Mammalian growth-associated H1 histone kinase: a homolog of cdc2+/CDC28 protein kinases controlling mitotic entry in yeast and frog cells.

Mammalian growth-associated H1 histone kinase, an enzyme whose activity is sharply elevated at mitosis, is similar to cdc2+ protein kinase from Schizosaccharomyces pombe and CDC28 protein kinase from Saccharomyces cerevisiae with respect to immunoreactivity, molecular size, and specificity for phosphorylation sites in H1 histone. Phosphorylation of specific growth-associated sites in H1 histone is catalyzed by yeast cdc2+/CDC28 kinase, as shown by the in vitro thermal lability of this activity in extracts prepared from temperature-sensitive mutants. In addition, highly purified Xenopus maturation-promoting factor catalyzes phosphorylation of the same sites in H1 as do the mammalian and yeast kinases. The data indicate that growth-associated H1 kinase is encoded by a mammalian homolog of cdc2+/CDC28 protein kinase, which controls entry into mitosis in yeast and frog cells. Since H1 histone is known to be an in vivo substrate of the mammalian kinase, this suggests that phosphorylation of H1 histone or an H1 histone counterpart is an important component of the mechanism for entry of cells into mitosis.     

Simian virus 40 large T antigen affects the Saccharomyces cerevisiae cell cycle and interacts with p34CDC28.

Simian virus 40 tumor (T) antigen, an established viral oncoprotein, causes alterations in cell growth control through interacting with, and altering the function of, cellular proteins. To examine the effects of T antigen on cell growth control, and to identify the cellular proteins with which it may functionally interact, T antigen was expressed in the budding yeast Saccharomyces cerevisiae. The yeast cells expressing T antigen showed morphological alterations as well as growth inhibition attributable, at least in part, to a lag in progression from G1 to S. This point in the cell cycle is also known to be affected by T antigen in mammalian cells. Both p34CDC28 and p34CDC2Hs were shown to bind to a chimeric T antigen-glutathione S-transferase fusion protein, indicating that T antigen interacts directly with cell cycle proteins which control the G1 to S transition. This interaction was confirmed by in vivo cross-linking experiments, in which T antigen and p34CDC28 were coimmunoprecipitated from extracts of T-antigen-expressing yeast cells. These immunoprecipitated complexes could phosphorylate histone H1, indicating that kinase activity was retained. In addition, in autophosphorylation reactions, the complexes phosphorylated a novel 60-kDa protein which appeared to be underphosphorylated (or underrepresented) in p34CDC28-containing complexes from cells which did not express T antigen. These results suggest that T antigen interacts with p34CDC28 and alters the kinase function of p34CDC28-containing complexes. These events correlate with alterations in the yeast cell cycle at the G1 to S transition.     

G1-specific cyclins: in search of an S-phase-promoting factor

In budding yeast, Saccharomyces cerevisiae, the two principal cell cycle transitions, from G1 to S phase and from G2 to M phase, are controlled by the same protein from G2 to M phase, are controlled by the same protein kinase, CDC28, a homolog of the cdc2 protein kinase in fission yeast and other organisms. The G1 to S phase activity of the kinase is associated with accumulation of a novel family of G1 cyclins, distinct from cyclins that are required to activate the kinase for G2 to M phase functions. It remains to be determined whether G1 cyclins with similar functions exist in higher cells.     

Properties of Saccharomyces cerevisiae wee1 and its differential regulation of p34CDC28 in response to G1 and G2 cyclins.

Wee1 is a protein kinase that negatively regulates p34cdc2 kinase activity. We have identified a Saccharomyces cerevisiae wee1 homolog encoded by the SWE1 gene. SWE1 overexpression arrests cells in G2 with short spindles whereas deletion of SWE1 did not alter the cell cycle but did eliminate the G2 delay observed in mih1- mutants. Swe1 immunoprecipitates were capable of tyrosine phosphorylating and inactivating p34CDC28 complexed with Clb2, a G2-type cyclin, but not p34CDC28 complexed with Cln2, a G1-type cyclin, consistent with the inability of Swe1 overexpression to inhibit the G1/S transition. These results suggest that specific cyclin subunits target p34CDC28 for distinct regulatory controls which may be important for ensuring proper p34CDC28 function during the cell cycle.     

Cdc28 activates exit from mitosis in budding yeast

The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast. CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis. Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis. CDC28-VF has reduced Cdc20- dependent APC activity in mitosis, but normal Hct1- dependent APC activity in the G1 phase of the cell cycle. The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC. The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast.     

Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle.

The yeast Cdc7 function is required for the G1/S transition and is dependent on passage through START, a point controlled by the Cdc28/cdc2/p34 protein kinase. CDC7 encodes a protein kinase activity, and we now show that this kinase activity varies in the cell cycle but that protein levels appear to remain constant. We present several lines of evidence that periodic activation of CDC7 kinase is at least in part through phosphorylation. First, the kinase activity of the Cdc7 protein is destroyed by dephosphorylation of the protein in vitro with phosphatase. Second, Cdc7 protein is hypophosphorylated and inactive as a kinase in extracts of cells arrested at START but becomes active and maximally phosphorylated subsequent to passage through START. The phosphorylation pattern of Cdc7 protein is complex. Phosphopeptide mapping reveals four phosphopeptides in Cdc7 prepared from asynchronous yeast cells. Both autophosphorylation and phosphorylation in trans appear to contribute to this pattern. Autophosphorylation is shown to occur by using a thermolabile Cdc7 protein. A protein in yeast extracts can phosphorylate and activate Cdc7 protein made in Escherichia coli, and phosphorylation is thermolabile in cdc28 mutant extracts. Cdc7 protein carrying a serine to alanine change in the consensus recognition site for Cdc28 kinase shows an altered phosphopeptide map, suggesting that this site is important in determining the overall Cdc7 phosphorylation pattern.     

Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells

The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X- irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.     

Activation of Ras-Ral pathway attenuates p53-independent DNA damage G2 checkpoint

Earlier we have found that in p53-deficient cells the expression of activated Ras attenuates the DNA damage-induced arrest in G(1) and G(2). In the present work we studied Ras-mediated effects on the G(2) checkpoint in two human cell lines, MDAH041 immortalized fibroblasts and Saos-2 osteosarcoma cells. The transduction of the H-Ras mutants that retain certain functions (V12S35, V12G37, and V12C40 retain the ability to activate Raf or RalGDS or phosphatidylinositol 3-kinase, respectively) as well as the activated or dominant-negative mutants of RalA (V23 and N28, respectively) has revealed that the activation of Ras-RalGEFs-Ral pathway was responsible for the attenuation of the G(2) arrest induced by ethyl metanesulfonate or doxorubicin. Noteworthy, the activated RalA V23N49 mutant, which cannot interact with RLIP76/RalBP1 protein, one of the best studied Ral effectors, retained the ability to attenuate the DNA damage-induced G(2) arrest. Activation of the Ras-Ral signaling affected neither the level nor the intracellular localization of cyclin B1 and CDC2 but interfered with the CDC2 inhibitory phosphorylation at Tyr(15) and the decrease in the cyclin B/CDC2 kinase activity in damaged cells. The revealed function of the Ras-Ral pathway may contribute to the development of genetic instability in neoplastic cells.     

PRP18, a protein required for the second reaction in pre-mRNA splicing.

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.     

cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication.

RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.     

Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae.

Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.     

Control of the yeast cell cycle is associated with assembly/disassembly of the Cdc28 protein kinase complex

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for progression from G1 to S phase in the cell cycle. We present evidence that the active form of the Cdc28 protein kinase is a complex of approximately 160 kd containing an endogenous substrate, p40, and possibly other polypeptides. This complex phosphorylates p40 and exogenous histone H1 in vitro. Cell cycle arrest during G1 results in inactivation of the protein kinase accompanied by the disassembly of the complex. Furthermore, assembly of the complex is regulated during the cell cycle, reaching a maximum during G1. Partial complexes thought to be intermediates in the assembly process phosphorylate histone H1 but not p40. Addition of soluble factors to these partial complexes in vitro restores p40 phosphorylation and causes the complex to increase to the mature size. A model is presented in which p40 phosphorylation is required during G1 for cells to initiate a new cell cycle.     

Activation of the p34 CDC2 protein kinase at the start of S phase in the human cell cycle.

Using a protocol for selecting cells on the basis of both size and age (with respect to the preceding mitosis), we isolated highly synchronous human G1 cells. With this procedure, we demonstrated that the p34 CDC2 kinase was activated at the start of S phase. Cyclin A synthesis began at the same time, and activation of the p34 CDC2 kinase at the start of S phase was, at least in part, due to its association with cyclin A. Furthermore, cells synchronized in late G1 by exposure to the drug mimosine contain active cyclin A/p34 CDC2 kinase, indicating that p34 CDC2 activation can occur before DNA synthesis begins. Thus, the cyclin A/CDC2 complex, which previously has been shown to be sufficient to start SV40 DNA synthesis in vitro, assembles and is activated at the start of S phase in vivo.     

Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family.

A new human cyclin, named cyclin E, was isolated by complementation of a triple cln deletion in S. cerevisiae. Cyclin E showed genetic interactions with the CDC28 gene, suggesting that it functioned at START by interacting with the CDC28 protein. Two human genes were identified that could interact with cyclin E to perform START in yeast containing a cdc28 mutation. One was CDC2-HS, and the second was the human homolog of Xenopus CDK2. Cyclin E produced in E. coli bound and activated the CDC2 protein in extracts from human G1 cells, and antibodies against cyclin E immunoprecipitated a histone H1 kinase from HeLa cells. The interactions between cyclin E and CDC2, or CDK2, may be important at the G1 to S transition in human cells.     

Cdc4, a Protein Required for the Onset of S Phase, Serves an Essential Function during G2/M Transition in Saccharomyces cerevisiae

Saccharomyces cerevisiae proteins Cdc4 and Cdc20 contain WD40 repeats and participate in proteolytic processes. However, they are thought to act at two different stages of the cell cycle: Cdc4 is involved in the proteolysis of the Cdk inhibitor, Sic1, necessary for G(1)/S transition, while Cdc20 mediates anaphase-promoting complex-dependent degradation of anaphase inhibitor Pds1, a process necessary for the onset of chromosome segregation. We have isolated three mutant alleles of CDC4 (cdc4-10, cdc4-11, and cdc4-16) which suppress the nuclear division defect of cdc20-1 cells. However, the previously characterized mutation cdc4-1 and a new allele, cdc4-12, do not alleviate the defect of cdc20-1 cells. This genetic interaction suggests an additional role for Cdc4 in G(2)/M. Reexamination of the cdc4-1 mutant revealed that, in addition to being defective in the onset of S phase, it is also defective in G(2)/M transition when released from hydroxyurea-induced S-phase arrest. A second function for CDC4 in late S or G(2) phase was further confirmed by the observation that cells lacking the CDC4 gene are arrested both at G(1)/S and at G(2)/M. We subsequently isolated additional temperature-sensitive mutations in the CDC4 gene (such as cdc4-12) that render the mutant defective in both G(1)/S and G(2)/M transitions at the restrictive temperature. While the G(1)/S block in both cdc4-12 and cdc4Delta mutants is abolished by the deletion of the SIC1 gene (causing the mutants to be arrested predominantly in G(2)/M), the preanaphase arrest in the cdc4-12 mutant is relieved by the deletion of PDS1. Collectively, these observations suggest that, in addition to its involvement in the initiation of S phase, Cdc4 may also be required for the onset of anaphase.     

Mutational analysis of Cak1p, an essential protein kinase that regulates cell cycle progression

In Saccharomyces cerevisiae, entry into S phase requires the activation of the protein kinase Cdc28p through binding with cyclin Clb5p or Clb6p, as well as the destruction of the cyclin-dependent kinase inhibitor Sic1p. Mutants that are defective in this activation event arrest after START, with unreplicated DNA and multiple, elongated buds. These mutants include cells defective in CDC4, CDC34 or CDC53, as well as cells that have lost all CLB function. Here we describe mutations in another gene, CAK1, that lead to a similar arrest. Cells that are defective in CAK1 are inviable and arrest with a single nucleus and multiple, elongated buds. CAK1 encodes a protein kinase most closely related to the Cdc2p family of protein kinases. Mutations that lead to the production of an inactive kinase that can neither autophosphorylate, nor phosphorylate Cdc28p in vitro are also incapable of rescuing a cell with a deletion of CAK1. These results underscore the importance of the Cak1p protein kinase activity in cell cycle progression. Received: 2 January 1997 / Accepted: 20 June 1997     

A new pair of B-type cyclins from Saccharomyces cerevisiae that function early in the cell cycle.

Two new B-type cyclin genes from Saccharomyces cerevisiae, called CLB5 and CLB6, are located in a tail to tail arrangement adjacent to the G2/M phase promoting cyclins CLB2 and CLB1, respectively. These genomic cyclin arrays are flanked by tRNAs and repeated sequences of Ty elements suggesting an intrachromosomal gene duplication followed by an interchromosomal gene duplication. Based on their deduced protein sequence the CLB5 and CLB6 genes form a new pair of B-type cyclins. They are most related to each other and then to the deduced protein sequence of their adjacent genes CLB1 and CLB2. Both genes are periodically expressed, peaking early in the cell cycle. Loss of function mutants are viable, but clb5- mutants exhibit a delay in S phase whereas clb6- mutants show a delay in late G1 and/or S phase. The clb5 mutant phenotype is somewhat more pronounced in a double null mutant. Both cyclins have the potential to interact with the p34CDC28 kinase in vivo.