title: A motif from Lys216 to Lys222 in human BUB3 protein is a nuclear localization signal and critical for BUB3 function in mitotic checkpoint.
abstract: Human BUB3 is a key mitotic checkpoint factor that recognizes centromeric components and recruits other mitotic checkpoint molecules to the unattached kinetochore. The key amino acid residues responsible for its localization are not yet defined. In this study, we identified a motif from Lys(216) to Lys(222) in BUB3 as its nuclear localization signal. A BUB3 mutant with deletion of this motif (Del216-222) was found to localize to both the cytoplasm and the nucleus, distinct from the exclusively nuclear distribution of wild-type BUB3. Further analysis revealed that residues Glu(213), Lys(216), Lys(217), Lys(218), Tyr(219), and Phe(221), but not Lys(222), contribute to nuclear localization. Interestingly, the nuclear localization signal was also critical for the kinetochore localization of BUB3. The deletion mutant Del216-222 and a subtle mutant with four residue changes in this region (E213Q/K216E/K217E/K218E (QE)) did not localize to the kinetochore efficiently or mediate mitotic checkpoint arrest. Protein interaction data suggested that the QE mutant was able to interact with BUB1, MAD2, and BubR1 but that its association with the centromeric components CENP-A and KNL1 was impaired. A motif from Leu(61) to Leu(65) in CENP-A was found to be involved in the association of BUB3 and CENP-A in cells; however, further assays suggested that CENP-A does not physically interact with BUB3 and does not affect BUB3 localization. Our findings help to dissect the mechanisms of BUB3 in mitotic checkpoint signaling.
authors: From the Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research at School of Life Science and Technology, Tongji University, 1239 Si-ping Road, Shanghai 200092, China zhusongcheng@tongji.edu.cn.
authors: Songcheng
authors: S
authors: Zhu
authors: From the Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research at School of Life Science and Technology, Tongji University, 1239 Si-ping Road, Shanghai 200092, China.
authors: Ruiqi
authors: R
authors: Jing
authors: From the Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research at School of Life Science and Technology, Tongji University, 1239 Si-ping Road, Shanghai 200092, China.
authors: Yiwei
authors: Y
authors: Yang
authors: From the Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research at School of Life Science and Technology, Tongji University, 1239 Si-ping Road, Shanghai 200092, China.
authors: Yitong
authors: Y
authors: Huang
authors: From the Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research at School of Life Science and Technology, Tongji University, 1239 Si-ping Road, Shanghai 200092, China.
authors: Xin
authors: X
authors: Wang
authors: From the Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research at School of Life Science and Technology, Tongji University, 1239 Si-ping Road, Shanghai 200092, China.
authors: Ye
authors: Y
authors: Leng
authors: From the Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research at School of Life Science and Technology, Tongji University, 1239 Si-ping Road, Shanghai 200092, China.
authors: Jiajie
authors: J
authors: Xi
authors: From the Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research at School of Life Science and Technology, Tongji University, 1239 Si-ping Road, Shanghai 200092, China.
authors: Guiying
authors: G
authors: Wang
authors: From the Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research at School of Life Science and Technology, Tongji University, 1239 Si-ping Road, Shanghai 200092, China.
authors: Wenwen
authors: W
authors: Jia
authors: From the Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research at School of Life Science and Technology, Tongji University, 1239 Si-ping Road, Shanghai 200092, China jhkang@tongji.edu.cn.
authors: Jiuhong
authors: J
authors: Kang
mesh_terms: D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001324:Autoantigens / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D008239:Lysine; D059566:M Phase Cell Cycle Checkpoints; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals; D000075223:Poly-ADP-Ribose Binding Proteins; D015398:Signal Transduction
chemical_list: D001324:Autoantigens; C489444:BUB3 protein, human; C000619700:CENPA protein, human; D018797:Cell Cycle Proteins; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D019913:Nuclear Localization Signals; D000075223:Poly-ADP-Ribose Binding Proteins; D008239:Lysine
keywords: BUB3 (Budding Uninhibited by Benzimidazole); Cell Signaling; Centromeric Protein A (CENP-A); Kinetochore; Kinetochore Localization; Mitotic Checkpoint; Mitotic Spindle; Nuclear Localization; Protein Motif; Protein Translocation