Epstein-Barr virus nuclear antigen 3C regulates cyclin A/p27 complexes and enhances cyclin A-dependent kinase activity

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for primary B-cell transformation. In this report we show that cyclin A, an activator of S phase progression, bound tightly to EBNA3C. EBNA3C interacted with cyclin A in vitro and associated with cyclin A complexes in EBV-transformed lymphoblastoid cell lines. Importantly, EBNA3C stimulated cyclin A-dependent kinase activity and rescued p27-mediated inhibition of cyclin A/Cdk2 kinase activity by decreasing the molecular association between cyclin A and p27 in cells. Additionally, phosphorylation of the retinoblastoma protein, a major regulator of cell cycle progression, was enhanced both in vitro and in vivo in the presence of EBNA3C. Cyclin A interacted with a region of the carboxy terminus of EBNA3C, shown to be important both for stimulation of cyclin A-dependent kinase activity and for cell cycle progression. This provides the first evidence of an essential EBV latent antigen's directly targeting a cell cycle regulatory protein and suggests a novel mechanism by which EBV deregulates the mammalian cell cycle, which is of critical importance in B-cell transformation.     

Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes.

We have recently shown that two proteins, proliferating cell nuclear antigen (PCNA) and p21, are associated with cyclin D. Here we show that PCNA and p21 are common components of a wide variety of cyclin/cyclin-dependent kinase complexes in nontransformed cells. These include kinase complexes containing cyclin A, cyclin B, and cyclin D, associated either with CDC2, CDK2, CDK4, or CDK5. We show that PCNA and p21 form separate quaternary complex with each cyclin/CDK and that these quaternary complexes contain a substantial, if not major, fraction of the cell cycle kinases in asynchronously growing cells. These results suggest that PCNA and p21 may perform a common function for all these kinases.     

Cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF

In the clam, Spisula, two previously described proteins known as cyclin A and B display the unusual property of selective proteolytic degradation at the end of each mitosis. We show here that clam oocytes and embryos contain a cdc2 protein kinase. This protein kinase is a component of the M phase promoting factor (MPF) in frog eggs and the M phase-specific histone H1 kinase in starfish. Clam cdc2 is found in association with both cyclin A and B, probably not as a trimolecular association, but as separate cdc2/cyclin A and cdc2/cyclin B complexes. Clam cdc2 and the associated cyclins bind to p13suc1-Sepharose. The p13-bound complex, and also anti-cyclin A or B immunoprecipitates, each display cell cycle-dependent histone H1 kinase activity. We suggest that in addition to the cdc2 protein kinase, the cyclins are further components of the M phase promoting factor and that cyclin proteolysis provides the mechanism of MPF inactivation and thus exit from mitosis.     

Accelerated cell cycle progression in osteoblasts overexpressing the c-fos proto-oncogene: induction of cyclin A and enhanced CDK2 activity

Transgenic mice overexpressing the c-Fos oncoprotein develop osteosarcomas that are associated with deregulated expression of cell cycle genes. Here we have generated osteoblast cell lines expressing c-fos under the control of a tetracycline-regulatable promoter to investigate the role of c-Fos in osteoblast cell cycle control in vitro. Three stable subclones, AT9.2, AT9.3, and AT9.7, derived from MC3T3-E1 mouse osteoblasts, expressed high levels of exogenous c-fos mRNA and protein in the absence of tetracycline. Functional contribution of ectopic c-Fos to AP-1 complexes was confirmed by electromobility shift assays and transactivation of AP-1 reporter constructs. Induction of exogenous c-Fos in quiescent AT9.2 cells caused accelerated S-phase entry following serum stimulation, resulting in enhanced growth rate. Ectopic c-Fos resulted in increased expression of cyclins A and E protein levels, and premature activation of cyclin A-, cyclin E-, and cyclin-dependent kinase (CDK) 2-associated kinase activities, although cyclin D levels and CDK4 activity were not affected significantly in these cell lines. The enhanced CDK2 kinase activity was associated with a rapid, concomitant dissociation of p27 from CDK2-containing complexes. Deregulated cyclin A expression and CDK2 activity was also observed in primary mouse osteoblasts overexpressing c-Fos, but not in fibroblasts, and c-Fos transgenic tumor-derived osteosarcoma cells constitutively expressed high levels of cyclin A protein. These data suggest that overexpression of c-Fos in osteoblasts results in accelerated S phase entry as a result of deregulated cyclin A/E-CDK2 activity. This represents a novel role for c-Fos in osteoblast growth control and may provide c-Fos-overexpressing osteoblasts with a growth advantage during tumorigenesis.     

Cyclin A- and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts.

Cyclins are proteins which are synthesized and degraded in a cell cycle-dependent fashion and form integral regulatory subunits of protein kinase complexes involved in the regulation of the cell cycle. The best known catalytic subunit of a cyclin-dependent protein kinase complex is p34cdc2. In the cell, cyclins A and B are synthesized at different stages of the cell cycle and induce protein kinase activation with different kinetics. The kinetics of activation can be reproduced and studied in extracts of Xenopus eggs to which bacterially produced cyclins are added. In this paper we report that in egg extracts, both cyclin A and cyclin B associate with and activate the same catalytic subunit, p34cdc2. In addition, cyclin A binds a less abundant p33 protein kinase related to p34cdc2, the product of the cdk2/Eg1 gene. When complexed to cyclin B, p34cdc2 is subject to transient inhibition by tyrosine phosphorylation, producing a lag between the addition of cyclin and kinase activation. In contrast, p34cdc2 is only weakly tyrosine phosphorylated when bound to cyclin A and activates rapidly. This finding shows that a given kinase catalytic subunit can be regulated in a different manner depending on the nature of the regulatory subunit to which it binds. Tyrosine phosphorylation of p34cdc2 when complexed to cyclin B provides an inhibitory check on the activation of the M phase inducing protein kinase, allowing the coupling of processes such as DNA replication to the onset of metaphase. Our results suggest that, at least in the early Xenopus embryo, cyclin A-dependent protein kinases may not be subject to this checkpoint and are regulated primarily at the level of cyclin translation.     

Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase.

Exit from metaphase of the cell cycle requires inactivation of MPF, a stoichiometric complex between the cdc2 catalytic and the cyclin B regulatory subunits, as well as that of cyclin A-cdc2 kinase. Inactivation of both complexes depends on proteolytic degradation of the cyclin subunit, yet cyclin proteolysis is not sufficient to inactivate the H1 kinase activity of cdc2. Genetic evidence strongly suggests that type 1 phosphatase plays a key role in the metaphase-anaphase transition of the cell cycle. Here we report that inhibition of both type 1 and type 2A phosphatases by okadaic acid allows cyclin degradation to occur, but prevents cdc2 kinase inactivation. Complete inhibition of type 2A phosphatase alone is not sufficient to prevent cdc2 kinase inactivation following cyclin proteolysis. We show further that residue 161 of cdc2 is phosphorylated in active cyclin A or cyclin B complexes at metaphase, whilst unassociated cdc2 is not phosphorylated. Proteolysis of cyclin releases a free cdc2 subunit, which subsequently undergoes dephosphorylation and then migrates more slowly than its Thr161 phosphorylated counterpart in Laemmli gels. Removal of phosphothreonine 161 requires cyclin proteolysis. However, it does not occur even after cyclin proteolysis, when both type 1 and type 2A phosphatases are inhibited. We conclude that both cyclin degradation and dephosphorylation of Thr161 on cdc2, catalysed at least in part by type 1 phosphatase, are required to inactivate either cyclin B- or cyclin A-cdc2 kinases and thus for cells to exit from M phase.     

Cyclin E2, a Novel G1 Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers

A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27^Kip1 and p21^Cip1. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G₁/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G₁, demonstrating that cyclin E2 may be rate limiting for G₁ progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.     

Roughex mediates G(1) arrest through a physical association with cyclin A

Differentiation in the developing Drosophila eye requires synchronization of cells in the G(1) phase of the cell cycle. The roughex gene product plays a key role in this synchronization by negatively regulating cyclin A protein levels in G(1). We show here that coexpressed Roughex and cyclin A physically interact in vivo. Roughex is a nuclear protein, while cyclin A was previously shown to be exclusively cytoplasmic during interphase in the embryo. In contrast, we demonstrate that in interphase cells in the eye imaginal disk cyclin A is present in both the nucleus and the cytoplasm. In the presence of ectopic Roughex, cyclin A becomes strictly nuclear and is later degraded. Nuclear targeting of both Roughex and cyclin A under these conditions is dependent on a C-terminal nuclear localization signal in Roughex. Disruption of this signal results in cytoplasmic localization of both Roughex and cyclin A, confirming a physical interaction between these molecules. Cyclin A interacts with both Cdc2 and Cdc2c, the Drosophila Cdk2 homolog, and Roughex inhibits the histone H1 kinase activities of both cyclin A-Cdc2 and cyclin A-Cdc2c complexes in whole-cell extracts. Two-hybrid experiments suggested that the inhibition of kinase activity by Roughex results from competition with the cyclin-dependent kinase subunit for binding to cyclin A. These findings suggest that Roughex can influence the intracellular distribution of cyclin A and define Roughex as a distinct and specialized cell cycle inhibitor for cyclin A-dependent kinase activity.     

A cdc2-related kinase oscillates in the cell cycle independently of cyclins G2/M and cdc2.

The Eg1 gene in Xenopus laevis is related in sequence to the cdc2+ gene. We show here that the Eg1 gene product (cdk2) possesses histone H1 protein kinase activity and binds to PSTAIR antibodies as well as to Sepharose beads linked to the 13-kDa product of the suc 1 gene (p13suc1). Eg1 protein kinase is active only in an Mr approximately 200,000 complex with other proteins but is not associated with any of the three known Xenopus mitotic cyclins or with any newly synthesized protein in egg extracts that exhibit cell cycle oscillations in vitro. The protein kinase activity of Eg1 oscillates in the mitotic cell cycle, being high in M-phase and low in interphase. Hyperactivation of cdc2 kinase by the addition of cyclin A has no effect on the activity or oscillatory behavior of Eg1. Inhibition of cdc2 kinase activation by emetine or RNase treatment of oscillating extracts does not inhibit the activation of Eg1 but does block deactivation normally seen during exit from mitosis. These results indicate that Eg1 is regulated by a cell cycle clock independently of cyclin and cdc2 kinase.     

Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.     

The adenovirus E1A-associated kinase consists of cyclin E-p33cdk2 and cyclin A-p33cdk2.

The adenovirus E1A oncoproteins form stable complexes with several cellular proteins. Association of E1A with these proteins has been shown to be important for the oncogenic potential of E1A. Several of these proteins have been identified and include the product of the retinoblastoma gene and a key cell cycle regulatory protein, cyclin A. E1A also associates with a potent histone H1 kinase. The two major components of this activity are the cyclin E-p33cdk2 and cyclin A-p33cdk2 complexes. Both the cyclin E-p33cdk2 and cyclin A-p33cdk2 complexes have been implicated in regulatory events controlling entry into or passage through DNA synthesis. Although the architecture of such interactions remains unclear, it is likely that by targeting such complexes, adenovirus is affecting some aspect of cell cycle control.     

Cyclin E2, the cycle continues

The eukaryotic cell cycle is regulated by a family of serine/threonine protein kinases known as cyclin-dependent kinases (CDKs). The activation of a CDK is dependent on its association with a cyclin regulatory subunit. The formation of distinct cyclin-CDK complexes controls the progression through the first gap phase (G(1)) and initiation of DNA synthesis (S phase). These complexes are in turn regulated by protein phosphorylation and cyclin-dependent kinase inhibitors (CKIs). Cyclin E2 has emerged as the second member of the E-type cyclin family. Cyclin E2-associated kinase activity is regulated in a cell cycle dependent manner with peak activity at the G(1) to S transition. Ectopic expression of cyclin E2 in human cells accelerates G(1), suggesting that cyclin E2 is rate limiting for G(1) progression. Although the pattern and level of cyclin E2 expression in some primary tumor and normal tissue RNAs are distinct from cyclin E1, both E-type cyclins appear to have inherent functional redundancies. This functional redundancy has facilitated the rapid characterization of cyclin E2 and uncovered unique features associated with each E-type cyclin.     

Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.     

Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport

We have used immunofluorescence staining to study the subcellular distribution of cyclin A and B1 during the somatic cell cycle. In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards. Cyclin A may associated with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin B1 accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown. In mitotic cells, cyclin B1 associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus. Cyclin A is degraded during metaphase and cyclin B1 is precipitously destroyed at the metaphase----anaphase transition. Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin B1 are associated with PSTAIRE-containing proteins. The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein. Cyclin B1 is associated with p34cdc2 in the cytoplasm. Thus we propose that the different localization of cyclin A and cyclin B1 in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIRE-containing protein kinase subunit.     

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.     

Activation of p34cdc2 kinase by cyclin A

Functional clam cyclin A and B proteins have been produced using a baculovirus expression system. Both cyclin A and B can induce meiosis I and meiosis II in Xenopus in the absence of protein synthesis. Half-maximal induction occurs at 50 nM for cyclin A and 250 nM for cyclin B. Addition of 25 nM cyclin A to activated Xenopus egg extracts arrested in the cell cycle by treatment with RNase or emetine activates cdc2 kinase to the normal metaphase level and stimulates one oscillatory cell cycle. High levels of cyclin A cause marked hyperactivation of cdc2 kinase and a stable arrest at the metaphase point in the cell cycle. Kinetic studies demonstrate the concentration of cyclin A added does not affect the 10 min lag period required for kinase activation or the timing of maximal activity, but does control the rate of deactivation of cdc2 kinase during exit from mitosis. In addition, exogenous clam cyclin A inhibits the degradation of both A- and B-type endogenous Xenopus cyclins. These results define a system for investigating the biochemistry and regulation of cdc2 kinase activation by cyclin A.     

The tumour suppressor gene product APC blocks cell cycle progression from G0/G1 to S phase.

The APC gene is mutated in familial adenomatous polyposis (FAP) as well as in sporadic colorectal tumours. The product of the APC gene is a 300 kDa cytoplasmic protein associated with the adherence junction protein catenin. Here we show that overexpression of APC blocks serum-induced cell cycle progression from G0/G1 to the S phase. Mutant APCs identified in FAP and/or colorectal tumours were less inhibitory and partially obstructed the activity of the normal APC. The cell-cycle blocking activity of APC was alleviated by the overexpression of cyclin E/CDK2 or cyclin D1/CDK4. Consistent with this result, kinase activity of CDK2 was significantly down-regulated in cells overexpressing APC although its synthesis remained unchanged, while CDK4 activity was barely affected. These results suggest that APC may play a role in the regulation of the cell cycle by negatively modulating the activity of cyclin-CDK complexes.     

Permanent growth arrest in irradiated human fibroblasts

Exposure of human fibroblasts to doses of ionizing radiation sufficient to cause a permanent growth arrest repressed the expression of genes induced late during G(0)/G(1)-phase traverse, including both cyclin A and cyclin E. In addition, radiation prevented the cell cycle-dependent activation of cyclin D1-associated kinase activity and the subsequent phosphorylation of the RB tumor suppressor protein. Exposure to radiation did not alter the cellular levels of cyclin D1 protein, nor did it alter the formation of cyclin D1-CDK4 complexes. Surprisingly, the repression of cyclin D1-associated kinase activity in damaged mitogen-stimulated quiescent cells could not be accounted for by a relative increase in the association of CDKN1A (also known as p21(Cip1)) with cyclin D1 complexes, nor was cyclin D1 activity targeted by increased levels of CDKN1A in irradiated, logarithmically growing cultures under conditions where cyclin A activity was acutely repressed. Therefore, a radiation-induced permanent growth arrest is mediated by pathways that are distinct from those that cause cell cycle delay in damaged cells involving repression of cyclin-dependent kinase activity by CDKN1A.