Progress 10/01/99 to 09/30/04
Outputs
The focus of the study is to understand mechanisms of the spindle checkpoint that inhibit the metaphase-to-anaphase transition until all chromosomes have attached properly to the spindle microtubules. We have investigated regulation & interaction of spindle checkpoint proteins Mad1, Mad2, Bub1, Bub3, and BubR1, using frog egg extracts & budding yeast as the main experimental systems. We have systematically isolated the frog spindle checkpoint components & generated many reagents for these proteins. Using these tools, we have studied the role of these proteins in the spindle checkpoint & the effect of specific mutations. 1) By systematic immunodepletion of individual checkpoint proteins, we have demonstrated that in the frog Xenopus there is mutual dependency among these proteins at kinetochores & that the proteins work in a complex network, rather than a simple pathway. We have provided the biochemical evidence to support the hypothesis that unattached kinetochores
recruit & activate checkpoint proteins that are then released to bind & inhibit Cdc20, an activator of the anaphase promoting complex (APC), in the cytosol. As the spindle checkpoint signal is generated from unattached kinetochores, characterization of the kinetochore-bound checkpoint proteins shall provide information on how the checkpoint is triggered. A simple protocol was established to purify mitotic chromosomes from egg extracts for biochemical analysis. Using this method, we demonstrated that Bub1 and BubR1 become hyperphosphorylated & activated specifically at unattached kinetochores in a Mad1- & MAPK-dependent manner. Upon microtubule attachment, proteins are dephosphorylated & inactivated. Further, hyperphosphorylation of Bub1 at unattached kinetochores facilitates the spindle checkpoint. Our work reveals a novel regulatory mechanism unique to kinetochores & establishes new biochemical functions for Mad1 & MAPK in the checkpoint. 2) Unattached kinetochores induce formation
of a checkpoint complex composed of BubR1, Bub3, & Mad2 that binds & inhibits Cdc20, thus preventing anaphase onset. We have discovered that Cdc20 is phosphorylated by both MAPK & Cdc2 during mitosis & phosphorylation of Cdc20 is required for its binding with & inhibition by the checkpoint complex. It was shown 10 years ago that MAPK is important for the spindle checkpoint, but its target in the checkpoint had not been found. Our study identifies the first spindle checkpoint substrate for MAPK & demonstrates for the first time the functional importance of Cdc20 phosphorylation. 3) We have demonstrated that the spindle checkpoint destabilizes Cdc20 in budding yeast cells, thus lowering the Cdc20 protein level. This process depends on the association of Cdc20 with Mad2. Degradation of Cdc20 might keep Cdc20 under a certain threshold level to ensure its complete inhibition by the checkpoint until the last kinetochore captures spindle microtubules. This study shows a dual control
mechanism in the spindle checkpoint: inhibition of Cdc20 by direct binding with the checkpoint proteins & Cdc20 degradation induced by the checkpoint proteins. The latter mechanism has never been found in any system before.
Impacts
Many cancer cells contain grossly abnormal chromosomes and continuously display chromosome instability. Aneuploidy (an abnormal number of chromosomes in cells) is also the major cause of miscarriage, with the incidence rate increasing with the maternal age. The abnormality may arise from defects in the spindle checkpoint. Thus, our studies on the biochemical mechanism of the spindle checkpoint not only advance our understanding of the fundamental process of the metaphase-to-anaphase transition, but may also provide biochemical markers for functional analysis of the spindle checkpoint in human disorders. Our efforts shall eventually help to design clinical reagents and be beneficial to humankind.
Publications
- Pan, J. and Chen, R.-H. 2004. Spindle checkpoint regulates Cdc20p stability in S. cerevisiae. Genes Dev. 18: 1439-1451.
- Chen, R.-H. 2004. Phosphorylation and activation of Bub1 on unattached chromosomes facilitate the spindle checkpoint. EMBO J. 23: 3113-3121.
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Progress 01/01/03 to 12/31/03
Outputs
We study the mechanisms of the spindle checkpoint that inhibits the metaphase-to-anaphase transition until all chromosomes have attached properly to the spindle microtubules. We have been investigating the regulation and interaction of the spindle checkpoint proteins Mad1, Mad2, Bub1, Bub3, and BubR1, in frog egg extracts and in budding yeast. In the past, we put much of our effort in the isolation of frog homologs of these spindle checkpoint proteins. We also characterized the order their localization to kinetochores and their roles in forming a checkpoint complex that binds and inhibits Cdc20, an activator of the anaphase-promoting complex (APC). In the past year, we focused on the post-translational modification of the checkpoint components, which is important for understanding the regulation of the checkpoint. It has been previously shown by two other laboratories that Cdc20 is a substrate for mitotic kinase Cdc2 and that phosphorylation is not required for Cdc20
to activate the APC. We hypothesized that phosphorylation of Cdc20 may instead regulate its response to the checkpoint signal. In addition, we found that Cdc20 contains several potential phosphorylation sites for both Cdc2 and MAP kinase (MAPK). The latter has been shown to be important for the spindle checkpoint for nine years, but its substrate has never been identified. In deed, we found that Cdc20 loses part, but not all, of its phosphorylation when MAPK is inhibited. By a combination of mutagenesis and two-dimensional tryptic phosphopeptide mapping, we located the phosphorylation sites to four evolutionarily conserved residues, one of them from MAPK and the other from Cdc2. We demonstrated that phosphorylation of all four sites is necessary for Cdc20 to be recognized and inhibited by the checkpoint proteins. By examining chromosomes isolated from egg extracts, we found that Bub1 and BubR1 are hyperphosphorylated specifically at unattached kinetochores, in a Mad1- and
MAPK-dependent manner. Hyperphosphorylation correlates with the activation of Bub1 and BubR1 at kinetochores. This is the first demonstration that checkpoint proteins are specifically activated at unattached kinetochroes where the checkpoint signal is generated. Interestingly, phosphorylation and the kinase activities of Bub1 are dispensible under optimal condition for checkpoint activation, whereas they become essential when there is weak kinetochore attachment defect. Our findings indicate that the kinase activity of Bub1 enhance the checkpoint signal and is crucial for maintaining the checkpoint toward late prometaphase when there are very few or even one single unattached kinetochore in the cell. The function of the kinase activity of BubR1 remains to be addressed in the future. In addition to Bub1, BubR1, and Cdc20, we suspect that checkpoint protein Mps1 might also be a direct target of MAPK. In the near future, we will investigate the question of how MAPK pathway is kept active
in response to unattached kinetochores, and how the pathway is inactivated to allow the metaphase-to-anaphase transition to occur.
Impacts
Our recent study reveals an important mechanism of the spindle checkpoint regulation that has never been studied before. We also identify the first MAPK substrate in the spindle checkpoint, nine years after MAPK was first shown to be essential for the spindle checkpoint in metazoans. Furthermore, our result of multiple phosphorylation of Cdc20 by Cdc2 and MAPK raises an interesting possibility that MAPK inactivation may initiate the termination of the spindle checkpoint signal after metaphase is achieved.
Publications
- Chung, E. and Chen, R.-H. 2003. Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint. Nature Cell Biol. 5, 748-753.
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Progress 01/01/02 to 12/31/02
Outputs
Maintenance of genetic integrity relies on even distribution of the newly duplicated sister chromatids into the daughter cells at anaphase of each cell division. This process is controlled by the spindle checkpoint that delays the anaphase onset until all kinetochores have attached properly to the mitotic apparatus. This checkpoint mechanism involves the evolutionarily conserved Mad and Bub proteins. During the past year, we made major progress in two areas of spindle checkpoint regulation. 1. Functional role of Mad1-free Mad2 in the spindle checkpoint We found that Mad2 molecules exist in the Mad1-bound and Mad1-free forms in frog egg extracts independently of the cell cycle stage. By changing the ratio between Mad1 and Mad2, we demonstrated that the pool of Mad1-free Mad2 molecules is important for the spindle checkpoint. Addition of excess Mad1 to egg extracts titrates out the fraction of Mad1-free Mad2, and abolishes the establishment and maintenance of the
spindle checkpoint. Adding excess Mad2 molecules to these extracts restores the fraction of Mad1-free Mad2 molecules and rescues the checkpoint. We further demonstrated that the association between Mad2 and Cdc20, an anaphase activator, is enhanced when the spindle checkpoint is provoked by unattached kinetochores, in comparison to that at metaphase. Interestingly, Mad1-free Mad2 molecules fail to localize to kinetochores and cannot bind Cdc20, suggesting that kinetochore-localization of Mad2 facilitates the formation of Mad2-Cdc20 complex. We propose that Mad2 may become activated and dissociated from Mad1 at kinetochores and is replenished by the pool of Mad1-free Mad2. This notion is consistent with the model that unattached kinetochores generate and then release active spindle checkpoint molecules to inhibit Cdc20. 2. Function and regulation of BubR1 at kinetochores We cloned a full-length cDNA for Xenopus BubR1, and showed that BubR1 enriches at unattached kinetochores in a
Bub1-dependent manner. BubR1-depleted extracts loses the spindle checkpoint and greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20. Mad2 depletion has no effect on kinetochore binding of BubR1, but it prevents BubR1 from binding Cdc20, indicating a mutual dependency between Mad2 and BubR1 in binding Cdc20. In addition, we showed that the kinase activity of BubR1 is not essential for the spindle checkpoint. These studies demonstrate that BubR1 regulates the kinetochore localization of other spindle checkpoint proteins independently of its kinase activity. Interestingly, we found that kinetochore BubR1 and Bub1 are hyperphosphorylated at unattached kinetochores. This process is dependent on Mad1, but not Mad2, showing that Mad1 facilitates Bub1 and BubR1 hyperphosphorylation at kinetochores. The complicated mutual dependency among the checkpoint proteins at kinetochores indicates that the
generation of the spindle checkpoint signal in higher organisms involves a regulatory network, rather than a simple linear pathway.
Impacts
Our studies provide insight into the biochemical and molecular mechanisms of the spindle checkpoint. The information will eventually lead to the understanding of aneuploidy (a wrong number of chromosomes in cells) that manifests human diseases such as cancers and Down's syndrome.
Publications
- Chung, E. and Chen, R.-H. 2002. Spindle checkpoint requires Mad1-bound and Mad1-free Mad2. Mol. Biol. Cell 13, 1501-1511.
- Chen, R.-H. 2002. BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1. J. Cell Biol. 158, 487-496.
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Progress 01/01/01 to 12/31/01
Outputs
We have isolated a Xenopus Bub1, and generated specific antibody to investigate its role in the spindle checkpoint in Xenopus egg extracts. We demonstrate that Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, which is similar to the spindle checkpoint complex Mad1-Mad2. However, Bub1 differs from Mad1-Mad2 in that Bub1 remains on kinetochores that have attached to microtubules. The protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, adding back either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of other checkpoint proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore and that its kinase activity is not
necessary for the spindle checkpoint in Xenopus egg extracts. Cdc20 is the activator for the anaphase promoting complex and has been shown to be a target of Mad2. We have generated anti-Cdc20 antibodies that allow us to examine the interaction between Mad2 and Cdc20 in frog egg extract. We find that the interaction requires recruitment of Mad2 to unattached kinetochores and that the level of interaction is dependent on the number of unattached kinetochores. Our results support the hypothesis that unattached kinetochores trigger the checkpoint signal that contains the Mad2-Cdc20 complex. Interestingly, we find that inhibition of MAP kinase reduces Cdc20 phosphorylation and the interaction between Mad2 and Cdc20. Furthermore, a Cdc20 mutant that is not phosphorylated during mitosis fails to respond to the checkpoint signal. Our study identifies the first spindle checkpoint target for MAP kinase. We have successfully isolated a Xenopus homologue of BubR1, which appears to be similar to
yeast Mad3. We also generate anti-BubR1 antibodies for functional analysis. The preliminary study shows that BubR1 associates with Cdc20. Similar to Mad2-Cdc20 interaction, binding of BubR1 to Cdc20 also requires unattached kinetochores.
Impacts
In the next funding period, we will further extend our studies. Specifically, we will map the functional domains of Bub1 to identify regions of the protein that are essential for kinetochore binding and for recruiting other checkpoint proteins to kinetochores. We will also determine the functional role of BubR1 in the spindle checkpoint by examining its interaction with other checkpoint proteins and any dependency of kinetochore binding among BubR1 and other checkpoint proteins. We will also examine any dependency of Mad2-Cdc20 interaction on other checkpoint proteins. In summary, we have generated valuable reagents for all the major players of the spindle checkpoint. The reagents shall provide us with tools for a mechanistic study of the checkpoint.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are correctly attached to the mitotic spindle. This control mechanism plays a crucial role in preventing aneuploidy that is often associated with cancers and genetic disorders. We have studied the spindle checkpoint in two model systems, the frog egg extracts and the budding yeast. During the past funding period, we have made three major progresses. I. Isolation and characterization of Xenopus Bub1 We isolated Xenopus Bub1 based on homology between Bub1 in other species. The protein is localized to kinetochores, similar to the spindle checkpoint complex Mad1-Mad2. However, Bub1 differs from Mad1-Mad2 in that Bub1 remains on kinetochores that have attached to microtubules, showing a difference in their kinetochore binding mechanism. Most importantly, we demonstrated that immunodepletion of Bub1 abolishes kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and
CENP-E. Immunodepletion of Mad1 results in loss of Mad2 and reduction of CENP-E at kinetochores without affecting Bub1 and Bub3. This study recognizes a central role for Bub1 in triggering the spindle checkpoint signal from the kinetochore through assembling checkpoint molecules at kinetochores. II. Dynamic interaction of Mad1 and Mad2 with kinetochores The checkpoint signal generated at kinetochores must propagate into the cytosol in order to stop the cell cycle progression. We hypothesize that checkpoint proteins such as Bub1, Bub3, Mad1, and Mad2, may become activated upon binding to kinetochores and then released into the cytosol to perform its function. We have demonstrated that there is a constant turnover of Mad1 and Mad2 at kinetochores, consistent with their kinetochore association being a dynamic event. Furthermore, Mad1 and Mad2 form a stable complex, while a fraction of Mad2 molecules is not bound to Mad1. Interestingly, the checkpoint is abolished upon titrating out
Mad1-free Mad2 with addition of excess Mad1 or a truncated Mad1 that contains Mad2-binding region. We propose that Mad1-free Mad2 may have a different function from the Mad1-Mad2 complex or that Mad2 cycles through kinetochores independently of Mad1. III. Isolation of conditional yeast mad2 mutants Mad2 molecules that have been activated at and released from kinetochores are thought to bind and inhibit the anaphase-promoting complex. Several central questions remain to be addressed. How is the activity of Mad2 regulated? What is the active state of the protein? These questions have been difficult to answer, due to a lack of enzymatic activity or post-translational modification in Mad2. Thus, we take a genetic approach to isolate a panel of yeast mad2 mutants that will be used in a suppressor screen to find molecules interacting physically or functionally with Mad2. Molecules that may activate or inactivate Mad2 are likely to be identified in the screen.
Impacts
During the next funding period, we will further extend the study. Specifically, we will generate a kinase-deficient Bub1 and study the role of the kinase activity in the checkpoint. We will also use the Mass Spectrometry facility at Cornell University to identify the phosphorylation sites on Xenopus Mad1 and to study the role of Mad1 phosphorylation. In addition, we will continue to characterize the conditional yeast mad2 mutants and initiate the suppressor screen in the near future. The parallel studies performed in the Xenopus egg extracts and in yeast have been and shall continue to be fruitful.
Publications
- No publications reported this period
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