Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.     

Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes.

The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types. Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene. Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S. At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity. As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole. The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA. Under these conditions, the activated cells failed to enter the S phase of the cell cycle. Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition.     

Cdk7- and Cdc25A-independent dephosphorylation of Cdk2 during phorbol ester-mediated cell cycle arrest in U937 cells

The molecular mechanism underlying protein kinase C (PKC)-mediated cell cycle arrest is poorly understood. We undertook to characterize phorbol ester-activated PKC-mediated cell cycle arrest. Treatment with phorbol ester inhibited cell growth of human histiocytic lymphoma U937 cells with 83% of the cells arrested in G1 phase. Reduced activity of cdk2 correlated with cdk2 dephosphorylation and accumulation of cdk2 inhibitor p21Waf in phorbol ester-treated cells. Dephosphorylation of cdk2 was not associated with cdk7 and cdc25A activity in phorbol ester-treated cells. Protein phosphatase inhibitor assays suggest that the dephosphorylation of cdk2 results in the activation of a specific protein tyrosine phosphatase. Thus, dephosphorylation of cdk2 as well as accumulation of cdk2 inhibitor is likely to contribute to the G1 phase arrest in phorbol ester-treated in U937 cells.     

Cyclin E-cdk2 activation is associated with cell cycle arrest and inhibition of DNA replication induced by the thymidylate synthase inhibitor Tomudex

Tomudex (ZD1694) is a specific antifolate-based thymidylate synthase inhibitor active in a variety of solid tumor malignancies. Studies were carried out in vitro to evaluate downstream molecular alterations induced as a consequence of the potent and sustained inhibition of thymidylate synthase by Tomudex. Twenty-four hours following the initial 2-h treatment with Tomudex, human A253 head and neck squamous carcinoma cells, not expressing p53 and p21(WAF1), were accumulated with DNA content characteristic of early S phase of the cell cycle with a concomitant reduction of cells in G1 and G2/M phases. The changes in cyclin and cdk protein expression and their kinase activities were examined in control and drug-treated A253 cells. Tomudex treatment resulted in the decrease in p27(kip1) expression, with an increase in cyclin E and cdk2 protein expression and kinase activities 24 h after a 2-h exposure. Although cyclin A protein expression was markedly increased, cyclin A kinase activity was only slightly increased. Cyclin D1, cyclin B, cdk4, and cdc2 protein expression and kinase activities remain constant. Lack of activation of cyclin A- and B-cdc2 was associated with a reduced proportion of cells in G2/M phases. Increased cyclin E-cdk2 protein expression was accompanied by the inhibition of DNA synthesis, with a decrease in E2F-1 expression. These results propose that cyclin E-cdk2 kinase can negatively regulate DNA replication. The studies with dThyd rescue from cyclin E-cdk2 protein overexpression and growth inhibition by Tomudex indicate that increased cyclin E-cdk2 protein expression is associated with effective inhibition of thymidylate synthase and resultant dNTP pool imbalance. Provision of dThyd more than 24 h after exposure to Tomudex allowed cells to replicate DNA for a single cycle back to G1, but did not prevent the profound growth-inhibitory effect manifested in the following 5 days. Tomudex treatment resulted in a time-dependent induction of the megabase DNA fragments, followed by secondary 50- to 300-kb DNA fragmentation. The 50- to 300-kb DNA fragmentation may be derived from the inhibition of DNA synthesis associated with cyclin E-cdk2 activation. These results suggest that the megabase DNA fragmentation is induced as a consequence of inhibition of thymidylate synthase by Tomudex and kilobase DNA fragmentation may correlate with the reduction of p27(kip1) expression and the increase in cyclin E and cdk2 kinase activities. Activation of cyclin E and cdk2 kinases allows cells to transit from G1 to S phase accompanied by the inhibition of DNA synthesis. The changes in cell cycle regulatory proteins associated with growth inhibition and DNA damage by Tomudex are not p53 dependent.     

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.     

Human Speedy: a novel cell cycle regulator that enhances proliferation through activation of Cdk2

The decision for a cell to self-replicate requires passage from G1 to S phase of the cell cycle and initiation of another round of DNA replication. This commitment is a critical one that is tightly regulated by many parallel pathways. Significantly, these pathways converge to result in activation of the cyclin-dependent kinase, cdk2. It is, therefore, important to understand all the mechanisms regulating cdk2 to determine the molecular basis of cell progression. Here we report the identification and characterization of a novel cell cycle gene, designated Speedy (Spy1). Spy1 is 40% homologous to the Xenopus cell cycle gene, X-Spy1. Similar to its Xenopus counterpart, human Speedy is able to induce oocyte maturation, suggesting similar biological characteristics. Spy1 mRNA is expressed in several human tissues and immortalized cell lines and is only expressed during the G1/S phase of the cell cycle. Overexpression of Spy1 protein demonstrates that Spy1 is nuclear and results in enhanced cell proliferation. In addition, flow cytometry profiles of these cells demonstrate a reduction in G1 population. Changes in cell cycle regulation can be attributed to the ability of Spy1 to bind to and prematurely activate cdk2 independent of cyclin binding. We demonstrate that Spy1-enhanced cell proliferation is dependent on cdk2 activation. Furthermore, abrogation of Spy1 expression, through the use of siRNA, demonstrates that Spy1 is an essential component of cell proliferation pathways. Hence, human Speedy is a novel cell cycle protein capable of promoting cell proliferation through the premature activation of cdk2 at the G1/S phase transition.     

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.     

The substrates of the cdc2 kinase.

The eukaryotic cell cycle is characterized by two major events, DNA replication (S phase) and mitosis (M phase). According to the current paradigm of the cell cycle as a cdc2 cycle, both of these events are driven by serine-threonine specific protein kinases encoded by functional homologs of the fission yeast cdc2 gene. To understand how cdc2 kinases function, it is necessary to identify their physiological substrates and to determine how phosphorylation of these substrates promotes cell cycle progression. Definitive information about substrates relevant to early stages of the cell cycle (G1 and S phases) remains scarce, but several likely physiological targets of the mitotic cdc2 kinase have recently been identified. Current evidence indicates that cdc2 kinase may trigger entry of cells into mitosis not only by initiating important regulatory pathways but also by direct phosphorylation of abundant structural proteins.     

SAPK/JNK regulates cdc2/cyclin B kinase through phosphorylation and inhibition of cdc25c

Cells undergo M phase arrest in response to stresses like UV irradiation or DNA damage. Stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase, JNK) is activated by such stress stimuli. We addressed the potential effects of SAPK activation on cell cycle regulatory proteins. Activation of SAPK strongly correlated with inhibition of cdc2/cyclin B kinase, an important regulator of G2/M phase. SAPK directly phosphorylated the cdc2 regulator, cdc25c, in vitro on serine 168 (S168). This residue was highly phosphorylated in vivo in response to stress stimuli. cdc25c phosphorylated on S168 in cells lacks phosphatase activity, and expression of a S168A mutant of cdc25c reversed the inhibition of cdc2/cyclin B kinase activity by cell stress. Antibodies directed against phosphorylated S168 detect increased phosphorylation of S168 after cell stress. We conclude that SAPK regulates cdc2/cyclin B kinase following stress events by a novel mechanism involving inhibitory phosphorylation of the cdc2-activating phosphatase cdc25c on S168.     

Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis.

The temperature-sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non-permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non-permissive temperature, p34cdc2 was dephosphorylated and M-phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non-permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase-microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.     

Cdc25 regulates the phosphorylation and activity of the Xenopus cdk2 protein kinase complex.

The Xenopus cdk2 gene encodes a 32-kDa protein kinase with sequence similarity to the 34-kDa product of the cdc2 gene. Previous studies have shown that the kinase activity of the protein product of the cdk2 gene oscillates in the Xenopus embryonic cell cycle with a high in M-phase and a low in interphase. In the present study cdk2 was found not to be associated with any newly synthesized proteins during the cell cycle, but the enzyme did undergo periodic changes in phosphorylation. Upon exit from metaphase, cdk2 became increasingly phosphorylated on both tyrosine and serine residues, and labeling on these residues increased progressively until entry into mitosis, when tyrosine residues were markedly dephosphorylated. Phosphopeptide mapping of cdk2 demonstrated the major sites of phosphorylation were in a phosphopeptide with a pI of 3.7 that contained both phosphoserine and phosphotyrosine. This phosphopeptide accumulated in egg extracts blocked in S-phase with aphidicolin and was not evident in cdc2 immunoprecipitated under the same conditions. Under the same conditions cdc2 was phosphorylated primarily on a phosphopeptide containing both phosphothreonine and phosphotyrosine residues, most likely threonine 14 and tyrosine 15. Affinity-purified human GST-cdc25 was able to dephosphorylate and activate cdk2 isolated from interphase cells. Phosphopeptide mapping demonstrated that the phosphate was specifically removed from the same phosphopeptide identified as the major in vivo site of phosphorylation. These results demonstrate that cdk2 is regulated in the cell cycle by phosphorylation and dephosphorylation on both serine and tyrosine residues. Moreover, the increased phosphorylation of cdk2 in aphidicolin-blocked extracts and the ability of cdc25 to mediate cdk2 dephosphorylation in vitro suggest the possibility that cdk2 is part of the mechanism ensuring mitosis is not initiated until completion of DNA replication. It also implies cdc25 may have other functions in addition to the regulation of cdc2 kinase activity.     

Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts

Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.     

Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites.

The cdc2 kinase is a key regulator of the eukaryotic cell cycle. The activity of its catalytic subunit, p34cdc2, is controlled by cell cycle dependent interactions with other proteins as well as by phosphorylation--dephosphorylation reactions. In this paper, we examine the phosphorylation state of chicken p34cdc2 at various stages of the cell cycle. By peptide mapping, we detect four major phosphopeptides in chicken p34cdc2; three phosphorylation sites are identified as threonine (Thr) 14, tyrosine (Tyr) 15 and serine (Ser) 277. Analysis of synchronized cells demonstrates that phosphorylation of all four sites is cell cycle regulated. Thr 14 and Tyr 15 are phosphorylated maximally during G2 phase but dephosphorylated abruptly at the G2/M transition, concomitant with activation of p34cdc2 kinase. This result suggests that phosphorylation of Thr 14 and/or Tyr 15 inhibits p34cdc2 kinase activity, in line with the location of these residues within the putative ATP binding site of the kinase. During M phase, p34cdc2 is also phosphorylated, but phosphorylation occurs on a threonine residue distinct from Thr 14. Finally, phosphorylation of Ser 277 peaks during G1 phase and drops markedly as cells progress through S phase, raising the possibility that this modification may contribute to control the proposed G1/S function of the vertebrate p34cdc2 kinase.     

Identification of G1 kinase activity for cdk6, a novel cyclin D partner.

A family of vertebrate cdc2-related kinases has been identified, and these kinases are candidates for roles in cell cycle regulation. Here, we show that the human PLSTIRE gene product is a novel cyclin-dependent kinase, cdk6. The cdk6 kinase is associated with cyclins D1, D2, and D3 in lysates of human cells and is activated by coexpression with D-type cyclins in Sf9 insect cells. Furthermore, we demonstrate that endogenous cdk6 from human cell extracts is an active kinase which can phosphorylate pRB, the product of the retinoblastoma tumor suppressor gene. The activation of cdk6 kinase occurs during mid-G1 in phytohemagglutinin-stimulated T cells, well prior to the activation of cdk2 kinase. This timing suggests that cdk6, and by analogy its homolog cdk4, links growth factor stimulation with the onset of cell cycle progression.     

The retinoblastoma protein physically associates with the human cdc2 kinase.

The protein product (pRB) of the retinoblastoma susceptibility gene functions as a negative regulator of cell proliferation, and its activity appears to be modulated by phosphorylation. Using a new panel of anti-human pRB monoclonal antibodies, we have investigated the biochemical properties of this protein. These antibodies have allowed us to detect a pRB-associated kinase that has been identified as the cell cycle-regulating kinase p34cdc2 or a closely related enzyme. Since this associated kinase phosphorylates pRB at most of the sites used in vivo, these results suggest that this kinase is one of the major regulators of pRB. The associated kinase activity follows the pattern of phosphorylation seen for pRB in vivo. The associated kinase activity is not seen in the G1 phase but appears in the S phase, and the levels continue to increase throughout the remainder of the cell cycle.     

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.