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Cell Cycle Control
Summary: James Maller studies the molecular regulation of checkpoints in the cell cycle that control entry into or exit from mitosis. Of particular interest is how checkpoints control the activity of protein kinases and protein phosphatases that underlie cell cycle progression and the etiology of cancer.
Two events mark the reproductive life of a cell: replication of genomic DNA in S phase and distribution of that replicated DNA to daughter cells at mitosis, or M phase. Both events occur only after major commitments by the cell that occur at discrete points in time known as checkpoints. Checkpoints essentially are proofreading functions that integrate extracellular and intracellular signals to ensure that late cell cycle events are not initiated before early events have been completed. A checkpoint has at least three components: a signal specific for a cellular event, such as DNA replication; a signal transduction pathway; and a target that receives the signal and is linked to key enzymes required for cell cycle progression. Checkpoints most evident in the cell cycle occur in G1, G2, and M phases, governing entry into S phase, entry into M phase, and exit from M phase, respectively. Many checkpoint signals are DNA based, monitoring the completion of chromosome replication, the presence of DNA damage, or the alignment of chromosomes on the metaphase spindle. The end points of checkpoint control pathways often regulate the activity of a particular type of cyclin-dependent protein kinase (Cdk). Defects in checkpoint control are implicated in the etiology of human cancer.
DNA Damage Checkpoint Signaling
An ongoing interest in our lab concerns how cell cycle checkpoints are set up in early embryos and in stem cells. As a general rule, early cell divisions after fertilization are rapid and synchronous, and cell cycle progression is not blocked even under conditions in which DNA is not replicated, DNA is damaged, or mitotic spindles are disrupted. In Xenopus embryos the DNA damage and DNA replication checkpoints appear abruptly just after the 12-cleavage division, a period known as the midblastula transition (MBT). Activation of these checkpoints can be monitored both morphologically and biochemically after addition of DNA synthesis inhibitors or DNA with double-stranded breaks. A critical requirement for appearance of checkpoint arrest appears to be the DNA-to-cytoplasmic ratio achieved after the 12 divisions, based on studies in which checkpoints can be activated by damaged or unreplicated DNA in much earlier stage embryos or in egg extracts merely by increasing the undamaged DNA concentration to the MBT level.
Our recent studies revealed that undamaged "threshold" DNA participates in and enhances DNA damage signaling. Hallmark events associated with the DNA damage checkpoint include activation of the ATM kinase, phosphorylation and activation of checkpoint kinases Chk1 and Chk2, and phosphorylation of the histone H2A variant, H2AX. All these events are generated initially on damaged DNA but become concentrated on undamaged threshold DNA physically separated from the damaged DNA. This concentration and enhancement of signaling is necessary for engagement of the cell cycle arrest machinery by checkpoint signaling. Remarkably, these signals sent by damaged DNA, including activated ATM, persist in the cytoplasm even after damaged DNA is physically removed before addition of threshold DNA, indicating the mere absence of damage in a cell is not sufficient to end damage checkpoint signaling. These results illustrate how the increase in the DNA-to-cytoplasmic ratio achieved by the maternally programmed cleavage divisions creates a permissive condition for the abrupt activation of the DNA damage checkpoint at the MBT in development.
Cytostatic Factor and Metaphase Arrest
A continuing interest in our lab is the control of second meiotic metaphase arrest in the vertebrate egg. This is a stable physiological cell cycle arrest that is essential for fertilization and the genomic stability of the egg. The reduction in fertility observed in older women can be partially accounted for by failure to establish or maintain second meiotic metaphase arrest. The activity responsible for metaphase arrest is known as cytostatic factor (CSF). All available evidence points to CSF arrest as a multifaceted set of pathways that inhibit the anaphase-promoting complex (APC). The APC is a multisubunit E3 ubiquitin ligase that targets for degradation mitotic regulators that control sister chromatid separation and exit from mitosis. At least three CSF pathways that inhibit APC activity and prevent anaphase II in eggs have been identified. One involves the Mos/MAP kinase pathway; another involves the protein kinase cyclin E/Cdk2; and the third involves a protein known as early mitotic inhibitor-2. One interesting and unique feature of CSF arrest is that inhibition of the APC by all three pathways is relieved by the elevation of free calcium that occurs at fertilization.
We recently identified the Aurora B pathway as also contributing to CSF arrest. Aurora B is a key element in successful chromosome segregation and integrity of the spindle assembly checkpoint in the somatic cell cycle. Aurora B is typically found localized on kinetochores of metaphase chromosomes bound to two associated "passenger" proteins, INCENP and survivin. Our studies in Xenopus oocytes have shown that monomeric Aurora B is present in resting G2 cell phase, but it is catalytically inactive. Aurora B protein kinase activity is generated late in meiosis I in concert with the synthesis and accumulation of both survivin and INCENP.
Immunodepletion experiments show that nearly all newly synthesized INCENP associates with Aurora B. Aurora B activity reaches its maximum at metaphase of meiosis II during CSF arrest, and the activity rapidly decreases after release of arrest by elevated calcium. The decreased activity follows the proteolytic degradation of the INCENP subunit of the Aurora B complex. The importance of this decrease for release of CSF arrest is indicated by the ability of wild-type but not kinase-dead Aurora B to maintain APC inhibition and metaphase arrest even in the presence of calcium. This Aurora B–dependent arrest can be observed in egg extracts lacking DNA, indicating that APC inhibition by Aurora B is distinct from its classical role on kinetochores in the spindle assembly checkpoint in the cell cycle. These results define a fourth biochemical pathway that contributes to APC inhibition and CSF arrest at metaphase II that must be inactivated by calcium at fertilization for development to proceed.
Membrane Progesterone Receptors
A classical question in meiosis concerns the membrane action of progesterone that releases the G2 arrest of the oocyte and promotes meiotic maturation. We recently established a role in the initiation of maturation for a novel class of membrane progestin receptor, termed mPR. Peter Thomas and his colleagues (University of Texas) first described mPRβ in fish, and orthologs have since been cloned in higher vertebrates, including humans. The predicted structures of mPRs are seven-transmembrane G protein–coupled receptors. Our studies showed that mPRβ is expressed on the plasma membrane in Xenopus oocytes, as well as in transfected mammalian cells. Overexpression of mPRβ in oocytes enhances progesterone action; injection of oocytes with antibodies to mPRβ blocks progesterone action. Binding studies demonstrate specific membrane binding by mPRβ to progesterone but not to testosterone or estradiol. These results indicate the novel mPR class of receptors is important for initiation of oocyte maturation and partially resolve the molecular basis of progestin action at the oocyte plasma membrane.
Last updated: April 6, 2007
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