RESEARCH ARTICLE

Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization

  • Jiao Wang 1 ,
  • Zhizhi Wang 2 ,
  • Tingting Yu 1 ,
  • Huan Yang 1 ,
  • David M. Virshup 3,4 ,
  • Geert J. P. L. Kops 5 ,
  • Sang Hyun Lee 3 ,
  • Weihong Zhou 1 ,
  • Xin Li 1 ,
  • Wenqing Xu , 2,6 ,
  • Zihe Rao , 1,6
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  • 1. College of Life Sciences, Nankai University, Tianjin 30071, China
  • 2. Department of Biological Structure, University of Washington, Seattle, WA 98195, USA
  • 3. Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore
  • 4. Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA
  • 5. Molecular Cancer Research and Cancer Genomics Centre, and Department of Medical Oncology, UMC Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
  • 6. National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100010, China

Received date: 13 Mar 2016

Accepted date: 05 May 2016

Published date: 18 Jul 2016

Copyright

2016 The Author(s) 2016. This article is published with open access at Springerlink.com and journal.hep.com.cn

Abstract

Protein phosphatase 2A (PP2A) accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a catalytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kinetochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interaction plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attachment. Here we present the crystal structure of a PP2A B56-BubR1 complex, which demonstrates that a conserved BubR1 LxxIxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxIxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.

Cite this article

Jiao Wang , Zhizhi Wang , Tingting Yu , Huan Yang , David M. Virshup , Geert J. P. L. Kops , Sang Hyun Lee , Weihong Zhou , Xin Li , Wenqing Xu , Zihe Rao . Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization[J]. Protein & Cell, 2016 , 7(7) : 516 -526 . DOI: 10.1007/s13238-016-0283-4

1
Arnold HK, Sears RC (2006) Protein phosphatase 2A regulatory subunit B56α associates with c-myc and negatively regulates c-myc accumulation. Mol Cell Biol 26:2832–2844

DOI

2
Cho US, Xu WQ (2007) Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature 445:53–57

DOI

3
Cho US, Morrone S, Sablina AA, Arroyo JD, Hahn WC, Xu WQ (2007) Structural basis of PP2A inhibition by small t antigen. PLoS Biol 5:1810–1819

4
Cohen P (2000) The regulation of protein function by multisite phosphorylation—a 25 year update. Trends Biochem Sci 25:596–601

DOI

5
DeLano WL, Brunger AT (1994) Helix packing in proteins: prediction and energetic analysis of dimeric, trimeric, and tetrameric GCN4 coiled coil structures. Proteins 20:105–123

DOI

6
Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS (2003) Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 161:267–280

DOI

7
Elowe S, Hummer S, Uldschmid A, Li X, Nigg EA (2007) Tensionsensitive Plk1 phosphorylation on BubR1 regulates the stability of kinetochore microtubule interactions. Genes Dev 21:2205–2219

DOI

8
Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D 66:486–501

DOI

9
Espert A, Uluocak P, Bastos RN, Mangat D, Graab P, Gruneberg U (2014) PP2A–B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing. J Cell Biol 206:833–842

DOI

10
Foley EA, Maldonado M, Kapoor TM (2011) Formation of stable attachments between kinetochores and microtubules depends on the B56–PP2A phosphatase. Nat Cell Biol 13:1265–1271

DOI

11
Funabiki H, Wynne DJ (2013) Making an effective switch at the kinetochore by phosphorylation and dephosphorylation. Chromosoma 122:135–158

DOI

12
Gao ZH, Seeling JM, Hill V, Yochum A, Virshup DM (2002) Casein kinase I phosphorylates and destabilizes the β-catenin degradation complex. Proc Natl Acad Sci USA 99:1182–1187

DOI

13
Hara K, Zheng G, Qu Q, Liu H, Ouyang Z, Chen Z, Tomchick D, Yu H (2014) Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion. Nat Struct Mol Biol 21:864–870

DOI

14
Kitajima TS, Kawashima SA, Watanabe Y (2004) The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis. Nature 427:510–517

DOI

15
Kitajima TS, Sakuno T, Ishiguro K, Iemura S, Natsume T, Kawashima SA, Watanabe Y (2006) Shugoshin collaborates with protein phosphatase 2A to protect cohesin. Nature 441:46–52

DOI

16
Kruse T, Zhang G, Larsen MSY, Lischetti T, Streicher W, Nielsen TK, Bjorn SP, Nilsson J (2013) Direct binding between BubR1 and B56–PP2A phosphatase complexes regulate mitotic progression. J Cell Sci 126:1086–1092

DOI

17
Lampson MA, Kapoor TM (2005) The human mitotic checkpoint protein BubR1 regulates chromosome-spindle attachments. Nat Cell Biol 7:93–98

DOI

18
Li X, Yost HJ, Virshup DM, Seeling JM (2001) A specific PP2A regulatory subunit, B56γ, mediates DNA damage-induced dephosphorylation of p53 at Thr55. EMBO J 20:4122–4131

19
Li HH, Cai X, Shouse GP, Piluso LG, Liu X (2007) Protein phosphatase 2A and its B56 regulatory subunit inhibit Wnt signaling in Xenopus. EMBO J 26:402–411

20
Liu H, Jia L, Yu H (2013a) Phospho-H2A and cohesin specify distinct tension-regulated Sgo1 pools at kinetochores and inner centromeres. Curr Biol 23:1927–1933

21
Liu H, Rankin S, Yu H (2013b) Phosphorylation-enabled binding of SGO1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis. Nat Cell Biol 15:40–49

22
Magnusdottir A, Stenmark P, Flodin S, Nyman T, Kotenyova T, Graslund S, Ogg D, Nordlund P (2009) The structure of the PP2A regulatory subunit B56 γ: the remaining piece of the PP2A jigsaw puzzle. Proteins Struct Funct Bioinform 74:212–221

DOI

23
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674

DOI

24
Musacchio A (2015) The molecular biology of spindle assembly checkpoint signaling dynamics. Curr Biol 25:3017–3017

DOI

25
Nijenhuis W, Vallardi G, Teixeira A, Kops GJ, Saurin AT (2014) Negative feedback at kinetochores underlies a responsive spindle checkpoint signal. Nat Cell Biol 16:1257–1264

DOI

26
Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode, vol 276. Academic, New York

27
Riedel CG, Katis VL, Katou Y, Mori S, Itoh T, Helmhart W, Galova M, Petronczki M, Gregan J, Cetin B et al (2006) Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I. Nature 441:53–61

DOI

28
Sangodkar J, Farrington CC, McClinch K, Galsky MD, Kastrinsky DB, Narla G (2016) All roads lead to PP2A: exploiting the therapeutic potential of this phosphatase. FEBS J 283:1004–1024

DOI

29
Sarangapani KK, Asbury CL (2014) Catch and release: How do kinetochores hook the right microtubules during mitosis? Trends Genet 30:150–159

30
Seeling JM, Miller JR, Gil R, Moon RT, White R, Virshup DM (1999) Regulation of β-catenin signaling by the B56 subunit of protein phosphatase 2A. Science 283:2089–2091

DOI

31
Suijkerbuijk SJ, Vleugel M, Teixeira A, Kops GJ (2012) Integration of kinase and phosphatase activities by BUBR1 ensures formation of stable kinetochore-microtubule attachments. Dev Cell 23:745–755

DOI

32
Tang ZY, Shu HJ, Qi W, Mahmood NA, Mumby MC, Yu HT (2006) PP2A is required for centromeric localization of sgol and proper chromosome segregation. Dev Cell 10:575–585

DOI

33
Virshup DM, Shenolikar S (2009) From promiscuity to precision:protein phosphatases get a makeover. Mol Cell 33:537–545

DOI

34
Winn MD, Murshudov GN, Papiz MZ (2003) Overview of the CCP4 suite and current developments. Methods Enzymol 374:300–321

35
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A et al (2011) Macromolecular TLS refinement in REFMAC at moderate resolutions. Acta CrystallogrD 67:235–242

36
Wlodarchak N, Xing Y (2016) PP2A as a master regulator of the cell cycle. Crit Rev Biochem Mol Biol 51:162–184

DOI

37
Wurzenberger C, Gerlich DW (2011) Phosphatases: providing safe passage through mitotic exit. Nat Rev Mol Cell Biol 12:469–482

DOI

38
Xing Y, Li Z, Chen Y, Stock JB, Jeffrey PD, Shi Y (2008) Structural mechanism of demethylation and inactivation of protein phosphatase 2A. Cell 133:154–163

DOI

39
Xu Y, Xing Y, Chen Y, Chao Y, Lin Z, Fan E, Yu JW, Strack S, Jeffrey PD, Shi Y (2006) Structure of the protein phosphatase 2A holoenzyme. Cell 127:1239–1251

DOI

40
Xu Z, Cetin B, Anger M, Cho US, Helmhart W, Nasmyth K, Xu W (2009) Structure and function of the PP2A–shugoshin interaction. Mol Cell 35:426–441

DOI

41
Xu P, Raetz EA, Kitagawa M, Virshup DM, Lee SH (2013) BUBR1 recruits PP2A via the B56 family of targeting subunits to promote chromosome congression. Biol Open 2:479–486

DOI

42
Xu P, Virshup DM, Lee SH (2014) B56–PP2A regulates motor dynamics for mitotic chromosome alignment. J Cell Sci 127:4567–4573

DOI

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