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PubMed1992

Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle.

Human cyclin E, originally identified on the basis of its ability to function as a G1 cyclin in budding yeast, associated with a cell cycle-regulated protein kinase in human cells. The cyclin E-associated kinase activity peaked during G1, before the appearance of cyclin A, and was diminished during exit from the cell cycle after differentiation or serum withdrawal. The major cyclin E-associated kinase in human cells was Cdk2 (cyclin-dependent kinase 2). The abundance of the cyclin E protein and the cyclin E-Cdk2 complex was maximal in G1 cells. These results provide further evidence that in all eukaryotes assembly of a cyclin-Cdk complex is an important step in the biochemical pathway that controls cell proliferation during G1.

Animals↗
PubMed1994

Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition.

Progression through the cell cycle is monitored at two major points: during the G1/S and the G2/M transitions. In most cells, the G2/M transition is regulated by the timing of p34cdc2 dephosphorylation which results in the activation of the kinase activity of the cdc2-cyclin B complex. The timing of p34cdc2 dephosphorylation is determined by the balance between the activity of the kinase that phosphorylates p34cdc2 (wee1 in human cells) and the opposing phosphatase (cdc25C). Both enzymes are regulated and it has been shown that cdc25C is phosphorylated and activated by the cdc2-cyclin B complex. This creates a positive feed-back loop providing a switch used to control the onset of mitosis. Here, we show that another member of the human cdc25 family, cdc25A, undergoes phosphorylation during S phase, resulting in an increase of its phosphatase activity. The phosphorylation of cdc25A is dependent on the activity of the cdc2-cyclin E kinase. Microinjection of anti-cdc25A antibodies into G1 cells blocks entry into S phase. These results indicate that the cdc25A phosphatase is required to enter S phase in human cells and suggest that this enzyme is part of an auto-amplification loop analogous to that described at the G2/M transition. We discuss the nature of the in vivo substrate of the cdc25A phosphatase in S phase and the possible implications for the regulation of S phase entry.

Bisbenzimidazole↗
PubMed1992

Phosphorylation of the retinoblastoma protein by cdk2.

The retinoblastoma gene product (the RB protein) is phosphorylated in a cell cycle-dependent manner and this modification is believed to be important for cells to progress through the cell cycle. We found that purified cdk2 (cyclin-dependent kinase/cell division kinase 2) can phosphorylate the RB protein in vitro at the sites phosphorylated in the cell. The timing of activation of cdk2 in the cell cycle was similar to that of the onset of phosphorylation of the RB protein. The kinase coprecipitated with the RB protein also exhibited a similar substrate specificity to cdk2 and a similar time course of activation during the cell cycle. We further showed that cdk2 formed a complex with the RB protein in vitro and that its formation was not competitively inhibited by the simian virus 40 large T antigen. These observations suggest that cdk2 or a cdk2-related protein is involved in the cell cycle-dependent phosphorylation of the RB protein.

CDC2-CDC28 Kinases↗
PubMed1992

Cyclin A recruits p33cdk2 to the cellular transcription factor DRTF1.

Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.

Base Sequence↗
PubMed1995

Identification of a Myc-dependent step during the formation of active G1 cyclin-cdk complexes.

Activation of conditional alleles of Myc can induce proliferation in quiescent cells. We now report that induction of Myc in density-arrested fibroblasts triggers rapid hyperphosphorylation of the retinoblastoma protein and activation of both cyclin D1- and cyclin E-associated kinase activities in the absence of significant changes in the amounts of cyclin-cdk complexes. Kinase activation by Myc is blocked by inhibitors of transcription and requires intact DNA binding and heterodimerization domains of Myc. Activation of cyclin E-cdk2 kinase in serum-starved cells occurs in two steps. The first is induced by Myc and involves the release of a 120 kDa cyclin E-cdk2 complex from a 250 kDa inactive complex that is present in starved cells. This is necessary, but not sufficient, to generate full kinase activity, as cdc25 phosphatase activity is limiting in the absence of external growth factors. In vivo cdc25 activity can be supplied by the addition of growth factors. In vitro recombinant cdc25a strongly activates the 120 kDa, but only poorly activates the 250 kDa cyclin E-cdk2 complex. Our data show that two distinct signals, one of which is supplied by Myc, are necessary for consecutive steps during growth factor-induced formation of active cyclin E-cdk2 complexes in G(o)-arrested rodent fibroblasts.

Animals↗
PubMed1995

Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells.

Studies on the molecular mechanisms underlying neuronal differentiation are frequently performed using cell lines established from neuroblastomas. In this study we have used mouse N1E-115 neuroblastoma cells that undergo neuronal differentiation in response to DMSO. During differentiation, cyclin-dependent kinase (cdk) activities decline and phosphorylation of the retinoblastoma gene product (pRb) is lost, leading to the appearance of a pRb-containing E2F DNA-binding complex. The loss of cdk2 activity is due to a decrease in cdk2 abundance whereas loss of cdk4 activity is caused by strong association with the cdk inhibitor (CKI) p27KIP1 and concurrent loss of cdk4 phosphorylation. Moreover, neuronal differentiation can be induced by overexpression of p27KIP1 or pRb, suggesting that inhibition of cdk activity leading to loss of pRb phosphorylation, is the major determinant for neuronal differentiation.

Animals↗
PubMed1994

Interferon treatment enhances the expression of underphosphorylated (biologically-active) retinoblastoma protein in human papilloma virus-infected cells through the inhibitory TGF beta 1/IFN beta cytokine pathway.

Interferons (IFN) regulate transcription of certain genes playing a role in cell proliferation. Targets of IFN action may include tumor suppressor genes such as the retinoblastoma (RB) gene and cytokines such as transforming growth factor beta 1 (TGF beta 1) and IFN beta which are inhibitors of epithelial cell proliferation. Using reverse transcription followed by PCR amplification, an increase of those growth inhibitory gene mRNA levels (TGF beta 1, IFN beta and RB) were found after interferon treatment in condylomas harboring non-oncogenic human papilloma virus (HPV 6/11) types, in an oncogenic HPV 16-containing cell line, and in a HPV negative, epidermoid carcinoma cell line. In addition, immunodetection by Western blot demonstrated a higher proportion of underphosphorylated (active form) retinoblastoma gene protein (pRB) after IFN treatment due to the decrease in the phosphorylating cdc2 kinase levels. Changes in the phosphorylation pattern of pRB together with the increased expression of those inhibitory genes represent a growth inhibited state in those cells as demonstrated by diminished c-myc expression. Since the extent of c-myc inhibition was significantly lower in the case of oncogenic HPV infection, a role of viral oncoproteins in abrogation of the antiproliferative effect of IFN therapy could be considered. These results demonstrate a new mechanism via which IFNs exert their antiproliferative effect on HPV-infected cells by affecting the expression and phosphorylation of the RB tumor suppressor gene, through the inhibitory TGF beta 1/IFN beta cytokine pathway.

Blotting, Western↗
PubMed1995

Radiation-induced cell cycle arrest compromised by p21 deficiency.

The protein p21 is a dual inhibitor of cyclin-dependent kinases and proliferating-cell nuclear antigen (PCNA), both of which are required for passage through the cell cycle. The p21 gene is under the transcriptional control of p53 (ref. 5), suggesting that p21 might promote p53-dependent cell cycle arrest or apoptosis. p21 has also been implicated in cell senescence and in cell-cycle withdrawal upon terminal differentiation. Here we investigate the role of p21 in these processes using chimaeric mice composed partly of p21-/- and partly of p21+/+ cells. Immunohistochemical studies of the p21+/+ and p21-/- components of adult small intestine indicated that deletion of p21 has no detectable effect on the migration-associated differentiation of the four principal intestinal epithelial cell lineages or on p53-dependent apoptosis following irradiation. However, p21-/- mouse embryo fibroblasts are impaired in their ability to undergo G1 arrest following DNA damage.

Animals↗
PubMed1995

Differences in substrate specificity between Cdk2-cyclin A and Cdk2-cyclin E in vitro.

Cyclin-dependent kinase 2 (Cdk2), when bound to either cyclin A or cyclin E, recognizes the Ser/Thr-Pro-X-basic amino acid (motif A) as a phosphorylation site. In this study, we designed several peptides based on motif A and examined the substrate specificity of Cdk2-cyclin A and Cdk2-cyclin E using these peptides. Peptides containing a proline residue in the sequence Pro-X-Thr-Pro-X-basic amino acid (motif B) had higher affinity for both Cdk2 complexes than peptides containing motif A. Furthermore, differences in substrate affinity between the two Cdk2 complexes were caused by a proline residue adjacent to or three positions before the threonine residue. Similarly, the presence of different basic amino acids in motif B also had different effects on affinity for each complex. We demonstrate the possibility that the substrate specificity of Cdk2 bound to cyclin might be regulated by the species of cyclin.

Amino Acid Sequence↗
PubMed1994

Inactivation of a Cdk2 inhibitor during interleukin 2-induced proliferation of human T lymphocytes.

Peripheral blood T lymphocytes require two sequential mitogenic signals to reenter the cell cycle from their natural, quiescent state. One signal is provided by stimulation of the T-cell antigen receptor, and this induces the synthesis of both cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through G1. Antigen receptor stimulation alone, however, is insufficient to promote activation of G1 cyclin-Cdk2 complexes. This is because quiescent lymphocytes contain an inhibitor of Cdk2 that binds directly to this kinase and prevents its activation by cyclins. The second mitogenic signal, which can be provided by the cytokine interleukin 2, leads to inactivation of this inhibitor, thereby allowing Cdk2 activation and progression into S phase. Enrichment of the Cdk2 inhibitor from G1 lymphocytes by cyclin-CDK affinity chromatography indicates that it may be p27Kip1. These observations show how sequentially acting mitogenic signals can combine to promote activation of cell cycle proteins and thereby cause cell proliferation to start. CDK inhibitors have been shown previously to be induced by signals that negatively regulate cell proliferation. Our new observations show that similar proteins are down-regulated by positively acting signals, such as interleukin 2. This finding suggests that both positive and negative growth signals converge on common targets which are regulators of G1 cyclin-CDK complexes. Inactivation of G1 cyclin-CDK inhibitors by mitogenic growth factors may be one biochemical pathway underlying cell cycle commitment at the restriction point in G1.

Adult↗
PubMed1995

Multiple modes of ligand recognition: crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors, olomoucine and isopentenyladenine.

Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(delta 2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties.

Adenine↗
PubMed1995

Immortalization of human fibroblasts by SV40 large T antigen results in the reduction of cyclin D1 expression and subunit association with proliferating cell nuclear antigen and Waf1.

Protein complexes containing cyclins and cyclin-dependent protein kinases (cdks) have been shown to be rearranged in both spontaneous and viral tumor antigen-transformed cells. We have examined G1- and S-phase cyclin/cdk complexes as a function of the neoplastic progression of human diploid fibroblasts transfected with the SV40 large T antigen. We find that the expression of cyclin D1 and its association with proliferating cell nuclear antigen (PCNA) and Waf1 remain unchanged in precrisis human fibroblasts transfected with SV40 large T antigen. However, in these same cells the association of cdk4 with cyclin D1, PCNA, and Waf1 is disrupted. Upon immortalization, cyclin D1 protein expression is decreased, and binding of both PCNA and Waf1 with the remaining cyclin D1 is reduced. In contrast, large T antigen increased the expression of cyclin A and cyclin E proteins in both precrisis and immortal cells and did not reduce the binding of PCNA or Waf1 to either cdk2 or cyclin A proteins. These results show that large T-antigen expression in human fibroblasts selectively uncouples cyclin D1 from cdk4, and subsequent immortalization of these cells results in additional changes to the cyclin D1-dependent cell cycle regulatory pathways.

Antigens, Polyomavirus Transforming↗
PubMed1992

Human cyclin-dependent kinase 2 is activated during the S and G2 phases of the cell cycle and associates with cyclin A.

We have analyzed the cell cycle regulation of human cyclin-dependent kinase 2 (CDK2), a protein closely related to the cell cycle-regulatory protein kinase CDC2. We find that CDK2 activity, like that of CDC2, oscillates during the cell cycle in cultured mammalian fibroblasts. Unlike CDC2 activity (which peaks during mitosis), CDK2 activity rises in late G1 or early S phase and declines during mitosis. Active S-phase CDK2 migrates in multiple large complexes on gel filtration, and CDK2 in one of these complexes is associated with cyclin A. These findings suggest that CDK2 and CDC2, in association with distinct cyclins, regulate separate functions in the mammalian cell cycle.

Amino Acid Sequence↗
PubMed1995

Deregulated expression of cyclin D1 and other cell cycle-related genes in carcinogen-induced rat mammary tumors.

Dysregulation of cyclin expression has been reported for several human malignancies, including breast cancer. To further investigate the role of cyclin genes in mammary tumorigenesis we analyzed the expression of cyclins D1, E and A and other cell cycle-related proteins in a series of nine N-methyl-N-nitrosourea-induced primary rat mammary tumors. Western blot analysis revealed a 10- to 15-fold increase in the level of cyclin D1 protein in most (7/9) of the tumors, when compared with normal rat mammary gland. The two tumors that did not show this increase also displayed negligible levels of the retinoblastoma protein. A moderate increase, 1.5- to 2-fold, in the level of cyclin E was observed in four tumors and three tumors displayed abnormal low molecular weight cyclin E-related proteins. None of the tumors showed amplification of the cyclin D1 or E genes when studied by Southern blot analysis. All nine tumors showed a 2- to 6-fold increase in the level of cyclin A protein. Most of the tumors also displayed a marked increase in levels of the CDK2 and CDK4 proteins. These changes did not appear to be simply a consequence of increased cell proliferation, as assessed by proliferating cell nuclear antigen analysis. Thus, aberrant expression of cyclins and other cyclin-related genes occurs frequently in mammary tumorigenesis in both rodents and humans.

Animals↗
PubMed1994

Selective repression of growth-regulating cdk2, cyclin E and E2F1 genes in human cell senescence.

Normal human diploid TIG-1 fibroblasts underwent replicative senescence around 64-68 population doubling levels (PDL) by the irreversible serum-unresponsive G1-growth arrest. Repression of growth-promoting genes was searched in this study. The RT-PCR and Western blot analyses have shown that in senescent TIG-1 cells at PDL64-67, cdk2 and cyclin E were selectively repressed at the mRNA and protein levels even after serum stimulation, and cdc2 and cyclin A were less repressed than cdk2 and cyclin E. Such a specific lack of cdk2 and cyclin E proteins correlated with unphosphorylation of the retinoblastoma gene product (pRB) in senescent cells. Transcription factor E2F1 was also completely repressed at the mRNA and protein levels in senescent TIG-1 cells. Middle-passage cells exhibited active expressions of all the above genes and pRB phosphorylation. Therefore, the present results have indicated the selective repressions of cdk2, cyclin E and E2F1 in senescent cells.

Base Sequence↗
PubMed1995

A model of the G1 phase of the cell cycle incorporating cyclin E/cdk2 complex and retinoblastoma protein.

A mathematical model of cyclin E, cdk2 and retinoblastoma protein control of the G1 phase of the human cell cycle is proposed. The model includes retinoblastoma (Rb) protein phosphorylation by a cyclin E/cdk2 complex and its subsequent dephosphorylation at the end of the cell cycle. The numerical solutions to this model demonstrates the cyclic behavior of the cyclin E/cdk2 complex, with and without Rb function, cell cycle. This model suggests an inhibition of cyclin E/cdk2 complex formation (or its activation) by hypophosphorylated retinoblastoma protein. The experimental results of cell cycle arrest upon injection of transforming growth factor-beta, alpha-interferon or D-erythro-sphingosine during G1 phase are reproduced. Cell cycle behavior predicted by this model for increasing the concentration of hypophosphorylated retinoblastoma protein during the G1 phase is discussed. Additional results are obtained by numerical simulation.

CDC2-CDC28 Kinases↗