Viral infections and cell cycle G2／M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyrl5) on Cdc2, which is phosphorylated by Weel kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two wellcharacterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-Ⅰ) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.     

Speedy: a novel cell cycle regulator of the G2/M transition

Stage VI Xenopus oocytes are suspended at the G2/M transition of meiosis I, and represent an excellent system for the identification and examination of cell cycle regulatory proteins. Essential cell cycle regulators such as MAPK, cyclins and mos have the ability to induce oocyte maturation, causing the resumption of the cell cycle from its arrested state. We have identified the product of a novel Xenopus gene, Speedy or Spy1, which is able to induce rapid maturation of Xenopus oocytes, resulting in the induction of germinal vesicle breakdown (GVBD) and activation of M-phasepromoting factor (MPF). Spy1 activates the MAPK pathway in oocytes, and its ability to induce maturation is dependent upon this pathway. Spy1-induced maturation occurs much more rapidly than maturation induced by other cell cycle regulators including progesterone, mos or Ras, and does not require any of these proteins or hormones, indicating that Spy1-induced maturation proceeds through a novel regulatory pathway. In addition, we have shown that Spy1 physically interacts with cdk2, and prematurely activates cdk2 kinase activity. Spy1 therefore represents a novel cell cycle regulatory protein, inducing maturation through the activation of MAPK and MPF, and also leading to the premature activation of cdk2.     

Beta-catenin regulation during the cell cycle: implications in G2/M and apoptosis

Beta-catenin is a multifunctional protein involved in cell-cell adhesion and Wnt signal transduction. Beta-catenin signaling has been proposed to act as inducer of cell proliferation in different tumors. However, in some developmental contexts and cell systems beta-catenin also acts as a positive modulator of apoptosis. To get additional insights into the role of beta-catenin in the regulation of the cell cycle and apoptosis, we have analyzed the levels and subcellular localization of endogenous beta-catenin and its relation with adenomatous polyposis coli (APC) during the cell cycle in S-phase-synchronized epithelial cells. Beta-catenin levels increase in S phase, reaching maximum accumulation at late G2/M and then abruptly decreasing as the cells enter into a new G1 phase. In parallel, an increased cytoplasmic and nuclear localization of beta-catenin and APC is observed during S and G2 phases. In addition, strong colocalization of APC with centrosomes, but not beta-catenin, is detected in M phase. Interestingly, overexpression of a stable form of beta-catenin, or inhibition of endogenous beta-catenin degradation, in epidermal keratinocyte cells induces a G2 cell cycle arrest and leads to apoptosis. These results support a role for beta-catenin in the control of cell cycle and apoptosis at G2/M in normal and transformed epidermal keratinocytes.     

Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression.

Background: In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus. This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk). Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S. cerevisiae. In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete.Results: We have identified the S. pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle. As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division. Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis. Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle. In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry. Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine.Conclusions: S. pombe Clp1p affects cell cycle progression in a markedly different manner than its S. cerevisiae homolog, Cdc14p. This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.     

Involvement of the interaction between p21 and proliferating cell nuclear antigen for the maintenance of G2/M arrest after DNA damage

Although a major effect of p21, a cyclin-dependent kinase inhibitor, is considered to be exerted during G(1) phase of the cell cycle, p21 gene knock-out studies suggested its involvement in G(2)/M checkpoint as well. Here we demonstrate evidence that p21 is required for the cell cycle arrest at G(2) upon DNA damage. We found that expression of wild-type p21 (p21(WT)), not mutant p21 (p21(PCNA-)) lacking the interaction with proliferating cell nuclear antigen (PCNA), caused G(2) cell cycle arrest in p53-deficient DLD1 colon cancer cell line after the DNA damage by treatment with cis-diamminedichloroplatinum (II). We also found that p21(WT) was associated with Cdc2/cyclin B1 together with PCNA. Furthermore, coimmunoprecipitation experiments revealed that PCNA interacted with Cdc25C at the G(2)/M transition, and this interaction was abolished when p21(WT) was expressed presumably due to the competition between p21(WT) and Cdc25C in the binding to PCNA. These findings suggest that p21 plays a regulatory role in the maintenance of cell cycle arrest at G(2) by blocking the interaction of Cdc25C with PCNA.     

Mitogen-activated protein kinase kinase 1-dependent Golgi unlinking occurs in G2 phase and promotes the G2/M cell cycle transition

Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G(2)/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle "checkpoint." A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G(2) to control G(2)/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G(2) Golgi unlinking and the G(2)/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.     

Involvement of the role of Chk1 in lithium-induced G2/M phase cell cycle arrest in hepatocellular carcinoma cells

Lithium, a therapeutic agent for bipolar disorder, can induce G2/M arrest in various cells, but the mechanism is unclear. In this article, we demonstrated that lithium arrested hepatocellular carcinoma cell SMMC-7721 at G2/M checkpoint by inducing the phosphorylation of cdc2 (Tyr-15). This effect was p53 independent and not concerned with the inhibition of glycogen synthase kinase-3 and inositol monophosphatase, two well-documented targets of lithium. Checkpoint kinase 1 (Chk1), a critical enzyme in DNA damage-induced G2/M arrest, was at least partially responsible for the lithium action. The lithium-induced phosphorylation of cdc2 and G2/M arrest was abrogated largely by SB218078, a potent Chk1 inhibitor, as well as by Chk1 siRNA or the over-expression of kinase dead Chk1. Furthermore, lithium-induced cdc25C phosphorylation in 7721 cells and in vitro kinase assay showed that the activity of Chk1 was enhanced after lithium treatment. Interestingly, the increase of Chk1 activity by lithium may be independent of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) kinase. This is because no elevated phosphorylation on Chk1 (Ser-317 and Ser-345) was observed after lithium treatment. Moreover, caffeine, a known ATM/ATR kinase inhibitor, relieved the phosphorylation of cdc2 (Tyr-15) by hydroxyurea, but not that by lithium. Our study's results revealed the role of Chk1 in lithium-induced G2/M arrest. Given that Chk1 has been proposed to be a novel tumor suppressor, we suggest that the effect of lithium on Chk1 and cell cycle is useful in tumor prevention and therapy.     

Phosphorylation of p21 in G2/M promotes cyclin B-Cdc2 kinase activity

Little is known about the posttranslational control of the cyclin-dependent protein kinase (CDK) inhibitor p21. We describe here a transient phosphorylation of p21 in the G2/M phase. G2/M-phosphorylated p21 is short-lived relative to hypophosphorylated p21. p21 becomes nuclear during S phase, prior to its phosphorylation by CDK2. S126-phosphorylated cyclin B1 binds to T57-phosphorylated p21. Cdc2 kinase activation is delayed in p21-deficient cells due to delayed association between Cdc2 and cyclin B1. Cyclin B1-Cdc2 kinase activity and G2/M progression in p21-/- cells are restored after reexpression of wild-type but not T57A mutant p21. The cyclin B1 S126A mutant exhibits reduced Cdc2 binding and has low kinase activity. Phosphorylated p21 binds to cyclin B1 when Cdc2 is phosphorylated on Y15 and associates poorly with the complex. Dephosphorylation on Y15 and phosphorylation on T161 promotes Cdc2 binding to the p21-cyclin B1 complex, which becomes activated as a kinase. Thus, hyperphosphorylated p21 activates the Cdc2 kinase in the G2/M transition.     

Phospholipids are synthesized in the G2/M phase of the cell cycle

Very little is known about the metabolism of phospholipids in the G2 and M phases of the cell cycle, but limited studies have led to the postulation that phospholipid synthesis ceases during this period. To investigate whether phospholipids are synthesized in the G2/M phase of the cell cycle, protocols were developed to produce synchronized MCF-7 cell populations with greater than 80% of the cells in G1/S or G2/M phases that moved in synchrony following removal of the blocking agent. Analysis of the activities of key phosphatidylcholine and phosphatidylethanolamine biosynthetic enzymes in subcellular fractions obtained from MCF-7 cells at different cell cycle phases revealed that there was robust activity of key enzymes in the fractions prepared from MCF-7 cells in G2/M phase. Radiolabeled choline and ethanolamine were rapidly incorporated into cells maintained at G2/M phase with nocodazole, and the rates of incorporation were similar to those obtained in cells allowed to progress into the G1 phase. Furthermore, radiolabeled glycerol was incorporated into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid in MCF-7 cells maintained at G2/M phase with nocodazole. Similar results were obtained in CHO cells. These results demonstrate that glycerophospholipid synthesis is very active in the G2/M phase of these cells. Therefore, the postulated cessation of phospholipid synthesis in G2/M phases is not applicable to all cell types.     

Relevance of histone H1 kinase activity to the G2/M transition during the cell cycle ofDictyostelium discoideum

The implication of histone H1 kinase activity for the G2/M transition during the cell cycle was investigated usingDictyostelium discoideum Ax-2. Histone H1 kinase with its activity was purified from cell extracts by the use of p13^suc1 affinity gel. In the vegetative cell cycle, the activity of histone H1 kinase including Cdc2 kinase was found using synchronized Ax-2 cells to be highest just before the entry into mitosis. The activity also was markedly enhanced just prior to the M phase from which developing cells (possibly prespore cells) reinitiate their cell cycle at the mound-tipped aggregate stage. These results strongly suggest the importance of Cdc2 kinase activity in the G2 to M phase transition during the cell cycle, as the case for other eukaryotic cells.     

Identification of a restriction point at the M/G1 transition during the ongoing cell cycle

Progression through the cell cycle is dependent upon numerous external factors (growth factors, extracellular matrix components) which exert their effects through the activation of signal transduction networks. During last years we have studied the regulation of progression through the ongoing CHO cell cycle. Recently, we have demonstrated that in CHO cells at least two serum dependent points exist in G1 phase that lead to different cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis, while the second is located in late G1 phase and probably corresponds to the classical restriction point R. Because of the suggested link with apoptosis of the restriction point in early G1 phase, we have studied the possible role of PI 3-K in cell cycle progression through the ongoing G1 phase of CHO cells. In the presence of the PI 3-K inhibitors wortmannin or LY294002, cells were arrested during early G1 phase, leading to the expression of cleaved caspase-3, a central mediator of apoptosis. Addition of AP-2, an inhibitor of PKB, the downstream substrate of PI 3-K, at several time points during G1 phase demonstrated that inhibition during early G1 phase caused cell cycle arrest, while addition of the inhibitors during mid or late G1 phase had no effect on S phase entry. As for inhibition of PI 3-K, also inhibition of PKB resulted in expression of cleaved caspase-3. These results clearly demonstrate that a decision point exists in the early G1 phase of the cell cycle; in the presence of PKB activity the cells are continuing cell cycle progression, while in the absence of PKB activity the cells are induced for apoptosis.     

NFBD1/MDC1 participates in the regulation of G2/M transition in mammalian cells

NFBD1/MDC1 is a large nuclear protein involved in the early cellular response to DNA damage. Upon DNA damage, NFBD1 has an ability to facilitate the efficient DNA repair. In the present study, we have found that, in addition to DNA damage response, NFBD1 plays a critical role in the regulation of G2/M transition. Expression study using synchronized HeLa cells demonstrated that, like the mitotic kinase Plk1, NFBD1 expression level is maximal in G2/M-phase of the cell cycle. siRNA-mediated knockdown of NFBD1 resulted in G2/M arrest as well as simultaneous apoptosis in association with a significant increase in the amounts of γH2AX and pro-apoptotic p73. Since a remarkable down-regulation of mitotic phospho-histone H3 was detectable in NFBD1-knocked down cells, it is likely that knocking down of NFBD1 inhibits G2/M transition. Taken together, our present findings suggest that NFBD1 has a pivotal role in the regulation of proper mitotic entry.     

Phosphorylation of Bcl-2 protein by CDC2 kinase during G2/M phases and its role in cell cycle regulation

Although it has been reported that Bcl-2 phosphorylation is associated with certain types of apoptosis, there is much controversy over the functional significance of and the kinases responsible for the phosphorylation. In this study, we examined whether Bcl-2 is phosphorylated by CDC2 kinase, a master regulator of G(2)/M transition in the eukaryotic cell cycle. When CDC2 was activated by okadaic acid in HL-60 cells, Bcl-2 phosphorylation was readily induced. The phosphorylation was correlated with the accumulation of cells in G(2)/M phases, but was not proportional to the level of apoptosis. Furthermore, we found that Bcl-2 was phosphorylated during G(2)/M phases of normal cell cycle. The ability of CDC2 to phosphorylate Bcl-2 was confirmed by in vitro kinase assay with a highly purified CDC2-cyclin B complex. Using synthetic peptides and mutant cell lines, we identified threonine 56, one of two consensus sites for CDC2 within the Bcl-2 sequence, as a residue phosphorylated by CDC2. Mutation at threonine 56 abrogated the cell cycle inhibitory effect of Bcl-2 without affecting anti-apoptotic function. These results suggest that two distinct functions of Bcl-2 (anti-apoptosis and cell cycle inhibition) are differentially regulated by post-translational mechanisms such as phosphorylation. CDC2-mediated phosphorylation of Bcl-2 may play some physiological roles in the negative regulatory events during mitosis.     

Involvement of cyclin B1 in progesterone-mediated cell growth inhibition, G2/M cell cycle arrest, and apoptosis in human endometrial cell

Background  

Progesterone plays an important role in the proliferation and differentiation of human endometrial cells (hECs). Large-dose treatment with progesterone has been used for treatment of endometrial proliferative disorders. However, the mechanisms behind remain unknown.     

Human NDR kinases control G(1)/S cell cycle transition by directly regulating p21 stability

The G(1) phase of the cell cycle is an important integrator of internal and external cues, allowing a cell to decide whether to proliferate, differentiate, or die. Multiple protein kinases, among them the cyclin-dependent kinases (Cdks), control G(1)-phase progression and S-phase entry. With the regulation of apoptosis, centrosome duplication, and mitotic chromosome alignment downstream of the HIPPO pathway components MST1 and MST2, mammalian NDR kinases have been implicated to function in cell cycle-dependent processes. Although they are well characterized in terms of biochemical regulation and upstream signaling pathways, signaling mechanisms downstream of mammalian NDR kinases remain largely unknown. We identify here a role for human NDR in regulating the G(1)/S transition. In G(1) phase, NDR kinases are activated by a third MST kinase (MST3). Significantly, interfering with NDR and MST3 kinase expression results in G(1) arrest and subsequent proliferation defects. Furthermore, we describe the first downstream signaling mechanisms by which NDR kinases regulate cell cycle progression. Our findings suggest that NDR kinases control protein stability of the cyclin-Cdk inhibitor protein p21 by direct phosphorylation. These findings establish a novel MST3-NDR-p21 axis as an important regulator of G(1)/S progression of mammalian cells.