EDITORIALS: CELL CYCLE FEATURES Cell Cycle 14:19, 2999--3000; October 1, 2015; © 2015 Taylor & Francis Group, LLC
Bub1/BubR1: swiss army knives at kinetochores Jakob Nilsson* The Novo Nordisk Foundation Center for Protein Research; Faculty of Health and Medical Sciences; University of Copenhagen; Copenhagen, Denmark
When you go hiking only bring things that can be used for at least 2 different purposes, I was once told. The same idea seems to apply in the design of Bub1 and BubR1, key components of the Spindle Assembly Checkpoint (SAC) controlling chromosome segregation. Here I highlight recent work that has uncovered novel important aspects of Bub1 and BubR1 further underscoring the numerous protein interaction motifs present in these large proteins. Equal distribution of the genetic material to the 2 new daughter cells is ensured by the SAC that delays the onset of anaphase until all kinetochores on sister chromatids have attached properly to microtubules. In the presence of unattached kinetochores, the SAC generates a diffusible inhibitor referred to as the mitotic checkpoint complex (MCC) that inhibits the APC/C-Cdc20 complex. The APC/C-Cdc20 complex is a large ubiquitin ligase that targets key proteins for degradation to promote entry into anaphase. The MCC is assembled by the cooperative binding of the checkpoint proteins Mad2 and BubR1 to Cdc20. Mad2 binds a short motif in Cdc20 while the N-terminus of BubR1 binds the propeller domain of Cdc20 and to Mad2. How the kinetochore stimulates MCC formation is still poorly understood but the observed localization of all MCC components to kinetochores is likely critical for this. The initiating event in recruitment of checkpoint proteins to unattached kinetochores is the phosphorylation of the outer kinetochore protein KNL1 by the Mps1 checkpoint kinase. The phosphorylations on KNL1 generate binding sites for the Bub1-Bub3 complex that facilitate recruitment of additional checkpoint
components. It is now found that distinct regions in Bub1 are involved in localizing MCC components. My own lab and that of Andrea Musacchio identified a short region in Bub1 just after the Bub3 binding site that is required for BubR1 kinetochore localization1,2 (Fig. 1). This region in Bub1 can bind directly to BubR1 but surprisingly is not needed for a functional SAC arguing that BubR1 kinetochore localization might be dispensable for the checkpoint. Rather BubR1 bound to Bub1 appears to silence the checkpoint through its association with the B56PP2A phosphatase complex. While BubR1 kinetochore localization might not be required for MCC formation, the localization of Cdc20 and Mad2 appears to be. Mad2 binds tightly to the Mad1 checkpoint protein and this localizes a stable pool of Mad2 at kinetochores that allows recruitment of soluble Mad2 through Mad2 dimerization. It is the soluble pool of Mad2 that binds to Cdc20 and is incorporated into the MCC. In more complex eukaryotes, Mad1-Mad2 localization depends
on the Rod-Zwilch-ZW10 (RZZ) complex and interestingly the localization of this complex also depends on Bub1.1 We mapped a central region in Bub1 that was critical for RZZ recruitment and SAC signaling but the molecular mechanism of this recruitment needs further dissection. Interestingly, this region encompasses a conserved domain that also contributes directly to checkpoint activity suggesting that Bub1 might link Mad1-Mad2 localization to its activation. In close proximity to the domain in Bub1 required for RZZ recruitment, the Pines lab identified a short motif, referred to as the ABBA motif, which directly binds to Cdc203 (Fig. 1). The ABBA motif in Bub1 is required for a functional checkpoint and recruits a pool of Cdc20 to kinetochores.3,4 Potentially the proximity of Cdc20 to the Mad1-Mad2 complex, coordinated by Bub1, facilitates the binding of Mad2 to Cdc20 the ratelimiting step in MCC formation. Interestingly, the ABBA motif is also present in BubR1 and here this motif also
Figure 1. Schematic of Bub1 and BubR1 with the recently discovered interaction motifs indicated. Both Bub1 and BubR1 bind stably to Bub3 that is required for kinetochore localization of the proteins. The ABBA motifs are indicated in red while the direct interaction between Bub1 and BubR1 is mediated by the light blue regions.
*Correspondence to: Jakob Nilsson; Email: [email protected] Submitted: 07/23/2015; Accepted: 07/28/2015 http://dx.doi.org/10.1080/15384101.2015.1080980 Comment on: Zhang G, et al. Distinct domains in Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint.Nat Commun 2015; 6:7162; PMID: 26031201; http://dx.doi.org/10.1038/ncomms8162
www.tandfonline.com
Cell Cycle
2999
contributes to Cdc20 kinetochore localization but only makes a minor contribution to checkpoint activity.3,5,6 The role of the BubR1 ABBA motif appears to be dual in that it also contributes to SAC silencing by an unknown mechanism.5 As part of the MCC, BubR1 binds stably to a Cdc20 molecule through its N-terminus so one possibility is that the ABBA motif weakens this interaction to allow efficient MCC disassembly upon checkpoint References 1. Zhang G, et al. Nat Commun 2015; 6:7162; PMID:26031201; http://dx.doi.org/10.1038/ ncomms8162 2. Overlack K, et al. Elife 2015; 4:e05269; PMID:25611342; http://dx.doi.org/10.7554/eLife.05269
3000
satisfaction. However, another aspect to consider is the recent realization that the mature MCC binds to an additional Cdc20 molecule to efficiently inhibit the APC/C-Cdc20 complex.7 This binding is mediated by D-box and KEN-box motifs in BubR1, motifs present in APC/CCdc20 substrates, arguing that BubR1 has 3 distinct ways of interacting with Cdc20. Understanding how the different BubR1Cdc20 interactions are regulated and how
they influence each other is an important future goal. Just like a Swiss army knife that can unfold many surprising tools the same holds true for Bub1 and BubR1 the more we learn of these proteins. As we are getting closer to fully understanding all the protein interactions of the Bubs, the challenge now becomes to understand how they influence each other and how this controls chromosome segregation.
3. Di Fiore B, et al. J. Dev. Cell 2015; 32:358-372; PMID: 25669885; http://dx.doi.org/10.1016/j.devcel.2015. 01.003 4. Vleugel M, et al. J Cell Sci 2015; 128(16):2975-82; PMID:26148513; http://dx.doi.org/10.1242/jcs.169821 5. Lischetti T, et al. Nilsson J Nat Commun 2014; 5:5563; PMID:25482201; http://dx.doi.org/10.1038/ ncomms6563
6. Han JS. Proc Natl Acad Sci USA 2014; 111:E4185-93; PMID:25246557; http://dx.doi.org/10.1073/ pnas.1416277111 7. Izawa D, Pines J. Nature 2014; 517(7536):631-4; PMID:25383541; http://dx.doi.org/10.1038/ nature13911
Cell Cycle
Volume 14 Issue 19
Bub1/BubR1: swiss army knives at kinetochores Jakob Nilsson* The Novo Nordisk Foundation Center for Protein Research; Faculty of Health and Medical Sciences; University of Copenhagen; Copenhagen, Denmark
When you go hiking only bring things that can be used for at least 2 different purposes, I was once told. The same idea seems to apply in the design of Bub1 and BubR1, key components of the Spindle Assembly Checkpoint (SAC) controlling chromosome segregation. Here I highlight recent work that has uncovered novel important aspects of Bub1 and BubR1 further underscoring the numerous protein interaction motifs present in these large proteins. Equal distribution of the genetic material to the 2 new daughter cells is ensured by the SAC that delays the onset of anaphase until all kinetochores on sister chromatids have attached properly to microtubules. In the presence of unattached kinetochores, the SAC generates a diffusible inhibitor referred to as the mitotic checkpoint complex (MCC) that inhibits the APC/C-Cdc20 complex. The APC/C-Cdc20 complex is a large ubiquitin ligase that targets key proteins for degradation to promote entry into anaphase. The MCC is assembled by the cooperative binding of the checkpoint proteins Mad2 and BubR1 to Cdc20. Mad2 binds a short motif in Cdc20 while the N-terminus of BubR1 binds the propeller domain of Cdc20 and to Mad2. How the kinetochore stimulates MCC formation is still poorly understood but the observed localization of all MCC components to kinetochores is likely critical for this. The initiating event in recruitment of checkpoint proteins to unattached kinetochores is the phosphorylation of the outer kinetochore protein KNL1 by the Mps1 checkpoint kinase. The phosphorylations on KNL1 generate binding sites for the Bub1-Bub3 complex that facilitate recruitment of additional checkpoint
components. It is now found that distinct regions in Bub1 are involved in localizing MCC components. My own lab and that of Andrea Musacchio identified a short region in Bub1 just after the Bub3 binding site that is required for BubR1 kinetochore localization1,2 (Fig. 1). This region in Bub1 can bind directly to BubR1 but surprisingly is not needed for a functional SAC arguing that BubR1 kinetochore localization might be dispensable for the checkpoint. Rather BubR1 bound to Bub1 appears to silence the checkpoint through its association with the B56PP2A phosphatase complex. While BubR1 kinetochore localization might not be required for MCC formation, the localization of Cdc20 and Mad2 appears to be. Mad2 binds tightly to the Mad1 checkpoint protein and this localizes a stable pool of Mad2 at kinetochores that allows recruitment of soluble Mad2 through Mad2 dimerization. It is the soluble pool of Mad2 that binds to Cdc20 and is incorporated into the MCC. In more complex eukaryotes, Mad1-Mad2 localization depends
on the Rod-Zwilch-ZW10 (RZZ) complex and interestingly the localization of this complex also depends on Bub1.1 We mapped a central region in Bub1 that was critical for RZZ recruitment and SAC signaling but the molecular mechanism of this recruitment needs further dissection. Interestingly, this region encompasses a conserved domain that also contributes directly to checkpoint activity suggesting that Bub1 might link Mad1-Mad2 localization to its activation. In close proximity to the domain in Bub1 required for RZZ recruitment, the Pines lab identified a short motif, referred to as the ABBA motif, which directly binds to Cdc203 (Fig. 1). The ABBA motif in Bub1 is required for a functional checkpoint and recruits a pool of Cdc20 to kinetochores.3,4 Potentially the proximity of Cdc20 to the Mad1-Mad2 complex, coordinated by Bub1, facilitates the binding of Mad2 to Cdc20 the ratelimiting step in MCC formation. Interestingly, the ABBA motif is also present in BubR1 and here this motif also
Figure 1. Schematic of Bub1 and BubR1 with the recently discovered interaction motifs indicated. Both Bub1 and BubR1 bind stably to Bub3 that is required for kinetochore localization of the proteins. The ABBA motifs are indicated in red while the direct interaction between Bub1 and BubR1 is mediated by the light blue regions.
*Correspondence to: Jakob Nilsson; Email: [email protected] Submitted: 07/23/2015; Accepted: 07/28/2015 http://dx.doi.org/10.1080/15384101.2015.1080980 Comment on: Zhang G, et al. Distinct domains in Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint.Nat Commun 2015; 6:7162; PMID: 26031201; http://dx.doi.org/10.1038/ncomms8162
www.tandfonline.com
Cell Cycle
2999
contributes to Cdc20 kinetochore localization but only makes a minor contribution to checkpoint activity.3,5,6 The role of the BubR1 ABBA motif appears to be dual in that it also contributes to SAC silencing by an unknown mechanism.5 As part of the MCC, BubR1 binds stably to a Cdc20 molecule through its N-terminus so one possibility is that the ABBA motif weakens this interaction to allow efficient MCC disassembly upon checkpoint References 1. Zhang G, et al. Nat Commun 2015; 6:7162; PMID:26031201; http://dx.doi.org/10.1038/ ncomms8162 2. Overlack K, et al. Elife 2015; 4:e05269; PMID:25611342; http://dx.doi.org/10.7554/eLife.05269
3000
satisfaction. However, another aspect to consider is the recent realization that the mature MCC binds to an additional Cdc20 molecule to efficiently inhibit the APC/C-Cdc20 complex.7 This binding is mediated by D-box and KEN-box motifs in BubR1, motifs present in APC/CCdc20 substrates, arguing that BubR1 has 3 distinct ways of interacting with Cdc20. Understanding how the different BubR1Cdc20 interactions are regulated and how
they influence each other is an important future goal. Just like a Swiss army knife that can unfold many surprising tools the same holds true for Bub1 and BubR1 the more we learn of these proteins. As we are getting closer to fully understanding all the protein interactions of the Bubs, the challenge now becomes to understand how they influence each other and how this controls chromosome segregation.
3. Di Fiore B, et al. J. Dev. Cell 2015; 32:358-372; PMID: 25669885; http://dx.doi.org/10.1016/j.devcel.2015. 01.003 4. Vleugel M, et al. J Cell Sci 2015; 128(16):2975-82; PMID:26148513; http://dx.doi.org/10.1242/jcs.169821 5. Lischetti T, et al. Nilsson J Nat Commun 2014; 5:5563; PMID:25482201; http://dx.doi.org/10.1038/ ncomms6563
6. Han JS. Proc Natl Acad Sci USA 2014; 111:E4185-93; PMID:25246557; http://dx.doi.org/10.1073/ pnas.1416277111 7. Izawa D, Pines J. Nature 2014; 517(7536):631-4; PMID:25383541; http://dx.doi.org/10.1038/ nature13911
Cell Cycle
Volume 14 Issue 19
