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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 sensor specific for a cellular event, such as DNA replication or DNA damage; 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 12th-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 DNA concentration to the MBT level.
Our earlier 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. Recent studies in Xenopus egg extracts show the enhancement of checkpoint signaling releases protein phosphatase 1 (PP1) from the chromatin, thereby enhancing accumulation of phosphorylated, active ATM, Chk1 and Chk2, and γ-H2AX on the chromatin. Release of PP1 appears to be regulated by an associated targeting subunit, Repo-Man. In the MCF10 cell model of breast cancer progression, advanced stages exhibit increasing levels of Repo-Man and decreased checkpoint responsiveness to DNA damage. Adenoviral-mediated elevation of Repo-Man in early-stage cancer cells decreased their responsiveness to DNA damage. Our findings suggest the elevation of Repo-Man in many human cancer cells may contribute to decreased sensitivity to DNA damage and facilitate progressive genomic instability.
Aurora Protein Kinases
The Aurora family of protein kinases is critically involved in multiple steps of mitosis, spindle formation, and cytokinesis. Both Aurora A and Aurora B have distinct functions and cellular localizations, and both are attractive drug targets for cancer therapy. We previously purified the major Aurora A activator from mitotic extracts and identified it as the microtubule-binding protein TPX2 (targeting protein against Xenopus kinesin-like protein 2). TPX2 has been reported to be overexpressed in numerous human cancers and has been suggested as a possible target for inhibition of Aurora A activity. Recently we identified a novel, Aurora A–independent domain of TPX2 that binds directly to the mitotic kinesin motor protein Eg5, which is responsible for separation of spindle poles in mitosis. Modest overexpression of this domain caused cytokinesis arrest in embryonic blastomeres and collapsed spindle poles in somatic tissue culture cells with activation of the spindle assembly checkpoint. Cytokinesis arrest by this domain could be prevented or reversed by expression of Eg5. Inasmuch as Eg5 has already been proposed as a drug target for cancer therapy, these findings highlight the attractiveness of TPX2 as a target to affect mitotic control by both the Aurora A and Eg5 proteins.
Centrosomes
A mayor focus in the laboratory is the control of centrosome duplication. Centrosomes are the major microtubule-organizing center in animal cells, and a centrosome organizes each spindle pole during mitosis. Like DNA, centrosomes duplicate once and only once in each cell cycle, at the G1- to S-phase transition. Cancer cells frequently duplicate additional centrosomes, however, leading to multipolar mitotic spindles, aneuploidy, and chromosomal instability. Our previous studies identified cyclin E/Cdk2 as a key regulator of centrosome duplication and characterized a centrosomal localization sequence (CLS) in cyclin E. Recently we employed a two-hybrid genetic screen to identify proteins that interact with the CLS. One such protein is MCM5, best known for its key role in DNA replication. Our studies demonstrate that MCM5 localization on the centrosome is mediated by interaction with the CLS of cyclin E. The domain in MCM5 that interacts with the CLS is highly conserved through evolution and distinct from any region of MCM5 known to be involved in DNA replication. In S-phase–arrested Chinese hamster ovary cells, centrosome overduplication was significantly blocked by full-length MCM5, as well as by the short 37–amino acid domain that interacts with the CLS. Unlike its role in DNA replication, control of centrosome duplication by MCM5 does not require interaction with other MCM family members. These findings highlight an emerging trend in which proteins previously thought to be specific for DNA replication are also involved in centrosome duplication.
Last updated November 24, 2008
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