Cks overexpression enhances chemotherapeutic efficacy by overriding DNA damage checkpoints.

Cdc kinase subunit (Cks) proteins Cks1 and Cks2 are adaptor-like proteins that bind many cyclin-dependent kinases. A wealth of clinical data has shown that Cks proteins are overexpressed in many types of human cancers and this often correlates with increased tumor aggressiveness. Previously, we showed that Cks overexpression abrogates the intra-S-phase checkpoint, a major barrier to oncogene-mediated transformation. Interestingly, the intra-S-phase checkpoint is crucial for the cellular response to replication stress, a major pathway of apoptosis induction by many chemotherapeutic agents. Here, we demonstrate cancer cells that overexpress Cks1 or Cks2 override the intra-S-phase checkpoint in the presence of replication stress-inducing chemotherapies such as 5-Fluorouracil (5-FU) and methotrexate (MTX) leading to enhanced sensitivity in vitro and in vivo. Furthermore, enforced expression of Cks1 in an MTX-resistant breast cancer cell line was found to restore drug sensitivity. Our results suggest that Cks proteins are important determinants of apoptosis induction of replication stress-inducing chemotherapies such as 5-FU.

Deregulated cyclin E promotes p53 loss of heterozygosity and tumorigenesis in the mouse mammary gland.

Deregulation of cyclin E expression and/or high levels have been reported in a variety of tumors and have been used as indicators of poor prognosis. Although the role that cyclin E plays in tumorigenesis remains unclear, there is evidence that it confers genomic instability when deregulated in cultured cells. Here we show that deregulated expression of a hyperstable allele of cyclin E in mice heterozygous for p53 synergistically increases mammary tumorigenesis more than that in mice carrying either of these markers individually. Most tumors and tumor-derived cell lines demonstrated loss of p53 heterozygosity. Furthermore, this tumor susceptibility is related to the number of times the transgene is induced indicating that it is directly attributable to the expression of the cyclin E transgene. An indirect assay indicates that loss of p53 function is an early event occurring in the mammary epithelia of midlactation mammary glands in which cyclin E is deregulated long before evidence of malignancy. These data support the hypothesis that deregulated expression of cyclin E stimulates p53 loss of heterozygosity by promoting genomic instability and provides specific evidence for this in vivo. Cyclin E deregulation and p53 loss are characteristics often observed in human breast carcinoma.

UBA domains mediate protein-protein interactions between two DNA damage-inducible proteins.

The Saccharomyces cerevisiae genes RAD23 and DDI1 were identified in a screen for multicopy suppressors of the temperature-sensitivity of a mutant allele of S. cerevisiae PDS1. Pds1 is a regulator of anaphase that needs to accumulate and then be degraded by the ubiquitin-proteasome pathway at the metaphase-anaphase transition for cells to progress normally through mitosis. Both the Rad23 and Ddi1 pds1 suppression phenotypes depend on a shared motif known as a UBA domain found in a variety of proteins associated with ubiquitin metabolism. UBA domains were found to be essential for homodimerization of Rad23 and heterodimerization between Rad23 and Ddi1, but not for homodimerization of Ddi1. This observation, coupled with the findings that Rad23 and Ddi1 UBA domains bind ubiquitin and that dimerization of Rad23 blocks ubiquitin binding, suggests a possible mechanism for regulating Rad23 and Ddi1 function.

Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line.

Cyclin E, one of the activators of the cyclin-dependent kinase Cdk2, is expressed near the G1-S phase transition and is thought to be critical for the initiation of DNA replication and other S-phase functions. Accumulation of cyclin E at the G1-S boundary is achieved by periodic transcription coupled with regulated proteolysis linked to autophosphorylation of cyclin E. The proper timing and amplitude of cyclin E expression seem to be important, because elevated levels of cyclin E have been associated with a variety of malignancies and constitutive expression of cyclin E leads to genomic instability. Here we show that turnover of phosphorylated cyclin E depends on an SCF-type protein-ubiquitin ligase that contains the human homologue of yeast Cdc4, which is an F-box protein containing repeated sequences of WD40 (a unit containing about 40 residues with tryptophan (W) and aspartic acid (D) at defined positions). The gene encoding hCdc4 was found to be mutated in a cell line derived from breast cancer that expressed extremely high levels of cyclin E.

Mec1p regulates Pds1p levels in S phase: complex coordination of DNA replication and mitosis.

Genetic evidence suggests that the securin Pds1p is the target of a late-S-phase checkpoint control. Here we show that Pds1p becomes essential once two-thirds of the genome has been replicated and that the coupling of the completion of genome replication with mitosis relies on the regulation of Pds1p levels. Mec1p is needed to maintain Pds1p levels under S-phase checkpoint conditions. In contrast, Rad53p and Chk1p, needed for the stabilization of Pds1p in the context of the G2 DNA-damage checkpoint pathway, are dispensable. Thus, the Pds1p-dependent late-S-phase checkpoint pathway couples replication with mitosis but is mechanistically distinct from the G2 DNA-damage checkpoint. Finally, we show that the inhibition of spindle elongation in early S phase, controlled by the Mec1p/Rad53p branch, is not regulated via Pds1p/Esp1p. This can mechanistically explain the need for branched S-phase checkpoint controls.

A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27Kip1.

The Cks/Suc1 proteins associate with CDK/cyclin complexes, but their precise function(s) is not well defined. Here we demonstrate that Cks1 directs the ubiquitin-mediated proteolysis of the CDK-bound substrate p27Kip1 by the protein ubiquitin ligase (E3) SCF(Skp2). Cks1 associates with the F box protein Skp2 and is essential for recognition of the p27Kip1 substrate for ubiquitination in vivo and in vitro. Using purified recombinant proteins, we reconstituted p27Kip1 ubiquitination activity and show that it is dependent on Cks1. CKS1-/- mice are abnormally small, and cells derived from them proliferate poorly, particularly under limiting mitogen conditions, possibly due to elevated levels of p27Kip1.

Accumulation of cyclin E is not a prerequisite for passage through the restriction point.

The restriction point (R) is defined as the point in G(1) after which cells can complete a division cycle without growth factors and divides G(1) into two physiologically different intervals in cycling cells, G(1)-pm (a postmitotic interval with a constant length of 3 to 4 h) and G(1)-ps (a pre-DNA-synthetic interval with a variable length of 1 to 10 h). Cyclin E is a G(1) regulatory protein whose accumulation has been suggested to be critical for passage through R. We have studied cyclin E protein levels in individual cells of asynchronously growing cell populations, with respect to both passage through R and entry into S phase. We found that the postmitotic G(1) cells that had not yet reached R were negative for cyclin E accumulation. On the other hand, cells that had passed R were found to accumulate cyclin E at variable times (1 to 8 h) after passage through R and 2 to 5 h before entry into S. These kinetic data rule out the hypothesis that passage through R is dependent on the accumulation of cyclin E but suggest, instead, the converse, that passage through R is a prerequisite for cyclin E accumulation. Furthermore, we found that most of the cyclin E protein is downregulated within 1 to 2 h after entry into S.

UBA domains of DNA damage-inducible proteins interact with ubiquitin.

Rad23 is a highly conserved protein involved in nucleotide excision repair (NER) that associates with the proteasome via its N-terminus. Its C-terminal ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to humans. However, the cellular function of UBA domains is not completely understood. Recently, RAD23 and DDI1, both DNA damage-inducible genes encoding proteins with UBA domains, were implicated genetically in Pds1-dependent mitotic control in yeast. The UBA domains of RAD23 and DDI1 are required for these interactions. Timely degradation of Pds1 via the ubiquitin/proteasome pathway allows anaphase onset and is crucial for chromosome maintenance. Here, we show that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction is dependent on their UBA domains, providing a possible mechanism for UBA-dependent cell cycle control. Moreover, we show that a hydrophobic surface on the UBA domain, which from structural work had been predicted to be a protein-protein interaction interface, is indeed required for ubiquitin binding. By demonstrating that UBA domains interact with ubiquitin, we have provided the first indication of a cellular function for the UBA domain.

Dosage suppressors of pds1 implicate ubiquitin-associated domains in checkpoint control.

In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1 mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated. To identify loci that genetically interact with PDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37 degrees C. Genetic and functional interactions of two suppressors are described. RAD23 and DDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128. rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128. UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them. Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling. Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.

A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1.

In Saccharomyces cerevisiae, the metaphase-anaphase transition is initiated by the anaphase-promoting complex-dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation. Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle. Here, we show that Esp1 has a novel role in promoting anaphase spindle elongation. The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B. The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase. Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1. Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding. In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells. Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

Multi-step control of spindle pole body duplication by cyclin-dependent kinase.

Organelles called centrosomes in metazoans or spindle pole bodies (SPBs) in yeast direct the assembly of a bipolar spindle that is essential for faithful segregation of chromosomes during mitosis. Abnormal accumulation of multiple centrosomes leads to genome instability, and has been observed in both tumour cells and cells with targeted mutations in tumour-suppressor genes. The defects that lead to centrosome amplification are not understood. We have recapitulated the multiple-centrosome phenotype in budding yeast by disrupting the activity of specific cyclin-dependent kinase (CDK) complexes. Our observations are reminiscent of mechanisms that govern DNA replication, and show that specific cyclin/CDK activities function both to promote SPB duplication and to prevent SPB reduplication.

Bud6 directs sequential microtubule interactions with the bud tip and bud neck during spindle morphogenesis in Saccharomyces cerevisiae.

In budding yeast, spindle polarity relies on a precise temporal program of cytoplasmic microtubule-cortex interactions throughout spindle assembly. Loss of Clb5-dependent kinase activity under conditions of attenuated Cdc28 function disrupts this program, resulting in diploid-specific lethality. Here we show that polarity loss is tolerated by haploids due to a more prominent contribution of microtubule-neck interactions to spindle orientation inherent to haploids. These differences are mediated by the relative partition of Bud6 between the bud tip and bud neck, distinguishing haploids from diploids. Bud6 localizes initially to the bud tip and accumulates at the neck concomitant with spindle assembly. bud6Delta mutant phenotypes are consistent with Bud6's role as a cortical cue for cytoplasmic microtubule capture. Moreover, mutations that affect Bud6 localization and partitioning disrupt the sequential program of microtubule-cortex interactions accordingly. These data support a model whereby Bud6 sequentially cues microtubule capture events at the bud tip followed by capture events at the bud neck, necessary for correct spindle morphogenesis and polarity.

Regulation of G(1) cyclin-dependent kinases in the mammalian cell cycle.

Cyclin-dependent kinases are the key regulators of cell-cycle transitions. In mammalian cells, Cdk2, Cdk4, Cdk6 and associated cyclins control the G(1) to S phase transition. Because proper regulation of this transition is critical for an organism's survival, these protein kinases are exquisitely regulated at different mechanistic levels and in response to a large variety of intrinsic and extrinsic signals.

Crystal structure and mutational analysis of the Saccharomyces cerevisiae cell cycle regulatory protein Cks1: implications for domain swapping, anion binding and protein interactions.

BACKGROUND: The Saccharomyces cerevisiae protein Cks1 (cyclin-dependent kinase subunit 1) is essential for cell-cycle progression. The biological function of Cks1 can be modulated by a switch between two distinct molecular assemblies: the single domain fold, which results from the closing of a beta-hinge motif, and the intersubunit beta-strand interchanged dimer, which arises from the opening of the beta-hinge motif. The crystal structure of a cyclin-dependent kinase (Cdk) in complex with the human Cks homolog CksHs1 single-domain fold revealed the importance of conserved hydrophobic residues and charged residues within the beta-hinge motif. RESULTS: The 3.0 A resolution Cks1 structure reveals the strict structural conservation of the Cks alpha/beta-core fold and the beta-hinge motif. The beta hinge identified in the Cks1 structure includes a novel pivot and exposes a cluster of conserved tyrosine residues that are involved in Cdk binding but are sequestered in the beta-interchanged Cks homolog suc1 dimer structure. This Cks1 structure confirms the conservation of the Cks anion-binding site, which interacts with sidechain residues from the C-terminal alpha helix of another subunit in the crystal. CONCLUSIONS: The Cks1 structure exemplifies the conservation of the beta-interchanged dimer and the anion-binding site in evolutionarily distant yeast and human Cks homologs. Mutational analyses including in vivo rescue of CKS1 disruption support the dual functional roles of the beta-hinge residue Glu94, which participates in Cdk binding, and of the anion-binding pocket that is located 22 A away and on an opposite face to Glu94. The Cks1 structure suggests a biological role for the beta-interchanged dimer and the anion-binding site in targeting Cdks to specific phosphoproteins during cell-cycle progression.

Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCF(Met30)-mediated inactivation of the transcription factor Met4.

Polyubiquitination of proteins by Cdc34/SCF complexes targets them for degradation by the 26S proteasome. The essential F-box protein Met30 is the substrate recognition subunit of the ubiquitin ligase SCF(Met30). The critical target of SCF(Met30) is the transcription factor Met4, as deletion of MET4 suppresses the lethality of met30 mutants. Surprisingly, Met4 is a relatively stable protein and its abundance is not influenced by Met30. However, transcriptional repression of Met4 target genes correlates with Cdc34/SCF(Met30)-dependent ubiquitination of Met4. Functionally, ubiquitinated Met4 associates with target promoters but fails to form functional transcription complexes. Our data reveal a novel proteolysis-independent function for Cdc34/SCF and indicate that ubiquitination of transcription factors can be utilized to directly regulate their activities.

Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast.

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Delta cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

Evidence that a free-running oscillator drives G1 events in the budding yeast cell cycle.

In yeast and somatic cells, mechanisms ensure cell-cycle events are initiated only when preceding events have been completed. In contrast, interruption of specific cell-cycle processes in early embryonic cells of many organisms does not affect the timing of subsequent events, indicating that cell-cycle events are triggered by a free-running cell-cycle oscillator. Here we present evidence for an independent cell-cycle oscillator in the budding yeast Saccharomyces cerevisiae. We observed periodic activation of events normally restricted to the G1 phase of the cell cycle, in cells lacking mitotic cyclin-dependent kinase activities that are essential for cell-cycle progression. As in embryonic cells, G1 events cycled on schedule, in the absence of S phase or mitosis, with a period similar to the cell-cycle time of wild-type cells. Oscillations of similar periodicity were observed in cells responding to mating pheromone in the absence of G1 cyclin (Cln)- and mitotic cyclin (Clb)-associated kinase activity, indicating that the oscillator may function independently of cyclin-dependent kinase dynamics. We also show that Clb-associated kinase activity is essential for ensuring dependencies by preventing the initiation of new G1 events when cell-cycle progression is delayed.

Functional characterization of rpn3 uncovers a distinct 19S proteasomal subunit requirement for ubiquitin-dependent proteolysis of cell cycle regulatory proteins in budding yeast.

By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated. In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit. Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G(1)/S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis. rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates. Thus, RPN3 function is required for the degradation of the G(1)-phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1. Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed. Surprisingly, but consistent with the lack of a G(1) arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype. In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit. While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators. The potential implications of these data are discussed in the context of possible Rpn3 function in multiubiquitin-protein conjugate recognition by the 19S proteasomal regulatory particle.

Deregulated cyclin E induces chromosome instability.

Cyclin E, a regulatory subunit of cyclin-dependent kinase 2 (Cdk2), is an important regulator of entry into S phase in the mammalian cell cycle. In normal dividing cells, cyclin E accumulates at the G1/S-phase boundary and is degraded as cells progress through S phase. However, in many human tumours cyclin E is overexpressed and the levels of protein and kinase activity are often deregulated relative to the cell cycle. It is not understood how alterations in expression of cyclin E contribute to tumorigenesis. Here we show that constitutive cyclin-E overexpression in both immortalized rat embryo fibroblasts and human breast epithelial cells results in chromosome instability (CIN). In contrast, analogous expression of cyclin D1 or A does not increase the frequency of CIN. Cyclin-E-expressing cells that exhibit CIN have normal centrosome numbers. However, constitutive overexpression of cyclin E impairs S-phase progression, indicating that aberrant regulation of this process may be responsible for the CIN observed. These results indicate that downregulation of cyclin-E/Cdk2 kinase activity following the G1/S-phase transition may be necessary for the maintenance of karyotypic stability.