|BUB1 mitotic checkpoint serine/threonine kinase|
Crystallographic structure of the human kinase BUB1 (rainbow colored cartoon, N-terminus = blue, C-terminus = red) complexed with ATP (space-filling model, carbon = white, oxygen = red, nitrogen = blue, phosphorus = orange).
|Symbols||; BUB1A; BUB1L; hBUB1|
|External IDs||ChEMBL: GeneCards:|
|RNA expression pattern|
Bub1 is a serine/threonine protein kinase first identified in genetic screens of Saccharomyces cerevisiae. The protein is bound to kinetochores and plays a key role in the establishment of the mitotic spindle checkpoint and chromosome congression. The mitotic checkpoint kinase is evolutionary conserved in organisms as diverse as Saccharomyces cerevisiae and humans. Loss-of-function mutations or absence of Bub1 has been reported to result in aneuploidy, chromosomal instability (CIN) and premature senescence.
Bub1p comprises a conserved N-terminal region, a central non-conserved region and a C-terminal serine/threonine kinase domain. The N-terminal region mediates binding of Hs-BUB1 to the mitotic kinetochore protein blinkin (a protein also commonly referred to as AF15q14). The latter interaction is essential for kinetochore localization of Bub1 and its function in cell cycle arrest induced by spindle assembly checkpoint (SAC) activation. The crystal structure of human Bub1 revealed the presence of a N-terminal tetratricopeptide repeat (TPR) domain and a C-terminal kinase domain (residues 784–1085), adopting a canonical kinase fold with two lobes. The ATP binding and the catalytic sites are located at the interface of the two lobes. The N-terminal extension contains three β-strands and an α-helix, wrapping around the N lobe of the kinase domain.:Figure1
In humans Bub1 accumulates gradually during G1 and S phase of the cell cycle, peaks at G2/M, and drops dramatically after mitosis. During prophase it localizes as one of the first proteins to the outer kinetochore, a process generally implicated in correct mitotic timing and checkpoint response to spindle damage.
The protein kinase Bub1 possesses versatile and distinct functions during the cell cycle, mainly in the SAC and chromosome alignment during metaphase. The protein’s interaction network currently identified is similarly complex (see Figure 1).
In eukaryotic cells the SAC serves as the central surveillance mechanism to ensure chromosomes are being passed on to the next generation in a reliable manner. Several components monitor correct bipolar attachment of microtubules to the kinetochore, presumably through detection of tension. Metaphase-to-anaphase transition is halted by the SAC as long as single kinetochores lack bipolar microtubule attachment, implying the need for a highly sensitive signaling pathway. Bub1 was claimed to be the master regulator of SAC formation and signaling. At least thirteen other proteins (Mad1, MAD2, MAD3/BubR1, BUB3, Mps1 etc.) are part of the check point, among which many have been identified to interact with Bub1.
Upon activation of the SAC Bub1 directly phosphorylates APC/C’s coactivator Cdc20. This phosphorylation event is probably achieved in complex with Bub3, which itself has been subjected to prior phosphorylation by Bub1. The phosphorylation of Cdc20 ultimately leads to decreased activity of APC/C which determines the metaphase-to-anaphase transition. In turn APC/C, now in complex with Cdh1, also acts on Bub1 by priming it for degradation to exit mitosis.
In addition, kinetochore localization of Bub1 early during G2 or prophase is another aspect of SAC functioning. Bub1 is thought to serve as a platform recruiting other checkpoint and motor proteins as Mad1, Mad2, BubR1, CENP-E and PLK1 to the kinetochore. Indeed, recent data suggest that the primary role of Bub1 during SAC activity is not Cdc20 phosphorylation but rather recruitment of BubR1, Mad1 and Mad2.
Upon spindle damage Bub1 is also triggered to phosphorylate Mad1 leading to dissociation of the Mad1-Mad2 complex and thereby rendering Mad2 accessible for inhibition of Cdc20. Bub1 generally protects sister chromatide cohesion by enhancing Shugoshin protein (Sgo1) localization to the centromeric region. Through recruitment of the phosphatase PP2A Bub1 inhibits the action of PLK1, which removes Sgo1 from the centromere.
Contrarily PLK1 localization, as mentioned, also depends on the activity of Bub1. Studies in Xenopus extracts using RNAi or antibody depletion have indicated a crucial function of Bub1 in the organization of the inner centromere. Similarly to its role in kinetochore assembly, it recruits members of the chromosomal passenger complex (CPC) like Aurora B kinase, Survivin and INCENP. Direct phosphorylation of INCENP by Bub1 has been observed.
RNAi mediated depletion of human Bub1 has indicated function in correct metaphase congression. Downstream targets identified are distinct kinetochore proteins as CENP-F, MCAK and the mentioned Sgo1.
Implications in cancer
Disturbed mitotic checkpoints are a common feature of many human cancers. More precisely, mutations in the spindle checkpoint can lead to chromosomal instability and aneuploidy, a feature present in over 90% of all solid tumors. Loss-of-function mutations or reduced gene expression of Bub1 have been identified in several human tumors as colon, esophageal, gastric, breast cancer and melanoma. A correlation between Bub1 expression levels and the localization of tumors along with their severity was found. For instance, low Bub1 expression levels resulted in more sarcomas, lymphomas and lung tumors, whereas higher ones caused sarcomas and tumors in the liver. Moreover, Bub1 has been identified as a target of the large T antigen of the SV-40 virus, possibly contributing to its potential for oncogenic transformation. Indications for possible Bub1 involvement in tumorigenesis also derive from animal experiments, where mice with reduced Bub1 expression showed an increase in tumor susceptibility. In vitro knockdown of Bub1 in p53 impaired cells (e.g. HeLa cells) caused aneuploidy. Whether aneuploidy alone is a sufficient driving cause during tumorigenesis or rather a mere consequence has been a matter of scientific debate.
Link to caspase-independent mitotic death (CIMD)
Recently Bub1 has been identified as a negative regulator of CIMD. Depletion of Bub1 results in increased CIMD in order to avoid aneuploidy caused by reduced SAC functioning. The transcriptional activity of p73 is thereby inhibited via phosphorylation. Direct interaction between these two players has not been visualized so far, therefore molecules linking Bub1 and p73 are yet to be determined. It has also been proposed that Bub1 binds p53 to prevent it from activating pro-apoptotic genes, therefore p53 is able to induce apoptosis when Bub1 is depleted. However, an interaction between p53 and Bub1 has not yet been shown while p53 binding BubR1 has been reported.
- PDB 3E7E; Kang J, Yang M, Li B, Qi W, Zhang C, Shokat KM, Tomchick DR, Machius M, Yu H (November 2008). "Structure and substrate recruitment of the human spindle checkpoint kinase Bub1". Mol. Cell 32 (3): 394–405. doi:10.1016/j.molcel.2008.09.017. PMC 2644263. PMID 18995837.
- "Entrez Gene: BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast)".
- Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B (March 1998). "Mutations of mitotic checkpoint genes in human cancers". Nature 392 (6673): 300–3. doi:10.1038/32688. PMID 9521327.
- Roberts BT, Farr KA, Hoyt MA (December 1994). "The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase". Mol. Cell. Biol. 14 (12): 8282–91. PMC 359367. PMID 7969164.
- Bolanos-Garcia VM, Kiyomitsu T, D'Arcy S, Chirgadze DY, Grossmann JG, Matak-Vinkovic D, Venkitaraman AR, Yanagida M, Robinson CV, Blundell TL (January 2009). "The Crystal Structure of the N-Terminal Region of BUB1 Provides Insight into the Mechanism of BUB1 Recruitment to Kinetochores". Structure 17 (1): 105–16. doi:10.1016/j.str.2008.10.015. PMC 2683949. PMID 19141287.
- Kiyomitsu T, Obuse C, Yanagida M (November 2007). "Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1". Dev. Cell 13 (5): 663–76. doi:10.1016/j.devcel.2007.09.005. PMID 17981135.
- "BUB1 protein (Homo sapiens) - STRING interaction network". STRING (Search Tool for the Retrieval of Interacting Genes/Proteins). European Molecular Biology Laboratory. Retrieved 2010-06-29.
- Tang Z, Shu H, Oncel D, Chen S, Yu H (November 2004). "Phosphorylation of Cdc20 by Bub1 provides a catalytic mechanism for APC/C inhibition by the spindle checkpoint". Mol. Cell 16 (3): 387–97. doi:10.1016/j.molcel.2004.09.031. PMID 15525512.
- Qi W, Yu H (February 2007). "KEN-box-dependent degradation of the Bub1 spindle checkpoint kinase by the anaphase-promoting complex/cyclosome". J. Biol. Chem. 282 (6): 3672–9. doi:10.1074/jbc.M609376200. PMID 17158872.
- Sharp-Baker H, Chen RH (June 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 (6): 1239–50. doi:10.1083/jcb.153.6.1239. PMC 2192030. PMID 11402067.
- Johnson VL, Scott MI, Holt SV, Hussein D, Taylor SS (March 2004). "Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression". J. Cell. Sci. 117 (Pt 8): 1577–89. doi:10.1242/jcs.01006. PMID 15020684.
- Meraldi P, Draviam VM, Sorger PK (July 2004). "Timing and checkpoints in the regulation of mitotic progression". Dev. Cell 7 (1): 45–60. doi:10.1016/j.devcel.2004.06.006. PMID 15239953.
- Klebig C, Korinth D, Meraldi P (June 2009). "Bub1 regulates chromosome segregation in a kinetochore-independent manner". J. Cell Biol. 185 (5): 841–58. doi:10.1083/jcb.200902128. PMC 2711590. PMID 19487456.
- Yu H (December 2002). "Regulation of APC-Cdc20 by the spindle checkpoint". Curr. Opin. Cell Biol. 14 (6): 706–14. doi:10.1016/S0955-0674(02)00382-4. PMID 12473343.
- Zhang Y, Lees E (August 2001). "Identification of an Overlapping Binding Domain on Cdc20 for Mad2 and Anaphase-Promoting Complex: Model for Spindle Checkpoint Regulation". Mol. Cell. Biol. 21 (15): 5190–9. doi:10.1128/MCB.21.15.5190-5199.2001. PMC 87243. PMID 11438673.
- Tang Z, Sun Y, Harley SE, Zou H, Yu H (December 2004). "Human Bub1 protects centromeric sister-chromatid cohesion through Shugoshin during mitosis". Proc. Natl. Acad. Sci. U.S.A. 101 (52): 18012–7. doi:10.1073/pnas.0408600102. PMC 539817. PMID 15604152.
- Tang Z, Shu H, Qi W, Mahmood NA, Mumby MC, Yu H (May 2006). "PP2A is required for centromeric localization of Sgo1 and proper chromosome segregation". Dev. Cell 10 (5): 575–85. doi:10.1016/j.devcel.2006.03.010. PMID 16580887.
- Jang YJ, Ji JH, Choi YC, Ryu CJ, Ko SY (January 2007). "Regulation of Polo-like kinase 1 by DNA damage in mitosis. Inhibition of mitotic PLK-1 by protein phosphatase 2A". J. Biol. Chem. 282 (4): 2473–82. doi:10.1074/jbc.M605480200. PMID 17121863.
- Pouwels J, Kukkonen AM, Lan W, Daum JR, Gorbsky GJ, Stukenberg T, Kallio MJ (July 2007). "Shugoshin 1 plays a central role in kinetochore assembly and is required for kinetochore targeting of Plk1". Cell Cycle 6 (13): 1579–85. doi:10.4161/cc.6.13.4442. PMID 17617734.
- Boyarchuk Y, Salic A, Dasso M, Arnaoutov A (March 2007). "Bub1 is essential for assembly of the functional inner centromere". J. Cell Biol. 176 (7): 919–28. doi:10.1083/jcb.200609044. PMC 2064078. PMID 17389228.
- Williams BR, Amon A (July 2009). "Aneuploidy –Cancer's Fatal Flaw?". Cancer Res. 69 (13): 5289–91. doi:10.1158/0008-5472.CAN-09-0944. PMC 2917070. PMID 19549887.
- King RW (September 2008). "When 2+2=5: The Origins and Fates of Aneuploid and Tetraploid Cells". Biochim. Biophys. Acta 1786 (1): 4–14. doi:10.1016/j.bbcan.2008.07.007. PMC 2581926. PMID 18703117.
- Hein J, Boichuk S, Wu J, Cheng Y, Freire R, Jat PS, Roberts TM, Gjoerup OV (January 2009). "Simian Virus 40 Large T Antigen Disrupts Genome Integrity and Activates a DNA Damage Response via Bub1 Binding". J. Virol. 83 (1): 117–27. doi:10.1128/JVI.01515-08. PMC 2612341. PMID 18922873.
- Jeganathan K, Malureanu L, Baker DJ, Abraham SC, van Deursen JM (October 2007). "Bub1 mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis". J. Cell Biol. 179 (2): 255–67. doi:10.1083/jcb.200706015. PMC 2064762. PMID 17938250.
- Schliekelman M, Cowley DO, O'Quinn R, Oliver TG, Lu L, Salmon ED, Van Dyke T (January 2009). "Impaired Bub1 function in vivo compromises tension-dependent checkpoint function leading to aneuploidy and tumorigenesis". Cancer Res. 69 (1): 45–54. doi:10.1158/0008-5472.CAN-07-6330. PMID 19117986.
- Williams GL, Roberts TM, Gjoerup OV (July 2007). "Bub1: escapades in a cellular world". Cell Cycle 6 (14): 1699–704. doi:10.4161/cc.6.14.4493. PMID 17643075.
- Kitagawa K, Niikura Y (April 2008). "Caspase-independent mitotic death (CIMD)". Cell Cycle 7 (8): 1001–5. doi:10.4161/cc.7.8.5720. PMID 18414023.
- Beeharry N, Yen TJ (April 2009). "p53-dependent apoptosis in response to spindle damage is linked to loss of Bub1". Cancer Biol. Ther. 8 (7): 645–7. doi:10.4161/cbt.8.7.8140. PMID 19270499.