Binding and catalytic properties of the Cdc2 and Crp proteins of Dictyostelium.

Dictyostelium expresses at least two proteins of the cyclin-dependent kinase (Cdk) family, Cdc2 and Crp. Cdc2 levels remain relatively constant during differentiation, whereas the levels of Crp increase dramatically as differentiation progresses. Crp is highly related to the mammalian Cdk5, and p25 (a truncated form of p35, the activating subunit of Cdk5 from mammalian brain) stimulates the histone H1 kinase activity of GST-Crp by several fold. In contrast, p25 does not stimulate the histone H1 kinase activity of GST-Cdc2 or the Cdc2 activity present in cell extracts from vegetative Dictyostelium cells. GST-Cdc2, in vitro translated Cdc2 and Cdc2 from all stages of differentiation bind to p13suc1. In contrast, GST-Crp, in vitro translated Crp and the Crp protein present in cell extracts do not bind to p13suc1. We have confirmed a previous report by Arakane and Maeda [J. Plant Res. (1997) 110, 81-85] that there is a peak of p13suc1 bound histone H1 kinase activity during late development, but we found that there was no corresponding peak of p13suc1 bound Cdc2 protein that corresponds to this activity. Taken together, these data suggest that neither Cdc2 nor Crp is responsible for the late developmental peak of histone H1 kinase activity that binds to p13suc1.     

Overexpression of an activated rasG gene during growth blocks the initiation of Dictyostelium development.

Transformants that expressed either the wild-type rasG gene, an activated rasG-G12T gene, or a dominant negative rasG-S17N gene, all under the control of the folate-repressible discoidin (dis1gamma) promoter, were isolated. All three transformants expressed high levels of Ras protein which were reduced by growth in the presence of folate. All three transformants grew slowly, and the reduction in growth rate correlated with the amount of RasG protein produced, suggesting that RasG is important in regulating cell growth. The pVEII-rasG transformant containing the wild-type rasG gene developed normally despite the presence of high levels of RasG throughout development. This result indicates that the down regulation of rasG that normally occurs during aggregation of wild-type strains is not essential for the differentiation process. Dictyostelium transformants expressing the dominant negative rasG-S17N gene also differentiated normally. Dictyostelium transformants that overexpressed the activated rasG-G12T gene did not aggregate. The defect occurred very early in development, since the expression of car1 and pde, genes that are normally induced soon after the initiation of development, was repressed. However, when the transformant cells were pulsed with cyclic AMP, expression of both genes returned to wild-type levels. The transformants exhibited chemotaxis to cyclic AMP, and development was synergized by mixing with wild-type cells. Furthermore, cells that were pulsed with cyclic AMP for 4 h before being induced to differentiate by plating on filters produced small, but otherwise normal, fruiting bodies. These results suggest that the rasG-G12T transformants are defective in cyclic AMP production and that RasG - GTP blocks development by interfering with the initial generation of cyclic AMP pulses.     

cAMP-dependent protein kinase regulates Polysphondylium pallidum development

In eukaryotic cells, the universal second messenger cAMP regulates various aspects of development and differentiation. The primary target for cAMP is the regulatory subunit of cAMP-dependent protein kinase A (PKA), which, upon cAMP binding, dissociates from the catalytic subunit and thus activates it. In the soil amoeba Dictyostelium discoideum, the function of PKA in growth, development and cell differentiation has been thoroughly investigated and substantial information is available. To obtain a more general view, we investigated the influence of PKA on development of the related species Polysphondylium pallidum. Cells were transformed to overexpress either a dominant negative mutant of the regulatory subunit (Rm) from Dictyostelium that cannot bind cAMP, or the catalytic subunit (PKA-C) from Dictyostelium. Cells overexpressing Rm rarely aggregated and the few multicellular structures developed slowly into very small fruiting bodies without branching of secondary sorogens, the prominent feature of Polysphondylium. Few round spores with reduced viability were formed. When mixed with wild-type cells and allowed to develop, the Rm cells were randomly distributed in aggregation streams, but were later found in the posterior region of the culminating slug or were left behind on the surface of the substratum. The PKA-C overexpressing cells exhibited precocious development and formed more aggregates of smaller size. Moreover, expression of PKA-C under the control of the prestalk-specific ecmB promoter of Dictyostelium leads to protrusions from aggregation streams. We conclude that Dictyostelium PKA subunits introduced into Polysphondylium cells are functional as signal components, indicating that a biochemically similar PKA mechanism works in Polysphondylium.     

Identification of a conserved sequence motif that promotes Cdc37 and cyclin D1 binding to Cdk4

Cdc37 is a molecular chaperone that is important for the stability and activity of several protein kinases, including Cdk4 and Raf1. We first determined, using in vitro assays, that Cdc37 binds to the amino-terminal lobe of Cdk4. Subsequent mutagenesis revealed that Gly-15 (G15A) and Gly-18 (G18A) were critical for Cdc37-Cdk4 complex formation. Gly-15 and Gly-18 of Cdk4 are within the conserved Gly-X-Gly-X-X-Gly motif that is required for ATP binding to the kinase. Mutation of either glycine at the equivalent positions of Raf1 (G358A and G361A) also inhibited Cdc37 binding to Raf1. Replacing another conserved residue critical for ATP binding and kinase activity, Lys-35 (K35A), reduced Cdc37-Cdk4 complex formation but to a lesser extent. The interaction of Cdk4 with Cdc37 in vitro was not sensitive to changes in ATP levels. Cell-based assays indicated that Cdk4(G15A) and Cdk4(G18A) were present at the same level as wild type Cdk4. Equivalent amounts of p16 bound to Cdk4(G15A) and Cdk4(G18A) relative to wild type Cdk4, suggesting that Cdk4(G15A) and Cdk4(G18A) adopt significant tertiary structure. However, in contrast to wild type Cdk4, Cdk4(G15A), and Cdk4(G18A) had greatly reduced binding of cyclin D1, Cdc37, and Hsp90. Importantly, overexpression of Cdc37 not only stimulated cyclin D1 binding to wild type Cdk4 but also restored its binding to Cdk4(G15A). Under the same conditions, p16 binding to wild type Cdk4 was suppressed. Our findings show that the interaction of Cdc37 with its client protein kinases requires amino acid residues within a motif that is present in many protein kinases.     

Isolation and characterization of a cdc 2 cDNA from Dictyostelium discoideum.

A cdc2 homologous sequence was amplified from Dictyostelium discoideum by the polymerase chain reaction and used to isolate several cDNA clones. The amino acid sequence encoded by these cDNAs exhibited approx. 60% identity to the Cdc2 proteins of other species. A cDNA containing the entire coding sequence complemented the temperature sensitive cdc28 mutant of Saccharomyces cerevisiae, although growth of the transformants was slow and limited. Southern blot analysis of restriction digests under high stringency conditions provided evidence that Dictyostelium contains a single cdc2 gene, although at lower stringency multiple fragments were detected, suggesting the existence of a cdc2 gene family. Northern blot analysis of RNA from different stages of Dictyostelium development showed that cdc2 mRNA levels increased during aggregation and then decreased to low levels by the pseudoplasmodial stage of development. By contrast, cdc2 mRNA levels remained relatively constant as cells passed from exponential growth to the stationary phase.     

A secondary disruption of the dmpA gene encoding a large membrane protein allows aggregation defective Dictyostelium rasC- cells to form multicellular structures

The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC, results in a strain that does not aggregate and has defects in both cAMP signal relay and cAMP chemotaxis. Disruption of a second gene in the rasC(-) strain by Restriction Enzyme Mediated Integration produced cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene (dmpA) encoded a novel membrane protein that was designated Dmp1. Although the rasC(-)/dmpA(-) cells progressed through early development, they did not form aggregation streams on a plastic surface under submerged starvation conditions. Phosphorylation of PKB in response to cAMP, which is significantly reduced in rasC(-) cells, remained low in the rasC(-)/dmpA(-) cells. However, in spite of this low PKB phosphorylation, the rasC(-)/dmpA(-) cells underwent efficient chemotaxis to cAMP in a spatial gradient. Cyclic AMP accumulation, which was greatly reduced in the rasC(-) cells, was restored in the rasC(-)/dmpA(-) strain, but cAMP relay in these cells was not apparent. These data indicate that although the rasC(-)/dmpA(-) cells were capable of associating to form multicellular structures, normal aggregative cell signaling was clearly not restored. Disruption of the dmpA gene in a wild-type background resulted in cells that exhibited a slight defect in aggregation and a more substantial defect in late development. These results indicate that, in addition to the role played by Dmp1 in aggregation, it is also involved in late development.     

The effect of the disruption of a gene encoding a PI4 kinase on the developmental defect exhibited by Dictyostelium rasC(-) cells

The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC results in a strain that fails to aggregate with defects in both cAMP signal relay and chemotaxis. Restriction enzyme mediated integration disruption of a second gene in the rasC(-) strain resulted in cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene, designated pikD(1), encodes a member of the phosphatidyl-inositol-4-kinase beta subfamily. Although the rasC(-)/pikD(1) cells were capable of progressing through early development, when starved on a plastic surface under submerged conditions, they did not form aggregation streams or exhibit pulsatile motion. The rasC(-)/pikD(1) cells were extremely efficient in their ability to chemotax to cAMP in a spatial gradient, although the reduced phosphorylation of PKB in response to cAMP observed in rasC(-) cells, was unchanged. In addition, the activation of adenylyl cyclase, which was greatly reduced in the rasC(-) cells, was only minimally increased in the rasC(-)/pikD(1) strain. Thus, although the rasC(-)/pikD(-) cells were capable of associating to form multicellular structures, normal cell signaling was clearly not restored. The disruption of the pikD gene in a wild type background resulted in a strain that was delayed in aggregation and formed large aggregation streams, when starved on a plastic surface under submerged conditions. This strain also exhibited a slight defect in terminal development. In conclusion, disruption of the pikD gene in a rasC(-) strain resulted in cells that were capable of forming multicellular structures, but which did so in the absence of normal signaling and aggregation stream formation.     

The cyclin-dependent kinase inhibitor roscovitine inhibits kinase activity, cell proliferation, multicellular development, and Cdk5 nuclear translocation in Dictyostelium discoideum

Roscovitine, a cyclin-dependent kinase (Cdk) inhibitor, inhibited kinase activity and the axenic growth of Dictyostelium discoideum at micromolar concentrations. Growth was almost fully rescued in 50 μM and ≈ 50% rescued in 100 μM roscovitine-treated cultures by the over-expression of Cdk5-GFP. This supports the importance of Cdk5 function during cell proliferation in Dictyostelium and indicates that Cdk5 is a primary target of the drug. Roscovitine did not affect the expression of Cdk5 protein during axenic growth but did inhibit its nuclear translocation. This novel result suggests that the effects of roscovitine could be due in part to altering Cdk5 translocation in other systems as well. Kinase activity was inhibited by roscovitine in assays using AX3 whole cell lysates, but not in assays using lysates from Cdk5-GFP over-expressing cells. At higher concentrations, roscovitine impaired slug and fruiting body formation. Fruiting bodies that did form were small and produced relatively fewer spores many of which were round. However, roscovitine did not affect stalk cell differentiation. Together with previous findings, these data reveal that roscovitine inhibits Cdk5 during growth and as yet undefined Cdks during mid-late development.     

Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration

The ampA gene has a role in cell migration in Dictyostelium discoideum. Cells overexpressing AmpA show an increase in cell migration, forming large plaques on bacterial lawns. A second-site suppressor of this ampA-overexpressing phenotype identified a previously uncharacterized gene, ndm, which is described here. The Ndm protein is predicted to contain a coiled-coil BAR-like domain-a domain involved in endocytosis and membrane bending. ndm-knockout and Ndm-monomeric red fluorescent protein-expressing cell lines were used to establish a role for ndm in suppressing endocytosis. An increase in the rate of endocytosis and in the number of endosomes was detected in ndm(-) cells. During migration ndm(-) cells formed numerous endocytic cups instead of the broad lamellipodia structure characteristic of moving cells. A second lamellipodia-based function-cell spreading-was also defective in the ndm(-) cells. The increase in endocytosis and the defect in lamellipodia formation were associated with reduced chemotaxis in ndm(-) cells. Immunofluorescence results and glutathione S-transferase pull-down assays revealed an association of Ndm with coronin and F-actin. The results establish ndm as a gene important in regulating the balance between formation of endocytic cups and lamellipodia structures.     

Cdk1 coordinates cell-surface growth with the cell cycle

The mechanisms that control cell growth during the cell cycle are poorly understood. In budding yeast, cyclin dependent kinase 1 (Cdk1) triggers polarization of the actin cytoskeleton and bud emergence in late G1 through activation of the Cdc42 GTPase. However, Cdk1 is not thought to be required for subsequent growth of the bud. Here, we show that Cdk1 has an unexpected role in controlling bud growth after bud emergence. Moreover, we show that G1 cyclin-Cdk1 complexes specifically phosphorylate multiple proteins associated with Cdc24, the guanine nucleotide-exchange factor (GEF) that activates the Cdc42 GTPase. A mutant form of a Cdc24-associated protein that fails to undergo Cdk1-dependent phosphorylation causes defects in bud growth. These results provide a direct link between Cdk1 activity and the control of polarized cell growth.     

Constitutive overexpression of the contact site A glycoprotein enables growth-phase cells of Dictyostelium discoideum to aggregate.

The contact site A (csA) glycoprotein is a developmentally regulated cell adhesion molecule which mediates EDTA-stable cell contacts during the aggregation stage of Dictyostelium discoideum. A transformation vector was constructed which allows overexpression of the csA protein during the growth phase. In that stage the csA protein is normally not expressed; in the transformants it was transported to the cell surface and carried all modifications investigated, including a phospholipid anchor and two types of oligosaccharide chain. csA expression enabled the normal non-aggregative growth-phase cells to form EDTA-stable contacts in suspension and to assemble into three-dimensional aggregates when moving on a substratum. After prolonged cultivation of csA overexpressing transformants in nutrient medium the developmental program was found to be turned on, as it normally occurs only in starving cells. During later development of transformed cells, the csA glycoprotein remained present on the cell surface, while it is down-regulated in the wild type. It was detected in both the prestalk and prespore regions of the multicellular slugs made from transformed cells.     

Differential regulation of Cdc2 and Cdk2 by RINGO and cyclins.

Cyclin-dependent kinases (Cdks) are key regulators of the eukaryotic cell division cycle. Cdk1 (Cdc2) and Cdk2 should be bound to regulatory subunits named cyclins as well as phosphorylated on a conserved Thr located in the T-loop for full enzymatic activity. Cdc2- and Cdk2-cyclin complexes can be inactivated by phosphorylation on the catalytic cleft-located Thr-14 and Tyr-15 residues or by association with inhibitory subunits such as p21(Cip1). We have recently identified a novel Cdc2 regulator named RINGO that plays an important role in the meiotic cell cycle of Xenopus oocytes. RINGO can bind and activate Cdc2 but has no sequence homology to cyclins. Here we report that, in contrast with Cdc2- cyclin complexes, the phosphorylation of Thr-161 is not required for full activation of Cdc2 by RINGO. We also show that RINGO can directly stimulate the kinase activity of Cdk2 independently of Thr-160 phosphorylation. Moreover, RINGO-bound Cdc2 and Cdk2 are both less susceptible to inhibition by p21(Cip1), whereas the Thr-14/Tyr-15 kinase Myt1 can negatively regulate the activity of Cdc2-RINGO with reduced efficiency. Our results indicate that Cdk-RINGO complexes may be active under conditions in which cyclin-bound Cdks are inhibited and can therefore play different regulatory roles.     

Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism

Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and thereby promote cell cycle progression. In vertebrates, Chk1 and Chk2 phosphorylate Cdc25A at multiple N-terminal sites and target it for rapid degradation in response to genotoxic stress. Here we show that Chk1, but not Chk2, phosphorylates Xenopus Cdc25A at a novel C-terminal site (Thr504) and inhibits it from C-terminally interacting with various Cdk-cyclin complexes, including Cdk1-cyclin A, Cdk1-cyclin B, and Cdk2-cyclin E. Strikingly, this inhibition, rather than degradation itself, of Cdc25A is essential for the Chk1-induced cell cycle arrest and the DNA replication checkpoint in early embryos. 14-3-3 proteins bind to Chk1-phosphorylated Thr504, but this binding is not required for the inhibitory effect of Thr504 phosphorylation. A C-terminal site presumably equivalent to Thr504 exists in all known Cdc25 family members from yeast to humans, and its phosphorylation by Chk1 (but not Chk2) can also inhibit all examined Cdc25 family members from C-terminally interacting with their Cdk-cyclin substrates. Thus, Chk1 but not Chk2 seems to inhibit virtually all Cdc25 phosphatases by a novel common mechanism.     

Meiotic nuclear divisions in budding yeast require PP2A(Cdc55)-mediated antagonism of Net1 phosphorylation by Cdk

During meiosis, one round of deoxyribonucleic acid replication is followed by two rounds of nuclear division. In Saccharomyces cerevisiae, activation of the Cdc14 early anaphase release (FEAR) network is required for exit from meiosis I but does not lead to the activation of origins of replication. The precise mechanism of how FEAR regulates meiosis is not understood. In this paper, we report that premature activation of FEAR during meiosis caused by loss of protein phosphatase PP2A(Cdc55) activity blocks bipolar spindle assembly and nuclear divisions. In cdc55 meiotic null (cdc55-mn) cells, the cyclin-dependent kinase (Cdk)-counteracting phosphatase Cdc14 was released prematurely from the nucleolus concomitant with hyperphosphorylation of its nucleolar anchor protein Net1. Crucially, a mutant form of Net1 that lacks six Cdk phosphorylation sites rescued the meiotic defect of cdc55-mn cells. Expression of a dominant mutant allele of CDC14 mimicked the cdc55-mn phenotype. We propose that phosphoregulation of Net1 by PP2A(Cdc55) is essential for preventing precocious exit from meiosis I.     

Mammalian target of rapamycin complex 1 signaling opposes the effects of anchorage loss, leading to activation of Cdk4 and Cdc6 stabilization

When deprived of an anchorage to the extracellular matrix, fibroblasts arrest in the G₁ phase with inactivation of Cdk4/6 and Cdk2 and destruction of Cdc6, the assembler of prereplicative complexes essential for S phase onset. How cellular anchorages control these kinases and Cdc6 stability is poorly understood. Here, we report that in rat embryonic fibroblasts, activation of mammalian target of rapamycin complex 1 by a Tsc2 mutation or overexpression of a constitutively active mutant Rheb overrides the absence of the anchorage and stabilizes Cdc6 at least partly via activating Cdk4/6 that induces Emi1, an APC/C^Cdh1 ubiquitin ligase inhibitor.

Structured summary

MINT-7890626: cdc27 (uniprotkb:Q4V8A2) physically interacts (MI:0915) with Cyclin-A (uniprotkb:Q6AY13) by anti bait coimmunoprecipitation (MI:0006)     

Two ubiquitin-conjugating enzymes,UbcP1/Ubc4 and UbcP4/Ubc11, have distinct functions for ubiquitination of mitotic cyclin

Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4+ cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11+ cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.     

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.     

Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr¹⁶¹ by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr¹⁴ and Tyr¹⁵ phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G₂/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr¹⁵. In addition, Tyr¹⁵-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G₂ and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr¹⁶¹ phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G₂/M is tightly coupled and regulated by Cdc25 phosphatases.     

Mechanisms governing maintenance of Cdk1/cyclin B1 kinase activity in cells infected with human cytomegalovirus

Previous work has demonstrated dysregulation of key cell cycle components in human cytomegalovirus (HCMV)-infected human fibroblasts, resulting in cell cycle arrest (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. L. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). The activation of the mitotic kinase Cdk1/cyclin B, which was detected as early as 8 h postinfection (p.i.) and maintained throughout the time course, was particularly interesting. To understand the mechanisms underlying the induction of this kinase activity, we have examined the pathways that regulate the activation of Cdk1/cyclin B1 complexes. The accumulation of the cyclin B1 subunit in HCMV-infected cells is the result of increased synthesis and reduced degradation of the protein. In addition, the catalytic subunit, Cdk1, accumulates in its active form in virus-infected cells. The decreased level of the Tyr15-phosphorylated form of Cdk1 in virus-infected fibroblasts is due in part to the down-regulation of the expression and activity of the Cdk1 inhibitory kinases Myt1 and Wee1. Increased degradation of Wee1 via the proteasome also accounts for its absence at 24 h p.i. At late times, we observed accumulation of the Cdc25 phosphatases that remove the inhibitory phosphates from Cdk1. Interestingly, biochemical fractionation studies revealed that the active form of Cdk1, a fraction of total cyclin B1, and the Cdc25 phosphatases reside predominantly in the cytoplasm of infected cells. Collectively, these data suggest that the maintenance of Cdk1/cyclin B1 activity observed in HCMV-infected cells can be explained by three mechanisms: the accumulation of cyclin B1, the inactivation of negative regulatory pathways for Cdk1, and the accumulation of positive factors that promote Cdk1 activity.