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.     

A systematic screen reveals new elements acting at the G2/M cell cycle control

Background

The major cell cycle control acting at the G2 to mitosis transition is triggered in all eukaryotes by cyclin-dependent kinases (CDKs). In the fission yeast Schizosaccharomyces pombe the activation of the G2/M CDK is regulated primarily by dephosphorylation of the conserved residue Tyr15 in response to the stress-nutritional response and cell geometry sensing pathways. To obtain a more complete view of the G2/M control we have screened systematically for gene deletions that advance cells prematurely into mitosis.

Results

A screen of 82% of fission yeast non-essential genes, comprising approximately 3,000 gene deletion mutants, identified 18 genes that act negatively at mitotic entry, 7 of which have not been previously described as cell cycle regulators. Eleven of the 18 genes function through the stress response and cell geometry sensing pathways, both of which act through CDK Tyr15 phosphorylation, and 4 of the remaining genes regulate the G2/M transition by inputs from hitherto unknown pathways. Three genes act independently of CDK Tyr15 phosphorylation and define additional uncharacterized molecular control mechanisms.

Conclusions

Despite extensive investigation of the G2/M control, our work has revealed new components of characterized pathways that regulate CDK Tyr15 phosphorylation and new components of novel mechanisms controlling mitotic entry.     

Ribosomal protein S3 is phosphorylated by Cdk1/cdc2 during G2/M phase

Ribosomal protein S3 (rpS3) is a multifunctional protein involved in translation, DNA repair, and apoptosis. The relationship between rpS3 and cyclin-dependent kinases (Cdks) involved in cell cycle regulation is not yet known. Here, we show that rpS3 is phosphorylated by Cdk1 in G2/M phase. Co-immunoprecipitation and GST pull-down assays revealed that Cdk1 interacted with rpS3. An in vitro kinase assay showed that Cdk1 phosphorylated rpS3 protein. Phosphorylation of rpS3 increased in nocodazole-arrested mitotic cells; however, treatment with Cdk1 inhibitor or Cdk1 siRNA significantly attenuated this phosphorylation event. The phosphorylation of a mutant form of rpS3, T221A, was significantly reduced compared with wild-type rpS3. Decreased phosphorylation and nuclear accumulation of T221A was much more pronounced in G2/M phase. These results suggest that the phosphorylation of rpS3 by Cdk1 occurs at Thr221 during G2/M phase and, moreover, that this event is important for nuclear accumulation of rpS3.     

New Cdc2 Tyr 4 phosphorylation by dsRNA‐activated protein kinase triggers Cdc2 polyubiquitination and G2 arrest under genotoxic stresses

Cell division cycle 2 (Cdc2) protein is an essential subunit of M‐phase kinase (MPK), which has a key role in G2/M transition. Even though the control of MPK activity has been well established with regard to the phosphorylation of Cdc2 at Thr 14 and/or Tyr 15 and Thr 161, little is known about the proteolytic control of Cdc2. In this study, we observed that Cdc2 was downregulated under genotoxic stresses and that double‐stranded RNA‐activated protein kinase (PKR) was involved in the process. The PKR‐mediated Tyr4 phosphorylation triggered Cdc2 ubiquitination. Phospho‐mimic mutations at the Tyr 4 residue (Y4D or Y4E) caused significant ubiquitination of Cdc2 even in the absence of PKR. Our findings demonstrate that (i) PKR, Ser/Thr kinase, phosphorylates its new substrate Cdc2 at the Tyr 4 residue, (ii) PKR‐mediated Tyr 4‐phosphorylation facilitates Cdc2 ubiquitination and proteosomal degradation, (iii) unphosphorylated Tyr 4 prevents Cdc2 ubiquitination, and (iv) downstream from p53, PKR has a crucial role in G2 arrest and triggers Cdc2 downregulation under genotoxic conditions.     

Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15.

We have examined the role of phosphorylation in the regulation of human cyclin-dependent kinase-2 (CDK2), a protein closely related to the cell cycle regulatory kinase CDC2. We find that CDK2 from HeLa cells contains three major tryptic phosphopeptides. Analysis of site-directed mutant proteins, expressed by transient transfection of COS cells, demonstrates that the two major phosphorylation sites are Tyr15 (Y15) and Thr160 (T160). Additional phosphorylation probably occurs on Thr14 (T14). Replacement of T160 with alanine abolishes the kinase activity of CDK2, indicating that phosphorylation at this site (as in CDC2) is required for kinase activity. Mutation of Y15 and T14 stimulates kinase activity, demonstrating that phosphorylation at these sites (as in CDC2) is inhibitory. Similarly, CDK2 is activated in vitro by dephosphorylation of Y15 and T14 by the phosphatase CDC25. Analysis of HeLa cells synchronized at various cell cycle stages indicates that CDK2 phosphorylation on T160 increases during S phase and G2, when CDK2 is most active. Phosphorylation on the inhibitory sites T14 and Y15 is also maximal during S phase and G2. Thus, the activity of a subpopulation of CDK2 molecules is inhibited at a time in the cell cycle when overall CDK2 activity is increased.     

Cell cycle regulation of the p34cdc2 inhibitory kinases.

In cells of higher eukaryotic organisms the activity of the p34cdc2/cyclin B complex is inhibited by phosphorylation of p34cdc2 at two sites within its amino-terminus (threonine 14 and tyrosine 15). In this study, the cell cycle regulation of the kinases responsible for phosphorylating p34cdc2 on Thr14 and Tyr15 was examined in extracts prepared from both HeLa cells and Xenopus eggs. Both Thr14- and Tyr15- specific kinase activities were regulated in a cell cycle-dependent manner. The kinase activities were high throughout interphase and diminished coincident with entry of cells into mitosis. In HeLa cells delayed in G2 by the DNA-binding dye Hoechst 33342, Thr14- and Tyr15-specific kinase activities remained high, suggesting that a decrease in Thr14- and Tyr15- kinase activities may be required for entry of cells into mitosis. Similar cell cycle regulation was observed for the Thr14/Tyr15 kinase(s) in Xenopus egg extracts. These results indicate that activation of CDC2 and entry of cells into mitosis is not triggered solely by activation of the Cdc25 phosphatase but by the balance between Thr14/Tyr15 kinase and phosphatase activities. Finally, we have detected two activities capable of phosphorylating p34cdc2 on Thr14 and/or Tyr15 in interphase extracts prepared from Xenopus eggs. An activity capable of phosphorylating Tyr15 remained soluble after ultracentrifugation of interphase extracts whereas a second activity capable of phosphorylating both Thr14 and Tyr15 pelleted. The pelleted fraction contained activities that were detergent extractable and that phosphorylated p34cdc2 on both Thr14 and Tyr15. The Thr14- and Tyr15-specific kinase activities co-purified through three successive chromatographic steps indicating the presence of a dual-specificity protein kinase capable of acting on p34cdc2.     

Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.     

S100A8/A9 (Calprotectin) Negatively Regulates G2/M Cell Cycle Progression and Growth of Squamous Cell Carcinoma

Malignant transformation results in abnormal cell cycle regulation and uncontrolled growth in head and neck squamous cell carcinoma (HNSCC) and other cancers. S100A8/A9 (calprotectin) is a calcium-binding heterodimeric protein complex implicated in cell cycle regulation, but the specific mechanism and role in cell cycle control and carcinoma growth are not well understood. In HNSCC, S100A8/A9 is downregulated at both mRNA and protein levels. We now report that downregulation of S100A8/A9 correlates strongly with a loss of cell cycle control and increased growth of carcinoma cells. To show its role in carcinogenesis in an in vitro model, S100A8/A9 was stably expressed in an S100A8/A9-negative human carcinoma cell line (KB cells, HeLa-like). S100A8/A9 expression increases PP2A phosphatase activity and p-Chk1 (Ser345) phosphorylation, which appears to signal inhibitory phosphorylation of mitotic p-Cdc25C (Ser216) and p-Cdc2 (Thr14/Tyr15) to inactivate the G2/M Cdc2/cyclin B1 complex. Cyclin B1 expression then downregulates and the cell cycle arrests at the G2/M checkpoint, reducing cell division. As expected, S100A8/A9-expressing cells show both decreased anchorage-dependent and -independent growth and mitotic progression. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increases growth and survival and reduces Cdc2 inhibitory phosphorylation at Thr14/Tyr15. The level of S100A8/A9 endogenous expression correlates strongly with the reduced p-Cdc2 (Thr14/Tyr14) level in HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B. S100A8/A9-mediated control of the G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may suggest new therapeutic approaches.     

Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding

The current concept regarding cell cycle regulation of DNA replication is that Cdt1, together with origin recognition complex and CDC6 proteins, constitutes the machinery that loads the minichromosome maintenance complex, a candidate replicative helicase, onto chromatin during the G(1) phase. The actions of origin recognition complex and CDC6 are suppressed through phosphorylation by cyclin-dependent kinases (Cdks) after S phase to prohibit rereplication. It has been suggested in metazoan cells that the function of Cdt1 is blocked through binding to an inhibitor protein, geminin. However, the functional relationship between the Cdt1-geminin system and Cdks remains to be clarified. In this report, we demonstrate that human Cdt1 is phosphorylated by cyclin A-dependent kinases dependent on its cyclin-binding motif. Cdk phosphorylation resulted in the binding of Cdt1 to the F-box protein Skp2 and subsequent degradation. In contrast, in vitro DNA binding activity of Cdt1 was inhibited by the phosphorylation. However, geminin binding to Cdt1 was not affected by the phosphorylation. Finally we provide evidence that inactivation of Cdk1 results in Cdt1 dephosphorylation and rebinding to chromatin in murine FT210 cells synchronized around the G(2)/M phase. Taken together, these findings suggest that Cdt1 function is also negatively regulated by the Cdk phosphorylation independent of geminin binding.     

Cell cycle arrest mediated by WEE1 is involved in the unfolded protein response in plants

Activation of the unfolded protein response (UPR) in mammalian cells leads to cell cycle arrest at the G1 phase (Thomas et al., J Biol Chem 288:7606–7617, 2013). However, how UPR signaling affects cell cycle arrest remains largely unknown in plants. Here, we examined UPR and endoreduplication in Col-0, wee1, and ER stress sensing-deficient ire1a&b plants during DNA replication and ER stress conditions. We found that WEE1, an essential negative regulator of the cell cycle, is involved in the maintenance of ER homeostasis during genotoxic stress and the ER stress hypersensitivity of ire1a&b is alleviated by loss-of-function mutation in WEE1. WEE1-mediated cell cycle arrest was required for IRE1–bZIP60 pathway activation during ER stress. In contrast, loss-of-function mutation in WEE1 caused increased expression of UPR-related genes during DNA replication stress. WEE1 and IRE1 were required for endoreduplication during DNA replication stress and ER stress, respectively. Taken together, these findings suggest that cell cycle regulation is associated with UPR activation in different manners during ER stress and DNA replication stress in Arabidopsis.     

Mathematical modeling of fission yeast Schizosaccharomyces pombe cell cycle: exploring the role of multiple phosphatases

Cell cycle is the central process that regulates growth and division in all eukaryotes. Based on the environmental condition sensed, the cell lies in a resting phase G0 or proceeds through the cyclic cell division process (G1??S??G2??M). These series of events and phase transitions are governed mainly by the highly conserved Cyclin dependent kinases (Cdks) and its positive and negative regulators. The cell cycle regulation of fission yeast Schizosaccharomyces pombe is modeled in this study. The study exploits a detailed molecular interaction map compiled based on the published model and experimental data. There are accumulating evidences about the prominent regulatory role of specific phosphatases in cell cycle regulations. The current study emphasizes the possible role of multiple phosphatases that governs the cell cycle regulation in fission yeast S. pombe. The ability of the model to reproduce the reported regulatory profile for the wild-type and various mutants was verified though simulations.     

Antiproliferative effect of bcl-2 gene does not concern the control of mitotic events

E1A + c-Ha-ras-transformants overexpressing bcl-2 oncogene are able to be arrested at the G1/S boundary of the cell cycle after DNA damage and upon serum starvation, this cell cycle blockage being accompanied by a decrease in the activity of cyclin E--Cdk2 complexes. Roscovitine-induced inhibition of cyclin-dependent kinases (Cdks) activity does not result in the G1/S arrest of E1A + c-Ha-ras + bcl-2-transformants. Roscovitine treatment causes an accumulation of G2/M cells, mainly at the expense of mitotic cells. However, the expression of Bcl-2 oncoproducts does not re-establish the regulation of mitotic events broken by introduction of E1A and c-Ha-ras oncogenes in normal cells, as revealed by the treatment of E1A + c-Ha-ras + bcl-2-transformants with nocodazole inducing mitotic arrest in normal cells. In spite of the elevated expression of antiapoptotic bcl-2 gene in transformants, nocodazole treatment results in mass apoptotic death preceded by polyploidy. Roscovitine also induces apoptosis with no polyploid cell accumulation being observed. Inhibition of Cdks activity with Roscovitine, as well as violation of microtubule depolymerization with nocodazole result in the apoptotic death in the tested cell lines sensitive (E1A + c-Ha-ras) and resistant (E1A + c-Ha-ras + bcl-2) to damaging agents. Thus, the application of Roscovitine, a specific inhibitor of Cdks, suggests that the decrease in Cdks activity in E1A + c-Ha-ras + bcl-2-transformants is not likely to be responsible for G1/S cell cycle arrest realization after damaging influences. Moreover, an antiproliferative effect of Bcl-2 in E1A + c-Ha-ras-transformants is restricted by restoration of cell cycle events at G1/S and G2/M boundaries, and does not concern the program of mitotic events regulation.     

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.     

Drosophila Wee1 kinase regulates Cdk1 and mitotic entry during embryogenesis

Cyclin-dependent kinases (Cdks) are the central regulators of the cell division cycle. Inhibitors of Cdks ensure proper coordination of cell cycle events and help regulate cell proliferation in the context of tissues and organs. Wee1 homologs phosphorylate a conserved tyrosine to inhibit the mitotic cyclin-dependent kinase Cdk1. Loss of Wee1 function in fission or budding yeast causes premature entry into mitosis. The importance of metazoan Wee1 homologs for timing mitosis, however, has been demonstrated only in Xenopus egg extracts and via ectopic Cdk1 activation . Here, we report that Drosophila Wee1 (dWee1) regulates Cdk1 via phosphorylation of tyrosine 15 and times mitotic entry during the cortical nuclear cycles of syncytial blastoderm embryos, which lack gap phases. Loss of maternal dwee1 leads to premature entry into mitosis, mitotic spindle defects, chromosome condensation problems, and a Chk2-dependent block of subsequent development, and then embryonic lethality. These findings modify previous models about cell cycle regulation in syncytial embryos and demonstrate that Wee1 kinases can regulate mitotic entry in vivo during metazoan development even in cycles that lack a G2 phase.     

G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast

Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear. We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2. This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase. We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset. This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2. Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.     

p38 MAP kinase plays a role in G2 checkpoint activation and inhibits apoptosis of human B cell lymphoma cells treated with etoposide

p38 MAPK is mainly activated by stress stimuli and mediates signals that regulate various cellular responses, including cell-cycle progression and apoptosis, depending on cell types and stimuli. Here we examine the role of p38 in regulation of apoptosis and cell cycle checkpoint in Daudi B-cell lymphoma cells treated with the topoisomerase II inhibitor etoposide. Etoposide activated p38, inhibited the G2/M transition with the persistent inhibitory phosphorylation of Cdc2 on Tyr15, and caused apoptosis of Daudi cells. Inducible expression of a dominant negative p38α mutant in Daudi cells reduced the inhibition of Cdc2 as well as G2/M arrest and augmented apoptosis induced by etoposide. SB203580, a specific inhibitor of p38α and p38β, similarly reduced the inhibitory phosphorylation of Cdc2 as well as G2/M arrest and augmented apoptosis of Daudi cells treated with etoposide. These results suggest that p38 plays a role in G2/M checkpoint activation through induction of the persistent inhibitory phosphorylation of Cdc2 and, thereby, inhibits apoptosis of Daudi cells treated with etoposide. The present study, thus, raises the possibility that p38 may represent a new target for sensitization of lymphoma cells to DNA-damaging chemotherapeutic agents.