Targets of the cyclin-dependent kinase Cdk1

The events of cell reproduction are governed by oscillations in the activities of cyclin-dependent kinases (Cdks). Cdks control the cell cycle by catalysing the transfer of phosphate from ATP to specific protein substrates. Despite their importance in cell-cycle control, few Cdk substrates have been identified. Here, we screened a budding yeast proteomic library for proteins that are directly phosphorylated by Cdk1 in whole-cell extracts. We identified about 200 Cdk1 substrates, several of which are phosphorylated in vivo in a Cdk1-dependent manner. The identities of these substrates reveal that Cdk1 employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylation of the molecular machines that drive cell-cycle events. Detailed analysis of these substrates is likely to yield important insights into cell-cycle regulation.     

Can sequencing shed light on cell cycling?

Every organism must have cells that can replicate indefinitely. Can the draft human genome sequence tell us how the cell cycle works and how it evolved? We studied two protein families--the cyclins and their partners the cyclin-dependent kinases (Cdks)--and a conserved regulatory circuit, the spindle checkpoint. Disappointingly, we discovered a few novel cyclins and no new Cdks or components of the spindle checkpoint, and could shed little light on the organization of the cell cycle.     

Role for cyclin A in the dependence of mitosis on completion of DNA replication.

THE cyclins were first identified by their cell-cycle-dependent synthesis and destruction and have a key role in the control of mitosis in Xenopus embryonic cell cycles. All higher eukaryotes have at least two types of cyclins, the A- and B-type, which can be distinguished by sequence motifs and the timing of their destruction in the cell cycle. The degradation of both cyclins is required for exit from mitosis, but the activation and destruction of cyclin A occur earlier in the cell cycle than with the B-type cyclins. This suggests that cyclin A has a distinct role in cell-cycle progression. We have used an antisense oligodeoxy-nucleotide directed against cyclin A to investigate this role. Ablation of cyclin A messenger RNA in cytostatic factor/metaphase-arrested extracts of Xenopus eggs, followed by in vitro progression into interphase, resulted in the premature appearance of cyclin B-cdc2-associated H1 kinase activity and premature entry into mitosis. Although cyclin A-ablated extracts could initiate DNA synthesis during interphase, S phase was not completed before entry into mitosis. The effects of cyclin A ablation were reversed by the addition of cyclin A mRNA or cyclin A protein to the extracts.     

Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates

Cell-cycle events are controlled by cyclin-dependent kinases (CDKs), whose periodic activation is driven by cyclins. Different cyclins promote distinct cell-cycle events, but the molecular basis for these differences remains unclear. Here we compare the specificity of two budding yeast cyclins, the S-phase cyclin Clb5 and the M-phase cyclin Clb2, in the phosphorylation of 150 Cdk1 (Cdc28) substrates. About 24% of these proteins were phosphorylated more efficiently by Clb5-Cdk1 than Clb2-Cdk1. The Clb5-specific targets include several proteins (Sld2, Cdc6, Orc6, Mcm3 and Cdh1) involved in early S-phase events. Clb5 specificity depended on an interaction between a hydrophobic patch in Clb5 and a short sequence in the substrate (the RXL or Cy motif). Phosphorylation of Clb5-specific targets during S phase was reduced by replacing Clb5 with Clb2 or by mutating the substrate RXL motif, confirming the importance of Clb5 specificity in vivo. Although we did not identify any highly Clb2-specific substrates, we found that Clb2-Cdk1 possessed higher intrinsic kinase activity than Clb5-Cdk1, enabling efficient phosphorylation of a broad range of mitotic Cdk1 targets. Thus, Clb5 and Clb2 use distinct mechanisms to enhance the phosphorylation of S-phase and M-phase substrates.     

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.     

Calcineurin is required to release Xenopus egg extracts from meiotic M phase

Fertilization induces a transient increase in cytoplasmic Ca2+ concentration in animal eggs that releases them from cell cycle arrest in the second meiotic metaphase. In frog eggs, Ca2+ activates Ca2+/calmodulin-activated kinase, which inactivates cytostatic factor, allowing the anaphase-promoting factor to turn on and ubiquitinate cyclins and securin, which returns the cell cycle to interphase. Here we show that the calcium-activated protein phosphatase calcineurin is also important in this process. Calcineurin is transiently activated after adding Ca2+ to egg extracts, and inhibitors of calcineurin such as cyclosporin A (ref. 8) delay the destruction of cyclins, the global dephosphorylation of M-phase-specific phosphoproteins and the re-formation of a fully functional nuclear envelope. We found that a second wave of phosphatase activity directed at mitotic phosphoproteins appears after the spike of calcineurin activity. This activity disappeared the next time the extract entered M phase and reappeared at the end of mitosis. We surmise that inhibition of this second phosphatase activity is important in allowing cells to enter mitosis, and, conversely, that its activation is required for a timely return to interphase. Calcineurin is required to break the deep cell cycle arrest imposed by the Mos-MAP (mitogen-activated protein) kinase pathway, and we show that Fizzy/Cdc20, a key regulator of the anaphase-promoting factor, is an excellent substrate for this phosphatase.     

A novel cyclin encoded by a bcl1-linked candidate oncogene

We have previously identified a candidate oncogene (PRAD1 or D11S287E) on chromosome 11q13 which is clonally rearranged with the parathyroid hormone locus in a subset of benign parathyroid tumours. We now report that a cloned human placental PRAD1 complementary DNA encodes a protein of 295 amino acids with sequence similarities to the cyclins. Cyclins can form a complex with and activate p34cdc2 protein kinase, thereby regulating progress through the cell cycle. PRAD 1 messenger RNA levels vary dramatically across the cell cycle in HeLa cells. Addition of the PRAD1 protein to interphase clam embryo lysates containing inactive p34cdc2 kinase and lacking endogenous cyclins allows it to be isolated using beads bearing p13suc1, a yeast protein that binds cdc2 and related kinases with high affinity and coprecipitates kinase-associated proteins. Addition of PRAD1 also induces phosphorylation of histone H1, a preferred substrate of cdc2. These data suggest that PRAD1 encodes a novel cyclin whose overexpression may play an important part in the development of various tumours with abnormalities in 11q13.     

Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.     

APC(Cdc20) promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5

Ubiquitin-mediated proteolysis due to the anaphase-promoting complex/cyclosome (APC/C) is essential for separation of sister chromatids, requiring degradation of the anaphase inhibitor Pds1, and for exit from mitosis, requiring inactivation of cyclin B Cdk1 kinases. Exit from mitosis in yeast involves accumulation of the cyclin kinase inhibitor Sic1 as well as cyclin proteolysis mediated by APC/C bound by the activating subunit Cdh1/Hct1 (APC(Cdh1)). Both processes require the Cdc14 phosphatase, whose release from the nucleolus during anaphase causes dephosphorylation and thereby activation of Cdh1 and accumulation of another protein, Sic1 (refs 4-7). We do not know what determines the release of Cdc14 and enables it to promote Cdk1 inactivation, but it is known to be dependent on APC/C bound by Cdc20 (APC(Cdc20)) (ref. 4). Here we show that APC(Cdc20) allows activation of Cdc14 and promotes exit from mitosis by mediating proteolysis of Pds1 and the S phase cyclin Clb5 in the yeast Saccharomyces cerevisiae. Degradation of Pds1 is necessary for release of Cdc14 from the nucleolus, whereas degradation of Clb5 is crucial if Cdc14 is to overwhelm Cdk1 and activate its foes (Cdh1 and Sic1). Remarkably, cells lacking both Pds1 and Clb5 can proliferate in the complete absence of Cdc20.     

An integrative model and analysis of cell cycle in fission yeast

Cell cycle is a programmed process, during which a cell proliferates to two daughter cells. The eukaryotic or-ganisms share the same characters, such as four cycle phases G1, S, G2 and M, the evolutionally conserved cell cycle proteins and its dependent kinases, and the check-points mechanism[1,2]. Due to the different functions and the complicated interactions of these proteins involved in cell cycle, it is very difficult to understand the regulatory mechanism of cell cycle in a whole sense …     

Release of mature starfish oocytes from interphase arrest by microinjection of human centrosomes

Mature oocytes (unfertilized eggs) are arrested at definite cell-cycle stages which vary from species to species. In frogs and mammals, the oocytes are arrested at the second metaphase of meiosis whereas in echinoderms they are blocked later, at the pronucleus stage. What causes the maturing oocytes to stop at some point in the cell cycle is not entirely clear. In frogs, the metaphase arrest seems to be maintained by a cytostatic factor. In echinoderms, which stop at interphase, no such a factor has so far been found. The fertilization process, beyond the introduction of paternal chromosomes, releases the oocyte from cell-cycle arrest and provides a functional centrosome to replace the endogenous centrosome which is apparently lost during oogenesis in most species. Several lines of evidence suggest that release from cell-cycle arrest is mediated by a Ca2+ burst which is associated with fertilization, and it is known that the functional centrosome provided by the sperm is necessary for mitotic spindle formation and cleavages. We report here that microinjection of purified human centrosomes into mature starfish oocytes is sufficient to release them from arrest at interphase and to support many cleavages leading to the occasional formation of normal embryos. In this species centrosome induced re-entry into the cell cycle does not require a transient calcium burst nor does it require intact microtubules.     

The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis

When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the IR-induced S-phase checkpoint cause 'radioresistant DNA synthesis', a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene. The Cdc25A phosphatase activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. Here we report a functional link between ATM, the checkpoint signalling kinase Chk2/Cds1 (Chk2) and Cdc25A, and implicate this mechanism in controlling the S-phase checkpoint. We show that IR-induced destruction of Cdc25A requires both ATM and the Chk2-mediated phosphorylation of Cdc25A on serine 123. An IR-induced loss of Cdc25A protein prevents dephosphorylation of Cdk2 and leads to a transient blockade of DNA replication. We also show that tumour-associated Chk2 alleles cannot bind or phosphorylate Cdc25A, and that cells expressing these Chk2 alleles, elevated Cdc25A or a Cdk2 mutant unable to undergo inhibitory phosphorylation (Cdk2AF) fail to inhibit DNA synthesis when irradiated. These results support Chk2 as a candidate tumour suppressor, and identify the ATM-Chk2-Cdc25A-Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis.     

Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

Somatic cell nuclear transfer has been succeeded in procedures of nuclear transfer. One is single nucleartransfer, the other is serial nuclear transfer. Viable animals have been cloned in different species using both me-thods[1—6]. Different nuclear recipients and donors wereused in serial nuclear transfer, namely, transferring thenuclear of reconstructed embryo into enucleated MⅡoocytes[7], transferring the nuclear of reconstructed em-bryos at one cell stage into enucleated zygote[4] and t…     

APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit

Cyclin degradation is central to regulation of the cell cycle. Mitotic exit was proposed to require degradation of the S phase cyclin Clb5 by the anaphase-promoting complex activated by Cdc20 (APC(Cdc20)). Furthermore, Clb5 degradation was thought to be necessary for effective dephosphorylation and activation of the APC regulatory subunit Cdh1 (also known as Hct1) and the cyclin-dependent kinase inhibitor Sic1 by the phosphatase Cdc14, allowing mitotic kinase inactivation and mitotic exit. Here we show, however, that spindle disassembly and cell division occur without significant APC(Cdc20)-mediated Clb5 degradation, as well as in the absence of both Cdh1 and Sic1. We find instead that destruction-box-dependent degradation of the mitotic cyclin Clb2 is essential for mitotic exit. APC(Cdc20) may be required for an essential early phase of Clb2 degradation, and this phase may be sufficient for most aspects of mitotic exit. Cdh1 and Sic1 may be required for further inactivation of Clb2-Cdk1, regulating cell size and the length of G1.     

Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells

Sensory hair cells of the mammalian organ of Corti in the inner ear do not regenerate when lost as a consequence of injury, disease, or age-related deafness. This contrasts with other vertebrates such as birds, where the death of hair cells causes surrounding supporting cells to re-enter the cell cycle and give rise to both new hair cells and supporting cells. It is not clear whether the lack of mammalian hair cell regeneration is due to an intrinsic inability of supporting cells to divide and differentiate or to an absence or blockade of regenerative signals. Here we show that post-mitotic supporting cells purified from the postnatal mouse cochlea retain the ability to divide and trans-differentiate into new hair cells in culture. Furthermore, we show that age-dependent changes in supporting cell proliferative capacity are due in part to changes in the ability to downregulate the cyclin-dependent kinase inhibitor p27(Kip1) (also known as Cdkn1b). These results indicate that postnatal mammalian supporting cells are potential targets for therapeutic manipulation.