Comment |
Address correspondence to M. Andrew Hoyt, Dept. of Biology, Mudd Hall, Rm. 36, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Tel.: (410) 516-7299. Fax: (410) 516- 5213. E-mail: hoyt{at}jhu.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() |
---|
The spindle assembly checkpoint is a quality control mechanism that prevents chromosome segregation errors. It acts to restrain cells from entering anaphase, the chromosome segregation step in mitosis and meiosis, until all replicated chromatids have formed proper attachments to a functional bipolar spindle. The checkpoint pathway transduces a signal generated by improperly attached kinetochores. This signal is presumably amplified and ultimately acts to halt the action of an enzyme required to drive cells into anaphase, the anaphase-promoting complex (APC*; otherwise known as cyclosome). It seemed as if the output of the spindle checkpoint was the best understood aspect of this pathway. Numerous studies suggested that an activated form of the Mad2 checkpoint protein generated specifically at unattached kinetochores binds and inhibits the actions of the APC (for review see Shah and Cleveland, 2000). However, two recent studies (Sudakin et al., 2001; Tang et al., 2001) indicate that Mad2 is not the whole story. Although these studies disagree on some conclusions, they strikingly agree on the most important issues. Another checkpoint protein, the BubR1 kinase, was found to bind and inhibit the APC and at least in vitro was a remarkably more potent inhibitor than Mad2 alone.
Studies of budding yeast revealed that the functions of six checkpoint proteins, Mad13, Bub1, Bub3, and Mps1, are required to prevent anaphase entry when spindle function is compromised (for review see Amon, 1999). These proteins appear conserved in all eukaryotic genomes sequenced to date with one key difference. In fungal species, Bub1 and Mad3 are sequence-related proteins. Both have similar NH2 termini with defined Cdc20-binding (an APC activator) and Bub3-binding domains (Hardwick et al., 2000; Murray and Marks, 2001). Bub1 uniquely possesses a COOH-terminal protein kinase domain. All multicellular animal genomes examined also encode two members of the Bub1/Mad3 family; however, both possess COOH-terminal kinase domains. The kinase with the Mad3-like NH2-terminus has been named BubR1. It has yet to be demonstrated that BubR1 is the functional ortholog of yeast Mad3, but this is the most reasonable presumption. Strikingly, localization studies have placed all six of the animal checkpoint proteins at kinetochores, and all but Mps1 are found with marked preference at those kinetochores that are improperly attached to spindle microtubules (Shah and Cleveland, 2000; Abrieu et al., 2001; Fisk and Winey, 2001).
Entry into anaphase requires the proteolytic destruction of certain cell cycle regulatory proteins. Most notable is securin (Pds1 in Saccharomyces cerevisiae), an anaphase inhibitor that acts by sequestering separin (Esp1) required for sister chromatid separation (Zachariae and Nasmyth, 1999). Securin and other proteins are targeted for destruction by the covalent addition of ubiquitin, catalyzed by the E3-type ligase activity of the APC. Attention was first focused upon Mad2 by the finding that it bound Cdc20, an activator of the APC that targets it to anaphase-specific substrates (Hwang et al., 1998; Kim et al., 1998). Subsequent studies showed that the interaction of Mad2 with Cdc20-bound APC inhibits its ubiquitin ligase activity (Li et al., 1997; Fang et al., 1998). The simple hypothesis that emerged from these studies is that Mad2 is converted to a diffusible inhibitory form during a transient association with unattached kinetochores. Support for this hypothesis came from the finding that Mad2 does in fact interact with unattached kinetochores in a dynamic fashion (Howell et al., 2000). Fluorescently labeled Mad2 repopulated photobleached kinetochores with a rapid recovery half time of 24 s. Therefore, the role of other checkpoint components in this simple formulation would be upstream of Mad2, acting at the unattached kinetochore to convert Mad2 to its APC inhibitory form.
Well...not so fast, say these new studies. An association of BubR1 with Cdc20 and the APC had been observed previously (Chan et al., 1999; Wu et al., 2000), a finding inconsistent with the Mad2 lone inhibitor hypothesis. Both new studies report the purification of a complex from HeLa cells containing BubR1 and other checkpoint proteins. The complex characterized by Sudakin et al. (2001) and named the mitotic checkpoint complex (MCC) contains BubR1, Bub3, Cdc20, and Mad2 in near equal stoichiometry. Normalized to the amount of Mad2, the MCC was found to inhibit APC ubiquitination activity in vitro 3,000-fold more effectively than purified recombinant Mad2 alone. Tang et al. (2001) examined the effects of the addition of individual recombinant protein preparations to an in vitro APC assay system. BubR1 added by itself was found to inhibit APC activity much more potently than Mad2 (KI = 40 nM versus 2 µM for BubR1 and Mad2, respectively). Mutant forms of BubR1 defective for kinase domain function were found to inhibit APC as effectively as the intact form, a finding that calls attention to the absence of a kinase domain in the yeast homologue Mad3. Both studies indicate that the BubR1 complex inhibits APC stoichiometrically (as opposed to catalytically) by binding to APC and either holding Cdc20 in an inactive form or preventing the binding of free activating Cdc20.
Both groups concluded that the intracellular concentration of Mad2 is too low for it to act effectively as an APC inhibitor alone. Therefore, Mad2 must act in cooperation with other checkpoint proteins to exert its effects. Whether Mad2 acts in a complex with other checkpoint proteins, such as the MCC, or alone in a parallel and perhaps additive pathway of APC inhibition is not clear. A major disagreement between these two studies concerns the composition of the BubR1-containing complex. Sudakin et al. (2001) report that BubR1, Bub3, Cdc20, and Mad2 are present and equally represented in the inhibitory MCC. Associations between Mad3 and Bub3, Cdc20, and Mad2 have also been observed in S. cerevisiae (Hardwick et al., 2000). In contrast, the complex purified by Tang et al. (2001) contains equal amounts of only BubR1 and Bub3; Cdc20 is present at substoichiometric levels, and Mad2 does not appear to be present at all. Sudakin et al. (2001) show that the MCC contains only a small fraction of the total cellular Mad2 (<5%), and it is possible this did not remain associated through the Tang et al. (2001) purification protocol. On the other hand, the requirement or the role for Mad2 in MCC function has not been demonstrated yet (as has the requirement for BubR1). This difference between the two studies is certainly the result of the in vitro protocols applied to analyze this complex system and can be expected to be resolved with further study. The reader is reminded here that yeast genetic studies demonstrated that all six checkpoint proteins (Mad13, Bub1, Bub3, and Mps1) are required for the proper response in vivo and that double mutants do not seem to show additive effects (Alexandru et al., 1999). Although in vitro checkpoint reconstitution experiments have revealed important activities for individual proteins such as Mad2 and BubR1, we ultimately must provide an explanation for the combined actions of the entire set of checkpoint proteins in vivo.
![]() |
Regulation of the inhibitory action of the BubR1 complex |
---|
![]() ![]() ![]() ![]() ![]() |
---|
How might the APC's sensitivity to BubR1/MCC inhibition be regulated? The most plausible mechanism involves phosphorylation. Mitosis-specific phosphorylation of APC subunits is well documented, and preferential association of BubR1, Cdc20, and Mad2 to phosphorylated APC has been reported (Sudakin et al., 2001). Perhaps phosphorylation events required for inhibition are mediated by one of the spindle checkpoint kinases, Mps1, Bub1, or even BubR1. Another possibility is that the 95% of cellular Mad2 not associated with MCC participates in APC sensitization. Future studies must determine the mechanism of APC sensitization to inhibition in order to solidify this new view of checkpoint action.
![]() |
The kinetochore motor connection |
---|
![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
Note added in proof. Studies by Guowei Fang (personal communication) similarly demonstrate that BubR1 is a more potent APC inhibitor than Mad2 and that Mad2 alone cannot inhibit at its physiological concentration. However, synergism between BubR1 and Mad2 with respect to APC inhibition was detected.
Submitted: 1 August 2001
Accepted: 10 August 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() |
---|
Abrieu, A., J.A. Kahana, K.W. Wood, and D.W. Cleveland. 2000. CENP-E as an essential component of the mitotic checkpoint in vitro. Cell. 102:817826.[Medline]
Abrieu, A., L. Magnaghi-Jaulin, J.A. Kahana, M. Peter, A. Castro, S. Vigneron, T. Lorca, D.W. Cleveland, and J. Labbe. 2001. Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint. Cell. 106:8393.[Medline]
Alexandru, G., W. Zachariae, A. Schleiffer, and K. Nasmyth. 1999. Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage. EMBO J. 18:27072721.
Amon, A. 1999. The spindle checkpoint. Curr. Opin. Genet. Dev. 9:6975.[Medline]
Campbell, M.S., J.R. Daum, M.S. Gersch, R.B. Nicklas, and G.J. Gorbsky. 2000. Kinetochore "memory" of spindle checkpoint signaling in lysed mitotic cells. Cell Motil. Cytoskel. 46:146156.[Medline]
Chan, G.T.K., B.T. Schaar, and T.J. Yen. 1998. Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1. J. Cell Biol. 143:4963.
Chan, G.K.T., S.A. Jablonski, V. Sudakin, J.C. Hittle, and T.J. Yen. 1999. Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions and binds the cyclosome/APC. J. Cell Biol. 146:941954.
Fang, G., H. Yu, and M.W. Kirschner. 1998. The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Genes Dev. 12:18711883.
Fisk, H.A., and M. Winey. 2001. The mouse Mps1p-like kinase regulates centrosome duplication. Cell. 106:95104.[Medline]
Hardwick, K.G., R.C. Johnston, D.L. Smith, and A.W. Murray. 2000. MAD3 encodes a novel component of the spindle checkpoint, which interacts with Bub3p, Cdc20p, and Mad2p. J. Cell Biol. 148:871882.
Howell, B.J., D.B. Hoffman, G. Fang, A.W. Murray, and E.D. Salmon. 2000. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells. J. Cell Biol. 150:12331250.
Hwang, L.H., L.F. Lau, D.L. Smith, C.A. Mistrot, K.G. Hardwick, E.S. Hwang, A. Amon, and A.W. Murray. 1998. Budding yeast Cdc20: a target of the spindle checkpoint. Science. 279:10411044.
Kim, S.H., D.P. Lin, S. Matsumoto, A. Kitazono, and T. Matsumoto. 1998. Fission yeast Slp1: an effector of the Mad2-dependent spindle checkpoint. Science. 279:10451047.
Li, Y., C. Gorbea, D. Mahaffey, M. Rechsteiner, and R. Benezra. 1997. MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity. Proc. Natl. Acad. Sci. USA. 94:1243112436.
Murray, A.W., and D. Marks. 2001. Can sequencing shed light on cell cycling? Nature. 409:844846.[Medline]
Shah, J.V., and D.W. Cleveland. 2000. Waiting for anaphase: Mad2 and the spindle assembly checkpoint. Cell. 103:9971000.[Medline]
Sharp-Baker, H., and R.H. Chen. 2001. Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity. J. Cell Biol. 153:12391250.
Skoufias, D.A., P.R. Andreassen, F.B. Lacroix, L. Wilson, and R.L. Margolis. 2001. Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachment and kinetochore-tension checkpoints. Proc. Natl. Acad. Sci. USA. 98:44924497.
Sudakin, V., G.K.T. Chan, and T.J. Yen. 2001. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, MAD2. J. Cell Biol. 154:925936.
Tang, Z., R. Bharadwaj, B. Li, and H. Yu. 2001. Mad2-independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1. Dev. Cell. 1:227237.[Medline]
Wu, H., Z. Lan, W. Li, S. Wu, J. Weinstein, K.M. Sakamoto, and W. Dai. 2000. p55CDC/hCDC20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase. Oncogene. 19:45574562.[Medline]
Yao, X., A. Abrieu, Y. Zheng, K.F. Sullivan, and D.W. Cleveland. 2000. CENP-E forms a link between attachment of spindle microtubules to kinetochores and the mitotic checkpoint. Nat. Cell Biol. 2:484491.[Medline]
Zachariae, W., and K. Nasmyth. 1999. Whose end is destruction: cell division and the anaphase-promoting complex. Genes Dev. 13:20392058.
Related Article