45 and gadd153 genes. However, staurosporine had no effect of the ionizing radiation (IR) induction of the human GADD45. Caffeine and 2-aminopurine, on the other hand, completely blocked this IR induction. Suramin, an antitumor drug that interferes with the interaction of growth factors with their receptors, inhibited the UV radiation induction of GADD45 and GADD153 but had no effect on the MMS and IR pathways. Elevated expression of gadd45 by medium depletion (starvation) was partially reduced by the addition of either genistein or tyrphostin, two protein tyrosine kinase inhibitors, while gadd153 was affected by tyrphostin only. Two inhibitors acting preferentially on cAMP-dependent protein kinase (PKA), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, HCl (H8) and protein kinase inhibitor (PKI), also had a moderate effect on the medium depletion-induced levels of both gadd genes. Thus, these varied effects of inhibitors on gadd gene responses point to important differences in the pathways controlling these responses.     

Increased ubiquitin expression suppresses the cell cycle defect associated with the yeast ubiquitin conjugating enzyme, CDC34 (UBC3). Evidence for a noncovalent interaction between CDC34 and ubiquitin

The yeast ubiquitin (Ub) conjugating enzyme CDC34 plays a crucial role in the progression of the cell cycle from the G1 to S phase. In an effort to identify proteins that interact with CDC34 we undertook a genetic screen to isolate genes whose increased expression suppressed the cell cycle defect associated with the cdc34-2 temperature-sensitive allele. From this screen, the poly-Ub gene UBI4 was identified as a moderately strong suppressor. The fact that the overexpression of a gene encoding a single Ub protein could also suppress the cdc34-2 allele indicated that suppression was related to the increased abundance of Ub. Ub overexpression was found to suppress two other structurally unrelated cdc34 mutations, in addition to the cdc34-2 allele. In all three cases, suppression depended on the expression of Ub with an intact carboxyl terminus. Only the cdc34-2 allele, however, could be suppressed by Ub with an amino acid substitution at lysine 48 which is known to be involved in multi-Ub chain assembly. Genetic results showing allele specific suppression of cdc34 mutations by various Ub derivatives suggested a specific noncovalent interaction between Ub and CDC34. Consistent with this prediction, we have shown by chemical cross-linking the existence of a specific noncovalent Ub binding site on CDC34. Together, these genetic and biochemical experiments indicate that Ub suppression of these cdc34 mutations results from the combined contributions of Ub-CDC34 thiol ester formation and a noncovalent interaction between Ub and CDC34 and therefore suggest that the correct positioning of Ub on a surface of the ubiquitin conjugating enzyme is a requirement of enzyme function.     

The Cdc14 phosphatase is functionally associated with the Dbf2 protein kinase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Cdc14 protein phosphatase and Dbf2 protein kinase have been implicated to act during late M phase, but their functions are not known. We report here that CDC14 is a low-copy suppressor of the dbf2-2 mutation at 37 degrees C. The kinase activity of Dbf2 accumulated at a high level, in vivo, during a cdc14 arrest and was also much higher in cdc14 mutant cells at the permissive temperature of growth, therefore in cycling mutant cells than in cycling wild-type cells. This correlated with the accumulation of the more slowly migrating form of Dbf2, previously shown to correspond to the hyperphosphorylated form of the protein. The finding that the dbf2-2 mutation could be rescued following overproduction of catalytically inactive forms of Cdc14 suggested that the control of Dbf2 activity by Cdc14 might be only indirect and independent of Cdc14 phosphatase activity. However, it was found that Cdc14 could form oligomers within the cell, thus leaving open the possibility that catalytically inactive Cdc14 might associate with wild-type Cdc14 and rescue dbf2-2 in a phosphatase-dependent manner. We confirmed that overexpression of CDC14 could rescue mutations in CDC15, which encodes another kinase also implicated to act in late M phase. Cells of a cdc15-2 dbf2-2 double mutant died at temperatures much lower than did either single mutant, whereas there was only a slight additive phenotype in the cdc14-1 dbf2-2 and cdc14-1 cdc15-2 double mutant cells. Finally, functional association between Cdc14 and Dbf2 (and also Cdc15) was confirmed by the finding that the cdc14, dbf2 and cdc15 mutations could be partially rescued by the addition of 1.2 M sorbitol to the culture medium. Our data are the first to demonstrate a functional link between Cdc14 and Dbf2 based on both biochemical and genetic information.