Wild-type Cdc2 Research Articles

The DNA replication protein Cdc6 inhibits the microtubule-organizing activity of the centrosome

The centrosome serves as a major microtubule-organizing center (MTOC). The Cdc6 protein is a component of the pre-replicative complex and a licensing factor for the initiation of chromosome replication and localizes to centrosomes during the S and G2 phases of the cfell cycle of human cells. This cell cycle-dependent localization of Cdc6 to the centrosome motivated us to investigate whether Cdc6 negatively regulates MTOC activity and to determine the integral proteins that comprise the pericentriolar material (PCM). Time-lapse live-cell imaging of microtubule regrowth revealed that Cdc6 depletion increased microtubule nucleation at the centrosomes and that expression of Cdc6 in Cdc6-depleted cells reversed this effect. This increase and decrease in microtubule nucleation correlated with the centrosomal intensities of PCM proteins such as γ-tubulin, pericentrin, CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), and centrosomal protein 192 (Cep192). The regulation of microtubule nucleation and the recruitment of PCM proteins to the centrosome required Cdc6 ATPase activity, as well as a centrosomal localization of Cdc6. These results suggest a novel function for Cdc6 in coordinating centrosome assembly and function.

DNA replication licensing factor Cdc6 and Plk4 kinase antagonistically regulate centrosome duplication via Sas-6

Centrosome number is tightly controlled during the cell cycle to ensure proper spindle assembly and cell division. However, the underlying mechanism that controls centrosome number remains largely unclear. We show herein that the DNA replication licensing factor Cdc6 is recruited to the proximal side of the centrioles via cyclin A to negatively regulate centrosome duplication by binding and inhibiting the cartwheel protein Sas-6 from forming a stable complex with another centriole duplication core protein, STIL. We further demonstrate that Cdc6 colocalizes with Plk4 at the centrosome, and interacts with Plk4 during S phase. Plk4 disrupts the interaction between Sas-6 and Cdc6, and suppresses the inhibitory role of Cdc6 on Sas-6 by phosphorylating Cdc6. Overexpressing wild-type Cdc6 or Plk4-unphosphorylatable Cdc6 mutant 2A reduces centrosome over-duplication caused by Plk4 overexpression or hydroxyurea treatment. Taken together, our data demonstrate that Cdc6 and Plk4 antagonistically control proper centrosome duplication during the cell cycle.

Two nuclear export signals of Cdc6 are differentially associated with CDK-mediated phosphorylation residues for cytoplasmic translocation

Cdc6 is cleaved at residues 442 and 290 by caspase-3 during apoptosis producing p49-tCdc6 and p32-tCdc6, respectively. While p32-tCdc6 is unable to translocate into the cytoplasm, p49-tCdc6 retains cytoplasmic translocation activity, but it has a lower efficiency than wild-type Cdc6. We hypothesized that a novel nuclear export signal (NES) sequence exists between amino acids 290 and 442. Cdc6 contains a novel NES in the region of amino acids 300–315 (NES2) that shares sequence similarity with NES1 at residues 462–476. In mutant versions of Cdc6, we replaced leucine with alanine in NES1 and NES2 and co-expressed the mutant constructs with cyclin A. We observed that the cytoplasmic translocation of these mutants was reduced in comparison to wild-type Cdc6. Moreover, the cytoplasmic translocation of a mutant in which all four leucine residues were mutated to alanine was significantly inhibited in comparison to the translocation of wild-type Cdc6. The Crm1 binding activities of Cdc6 NES mutants were consistent with the efficiency of its cytoplasmic translocation. Further studies have revealed that L468 and L470 of NES1 are required for cytoplasmic translocation of Cdc6 phosphorylated at S74, while L311 and L313 of NES2 accelerate the cytoplasmic translocation of Cdc6 phosphorylated at S54. These results suggest that the two NESs of Cdc6 work cooperatively and distinctly for the cytoplasmic translocation of Cdc6 phosphorylated at S74 and S54 by cyclin A/Cdk2.

Cell division cycle 6, a mitotic substrate of polo-like kinase 1, regulates chromosomal segregation mediated by cyclin-dependent kinase 1 and separase

Defining the links between cell division and DNA replication is essential for understanding normal cell cycle progression and tumorigenesis. In this report we explore the effect of phosphorylation of cell division cycle 6 (Cdc6), a DNA replication initiation factor, by polo-like kinase 1 (Plk1) on the regulation of chromosomal segregation. In mitosis, the phosphorylation of Cdc6 was highly increased, in correlation with the level of Plk1, and conversely, Cdc6 is hypophosphorylated in Plk1-depleted cells, although cyclin A- and cyclin B1-dependent kinases are active. Binding between Cdc6 and Plk1 occurs through the polo-box domain of Plk1, and Cdc6 is phosphorylated by Plk1 on T37. Immunohistochemistry studies reveal that Cdc6 and Plk1 colocalize to the central spindle in anaphase. Expression of T37V mutant of Cdc6 (Cdc6-TV) induces binucleated cells and incompletely separated nuclei. Wild-type Cdc6 but not Cdc6-TV binds cyclin-dependent kinase 1 (Cdk1). Expression of wild-type Plk1 but not kinase-defective mutant promotes the binding of Cdc6 to Cdk1. Cells expressing wild-type Cdc6 display lower Cdk1 activity and higher separase activity than cells expressing Cdc6-TV. These results suggest that Plk1-mediated phosphorylation of Cdc6 promotes the interaction of Cdc6 and Cdk1, leading to the attenuation of Cdk1 activity, release of separase, and subsequent anaphase progression.

Cdc6 is a rate-limiting factor for proliferative capacity during HL60 cell differentiation

The DNA replication (or origin) licensing pathway represents a critical step in cell proliferation control downstream of growth signalling pathways. Repression of origin licensing through down-regulation of the MCM licensing factors (Mcm2–7) is emerging as a ubiquitous route for lowering proliferative capacity as metazoan cells exit the cell division cycle into quiescent, terminally differentiated and senescent “out-of-cycle” states. Using the HL60 monocyte/macrophage differentiation model system and a cell-free DNA replication assay, we have undertaken direct biochemical investigations of the coupling of origin licensing to the differentiation process. Our data show that down-regulation of the MCM loading factor Cdc6 acts as a molecular switch that triggers loss of proliferative capacity during early engagement of the somatic differentiation programme. Consequently, addition of recombinant Cdc6 protein to in vitro replication reactions restores DNA replication competence in nuclei prepared from differentiating cells. Differentiating HL60 cells over-expressing either wild-type Cdc6 or a CDK phosphorylation-resistant Cdc6 mutant protein (Cdc6A4) exhibit an extended period of cell proliferation compared to mock-infected cells. Notably, differentiating HL60 cells over-expressing the Cdc6A4 mutant fail to down-regulate Cdc6 protein levels, suggesting that CDK phosphorylation of Cdc6 is linked to its down-regulation during differentiation and the concomitant decrease in cell proliferation. In this experimental model, Cdc6 therefore plays a key role in the sequential molecular events leading to repression of origin licensing and loss of proliferative capacity during execution of the differentiation programme.

Cdc2-mediated Schwann cell migration during peripheral nerve regeneration

Schwann cell migration facilitates peripheral nerve regeneration after injury. We have recently found increased activation of Cdc2 kinase in regenerating sciatic nerves. Here we show that Cdc2 phosphorylation of caldesmon regulates Schwann cell migration and nerve regeneration. A robust but transient increase in Cdc2 expression was found in cultured Schwann cells prepared from the sciatic nerve in rats that had undergone crush injury for 7 days. These ;injury-preconditioned' Schwann cells exhibited enhanced migration compared with non-preconditioned control cells and treatment with the cdk inhibitor roscovitine prevented cell migration. After transduction with recombinant Cdc2 DNA adenoviral vectors, Schwann cells were implanted into sciatic nerves; those expressing wild-type Cdc2 migrated further in the distal direction than those expressing dominant-negative Cdc2. We identified caldesmon as a downstream substrate of Cdc2 in Schwann cells and its phosphorylation by Cdc2 changed its subcellular localization. Overexpression of dominant-negative caldesmon significantly counteracted the migration effect caused by Cdc2. Finally, neurite outgrowth of cultured DRG sensory neurons, facilitated by co-culture with injury-preconditioned Schwann cells, was suppressed by roscovitine treatment. The results indicate that activation of the Cdc2-caldesmon pathway is necessary for Schwann cell migration and suggest a role for this pathway in peripheral axonal growth.

Safe from Degradation

DNA replication is initiated through the assembly of prereplication complexes (pre-RCs); replication does not occur in quiescent cells and is limited to once per cell cycle because, outside of the G 1 phase of the cell cycle, inhibitors prevent assembly of the pre-RC. During G 1 , APC Cdh1 (the form of the anaphase-promoting complex E3 ubiquitin ligase that contains Cdh1) targets the pre-RC assembly inhibitors geminin and cyclin A for degradation. Paradoxically, however, APC Cdh1 targets Cdc6, which is essential to pre-RC assembly, for destruction as well (see Ayad). Mailand and Diffley noted that, in cultured human cells treated with the cyclin-dependent kinase (CDK) inhibitor roscovitine, Cdc6 abundance was reduced, whereas the abundance of geminin and cyclin A was unaffected. Knockdown of Cdk2 with small interfering RNA (siRNA) also decreased Cdc6 abundance. Roscovitine accelerated Cdc6 degradation, as did depletion of Cdk2 or cyclins E1 and E2, whereas overexpression of cyclin E (which forms an active CDK complex with Cdk2) promoted Cdc6 accumulation. Mutational analysis indicated that the effects of cyclin E on Cdc6 stability depended on Cdc6 phosphorylation. Cdc6 phosphorylation inhibited its APC Cdh1 -dependent ubiquitination and degradation; further, whereas wild-type Cdc-6 or an unphosphorylatable mutant coimmunoprecipitated with Cdh1, a phospho-mimicking Cdc6 mutant did not. In serum-starved (noncycling) cells, Cdc6, geminin, and cyclin A were undetectable. After serum stimulation, and reentry into the cell cycle, Cdc6 abundance increased 3 to 4 hours before the other APC Cdh1 targets became detectable. When Cdc6 was detected, it was phosphorylated, and cyclin E-associated kinase activity appeared simultaneously with Cdc6. Thus, the authors propose that, after entry into the cell cycle, cyclin E-Cdk2-dependent phosphorylation of Cdc6 allows it to accumulate (and promote pre-RC assembly) before inactivation of APC Cdh1 and the ensuing accumulation of pre-RC assembly inhibitors such as geminin and cyclin A. N. Mailand, J. F. X. Diffley, CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis. Cell 122 , 915-926 (2005). [PubMed] N. G. Ayad, CDKs give Cdc6 a license to drive into S phase. Cell 122 , 825-827 (2005). [PubMed]

Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1.

CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. Here, we show that, in addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G(1). A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G(1) and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent proteolysis of CDC6 in early G(1) and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G(1).

Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP motif.

Saccharomyces cerevisiae Cdc6 plays an essential role in establishing and maintaining the prereplicative complex (pre-RC) by interacting with the origin recognition complex (ORC) and associating with chromatin origins. These interactions are required to load minichromosome maintenance proteins (MCMs) and other initiator proteins onto replication origins. Although the temperature-sensitive cdc6 mutant, cdc6-1, has been widely used for these studies, the molecular mechanism of the cdc6-1 mutation has been unclear. In this study, we have identified a base substitution at Gly260-->Asp, near the CDC-NTP motif. Using a chromatin immunoprecipitation assay (CHIP), we found that cdc6-1 fails to load Mcm5 onto the replication origins. Chromatin fractions were used to study Mcm5 binding in both the wildtype and mutant background. These studies indicated that Cdc6 is also involved in unloading Mcm5 from chromatin. Specifically, the cdc6-1 mutation protein, cdc6(G260D), which failed to load Mcm5 onto replication origins, also failed to unload the Mcm5 protein. Furthermore, the overexpression of wildtype CDC6 accelerated the unloading of Mcm5 from chromatin fractions. In the absence of functional Cdc6, the Mcm5 protein showed nonorigin binding to chromatin with the cell cycle arrested at the G1S phase transition. Our results suggested that the cdc6(G260D) mutant protein fails to assemble an operational replicative complex and that wildtype Cdc6 plays a role in preventing re-replication by controlling the unloading the MCMs from chromatin origins.

The Stability of the Cdc6 Protein Is Regulated by Cyclin-dependent Kinase/Cyclin B Complexes inSaccharomyces cerevisiae

The Saccharomyces cerevisiae Cdc6 protein is necessary for the formation of prereplicative complexes that are a prerequisite for firing origins during DNA replication in the S phase. In budding yeast, the presence of Cdc6 protein is normally restricted to the G(1) phase of the cell cycle, at least partly because of its proteolytic degradation in the late G(1)/early S phase. Here we show that a Cdc28-dependent mechanism targets p57(CDC6) for degradation in mitotic-arrested budding yeast cells. Consistent with this observation, Cdc6-7 and Cdc6-8 proteins, mutants lacking Cdc28 phosphorylation sites, are stabilized relative to wild-type Cdc6. Our data also suggest a correlation between the absence of Cdc28/Clb kinase activity and Cdc6 protein stabilization, because a drop in Cdc28/Clb-associated kinase activity allows mitotic-arrested cells to accumulate Cdc6 protein. Finally, we also show that cdc28 temperature-sensitive G(1) mutants accumulate Cdc6 protein because of a post-transcriptional mechanism. Our data suggest that budding yeast cells target Cdc6 for degradation through a Cdc28-dependent mechanism in each cell cycle.

The Essential Role of Saccharomyces cerevisiae CDC6 Nucleotide-binding Site in Cell Growth, DNA Synthesis, and Orc1 Association

Saccharomyces cerevisiae Cdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6 temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G1/S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition.

UCN-01 Abrogates G2 Arrest through a Cdc2-dependent Pathway That Is Associated with Inactivation of the Wee1Hu Kinase and Activation of the Cdc25C Phosphatase

We have previously demonstrated that UCN-01, a potent protein kinase inhibitor currently in phase I clinical trials for cancer treatment, abrogates G2 arrest following DNA damage. Here we used murine FT210 cells, which contain temperature-sensitive Cdc2 mutations, to determine if UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the non-permissive temperature for Cdc2 function. Failure to abrogate G2 arrest was not due to UCN-01-inactivation at the elevated temperature because parental FM3A cells, which have wild-type Cdc2, were sensitive to UCN-01-induced G2 checkpoint abrogation. Having established that UCN-01 acted through Cdc2, we next assessed UCN-01's effect on the Cdc2-inhibitory kinase, Wee1Hu, and the Cdc2-activating phosphatase, Cdc25C. We found that Wee1Hu was indeed inactivated in UCN-01-treated cells, possibly just prior to Cdc2 activation and entry of DNA-damaged cells into mitosis. This inhibition appeared, however, to be a consequence of a further upstream action since in vitro studies revealed purified Wee1Hu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action, UCN-01 also promoted the hyperphosphorylation (activation) of Cdc25C in DNA-damaged cells. Our results suggest that UCN-01 abrogates G2 checkpoint function through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C, and that changes observed in these mitotic regulators are downstream consequences of UCN-01's actions.

The Cell Cycle Program in Germ Cells of theDrosophilaEmbryo

The germ cells of metazoans follow a program of proliferation that is distinct from proliferation programs of somatic cells. Despite their developmental importance, the cell proliferation program in the metazoan primordial germ cells is not well characterized and the regulatory controls are not understood. InDrosophila melanogaster,germ cell precursors (called pole cells) proliferate early in embryogenesis and then enter a prolonged quiescence. We found that polar nuclear divisions are asynchronous and lag behind somatic nuclear divisions during syncytial cycles 9 and 10. Thus, the polar division program deviates from the somatic division program when pole nuclei and somatic nuclei still share a common cytoplasm, earlier than previously thought. The lag in polar nuclear divisions is independent ofgrapes,which is required for lengthening somatic cell cycles 10–13. Mapping of the last S phase in pole cells and measurement of their DNA content indicate that pole cells become quiescent in G2 phase of the cell cycle. We were able to drive quiescent pole cells into mitosis by induction of either an activator of Cdc2 (Cdc25stringphosphatase) or a mutant form of Cdc2 that cannot be inhibited by phosphorylation. In contrast, induction of wild-type Cdc2 with a mitotic cyclin did not induce mitosis in pole cells. We propose that inhibition of Cdc2 by phosphorylation contributes to G2 arrest in pole cells during embryogenesis. Furthermore, pole cells enter G1 following induced mitoses, indicating that entry into both mitosis and S phase is blocked in quiescent pole cells. These studies represent the first molecular characterization of proliferation in embryonic germ cells of Drosophila

Saccharomyces cerevisiae Cdc6 Stimulates Abf1 DNA Binding Activity

In budding yeast Saccharomyces cerevisiae, an ARS binding factor 1 (Abf1) binds to the sequence-specific DNA element involved in DNA replication and transcription. We describe in this study how yeast Cdc6 protein stimulates Abf1 protein DNA binding activities. The Abf1 binding activity was reduced approximately 20-fold in a cdc6-1 mutant than in the wild-type strain. Introducing a copy of the wild-type CDC6 gene into the cdc6-1 mutant strain restored the Abf1 DNA binding activity. We demonstrated that recombinant Abf1 binds to ARS1 in vitro, and its DNA binding activity can be highly stimulated by the addition of a fusion glutathione S-transferase (GST)-Cdc6 protein. Deletion analysis revealed that the stimulating region is located at the amino terminus of the Cdc6 protein. However, we could not find the direct physical interaction between Cdc6 and Abf1. Instead, we found that the GST-Cdc6 can compete with distamycin A for binding to the DNA molecule. As distamycin A is a specific reagent that binds noncovalently to DNA at (A + T)-rich tracks, the stimulation of Abf1 DNA binding activity may be mediated by the Cdc6/DNA interaction. Our results favor a hypothesis that Cdc6 may function as an architectural factor in the assembly of a functional initiation replication complex.

Interaction between yeast Cdc6 protein and B-type cyclin/Cdc28 kinases.

During purification of recombinant Cdc6 expressed in yeast, we found that Cdc6 interacts with the critical cell cycle, cyclin-dependent protein kinase Cdc28. Cdc6 and Cdc28 can be coimmunoprecipitated from extracts, Cdc6 is retained on the Cdc28-binding matrix p13-agarose, and Cdc28 is retained on an affinity column charged with bacterially produced Cdc6. Cdc6, which is a phosphoprotein in vivo, contains five Cdc28 consensus sites and is a substrate of the Cdc28 kinase in vitro. Cdc6 also inhibits Cdc28 histone H1 kinase activity. Strikingly, Cdc6 interacts preferentially with B-type cyclin/Cdc28 complexes and not Cln/Cdc28 in log-phase cells. However, Cdc6 does not associate with Cdc28 when cells are blocked at the restrictive temperature in a cdc34 mutant, a point in the cell cycle when the B-type cyclin/Cdc28 inhibitor p40Sic1 accumulates and purified p40Sic1 inhibits the Cdc6/Cdc28 interaction. Deletion of the Cdc28 interaction domain from Cdc6 yields a protein that cannot support growth. However, when overproduced, the mutant protein can support growth. Furthermore, whereas overproduction of wild-type Cdc6 leads to growth inhibition and bud hyperpolarization, overproduction of the mutant protein supports growth at normal rates with normal morphology. Thus, the interaction may have a role in the essential function of Cdc6 in initiation and in restraining mitosis until replication is complete.

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.

SV40 Large T Antigen Transactivates the Human cdc2 Promoter by Inducing a CCAAT Box Binding Factor

Cyclin-dependent protein kinases (Cdks) play a key role in the cell division cycle of eukaryotic cells. Cdc2, the first mammalian Cdk that was discovered, is expressed in S phase and functions in the G2 to M phase transition. By transfecting segments of the human cdc2 promoter linked to a reporter gene into monkey kidney (CV-1) cells, we identified the region containing the Sp1, E2F, and two CCAAT box binding sites as essential and sufficient for basal transcription. SV40 large T antigen (SV40-LT) is a viral oncoprotein that transactivates viral and cellular promoters and induces DNA synthesis in quiescent cells. SV40-LT transactivated wild-type cdc2 promoter/reporter constructs in a dose-dependent manner, coinciding with an increase in endogenous cdc2 mRNA. A mutant promoter from which the two CCAAT box motifs were deleted was 8-fold less sensitive to SV40-LT. Activation by SV40-LT did not require its ability to bind the retinoblastoma or p53 tumor suppressor proteins. SV40-LT induced a specific CCAAT box-binding factor (CBF) in CV-1 and COS-7 cells, as judged by gel shift and Southwestern analyses. Similar results were obtained in human fibroblasts expressing a conditional SV40-LT. The SV40-LT-inducible CBF appears to be novel and differs from the CBF that activates heat shock protein 70 gene expression.

The Wee1 protein kinase regulates T14 phosphorylation of fission yeast Cdc2.

The Cdc2 protein kinase is a key regulator of the G1-S and G2-M cell cycle transitions in the fission yeast Schizosaccharomyces pombe. The activation of Cdc2 at the G2-M transition is triggered by dephosphorylation at a conserved tyrosine residue Y15. The level of Y15 phosphorylation is controlled by the Wee1 and Mik1 protein kinases acting in opposition to the Cdc25 protein phosphatase. Here, we demonstrate that Wee1 overexpression leads to a high stoichiometry of phosphorylation at a previously undetected site in S. pombe Cdc2, T14. T14 phosphorylation was also detected in certain cell cycle mutants blocked in progression through S phase, indicating that T14 phosphorylation might normally occur at low stoichiometry during DNA replication or early G2. Strains in which the chromosomal copy of cdc2 was replaced with either a T14A or a T14S mutant allele were generated and the phenotypes of these strains are consistent with T14 phosphorylation playing an inhibitory role in the activation of Cdc2 as it does in higher eukaryotes. We have also obtained evidence that Wee1 but not Mik1 or Chk1 is required for phosphorylation at this site, that the Mik1 and Chk1 protein kinases are unable to drive T14 phosphorylation in vivo, that residue 14 phosphorylation requires previous phosphorylation at Y15, and that the T14A mutant, unlike Y15F, is recessive to wild-type Cdc2 activity. Finally, the normal duration of G2 delay after irradiation or hydroxyurea treatment in a T14A mutant strain indicates that T14 phosphorylation is not required for the DNA damage or replication checkpoint controls.

A dominant negative allele of p34cdc2 shows altered phosphoamino acid content and sequesters p56cdc13 cyclin.

The cdc2 gene product, a 34-kDa phosphoprotein with serine/threonine protein kinase activity, has been implicated as the key component in the regulation of the eucaryotic cell cycle. Activation of the cdc2 protein kinase is regulated by its phosphorylation state and by interaction with other proteins. We have mutagenized the fission yeast cdc2 gene to obtain conditionally dominant negative alleles. One of these mutants, named DL2, is characterized in this report. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and leads to arrest in the G2 phase of the cell cycle. The mutant phenotype is the result of a single amino acid change in the GDSEID motif of the protein, a region of identity in all cdc2 homologs, and results in a nonfunctional protein that shows an altered content of phosphothreonine. Multicopy suppressors of the dominant negative phenotype have been isolated, and one of these has been shown to encode the cdc13 cyclin B gene product.

Expression of a dominant negative allele of cdc2 prevents activation of the endogenous p34cdc2 kinase

The cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a 34 kDa phosphoprotein with serine/threonine protein kinase activity that acts as the key component in regulation of the eukaryotic cell cycle. We used a repressible promoter fused to the cdc2 cDNA to isolate conditionally dominant negative mutants of cdc2. One of these mutants, DL5, is described in this paper. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and confers cell cycle arrest with a typical cdc- phenotype. Sequencing of the mutant cdc2 gene revealed a single amino acid substitution in a region highly conserved in cdc2-like proteins. The mutant protein exhibits no protein kinase activity, but is able to bind a component(s) required for an active protein kinase complex and thereby prevents binding of this component(s) to the co-existing wild-type cdc2 protein. We also demonstrate that S. pombe p34cdc2 contains no phosphoserine.