Molecular and Cellular Biochemistry

, Volume 306, Issue 1–2, pp 53–57

Association of Rpn10 with high molecular weight complex is enhanced during retinoic acid-induced differentiation of neuroblastoma cells

  • Yoko Tayama
  • Hiroyuki Kawahara
  • Ryosuke Minami
  • Masumi Shimada
  • Hideyoshi Yokosawa


The ubiquitin-binding Rpn10 protein serves as an ubiquitin receptor that delivers client proteins to the 26S proteasome, the protein degradation complex. It has been suggested that the ubiquitin-dependent protein degradation is critical for neuronal differentiation and for preventing neurodegenerative diseases. Our previous study indicated the importance of Rpn10 in control of cellular differentiation (Shimada et al., Mol Biol Cell 17:5356–5371, 2006), though the functional relevance of Rpn10 in neuronal cell differentiation remains a mystery to be uncovered. In the present study, we have examined the level of Rpn10 in a proteasome-containing high molecular weight (HMW) protein fraction prepared from the mouse neuroblastoma cell line Neuro2a. We here report that the protein level of Rpn10 in HMW fraction from un-differentiated Neuro2a cells was significantly lower than that of other cultured cell lines. We have found that retinoic acid-induced neural differentiation of Neuro2a cells significantly stimulates the incorporation of Rpn10 into HMW fractions, although the amounts of 26S proteasome subunits were not changed. Our findings provide the first evidence that the modulation of Rpn10 is linked to the control of retinoic acid-induced differentiation of neuroblastoma cells.


Rpn10 Ubiquitin 26S proteasome Retinoic acid Neuron 



High molecular weight protein fraction


von Willebrand A


4,2-Hydroxyethyl-1-piperazineethanesulfonic acid


Phosphate buffered saline




Ubiquitin interacting motif


all-trans Retinoic acid


  1. 1.
    Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479PubMedCrossRefGoogle Scholar
  2. 2.
    Herschko A, Chichanover A, Varshavsky A (2001) The ubiquitin system. Nat Med 6:1073–1081CrossRefGoogle Scholar
  3. 3.
    Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–533PubMedCrossRefGoogle Scholar
  4. 4.
    Finley D, Ciechanover A, Varshavsky A (2004) Ubiquitin as a central cellular regulator. Cell 116:S29–S32PubMedCrossRefGoogle Scholar
  5. 5.
    DeMartino GN, Slaughter CA (1999) The proteasome, a novel protease regulated by multiple mechanisms. J Biol Chem 274:22123–22126PubMedCrossRefGoogle Scholar
  6. 6.
    Voges D, Zwickl P, Baumeister W (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem 68:1015–1068PubMedCrossRefGoogle Scholar
  7. 7.
    Pickart CM, Cohen RE (2004) Proteasomes and their kin: proteases in the machine age. Nat Rev Mol Cell Biol 5:177–187PubMedCrossRefGoogle Scholar
  8. 8.
    Almeida A, Bolanos JP, Moreno S (2005) Cdh1/Hct1-APC is essential for the survival of postmitotic neurons. J Neurosci 25:8115–8121PubMedCrossRefGoogle Scholar
  9. 9.
    Bossy-Wetzel E, Schwarzenbacher R, Lipton SA (2004) Molecular pathways to neurodegeneration. Nat Med 10 Suppl:S2–S9PubMedCrossRefGoogle Scholar
  10. 10.
    Jackson PK (2006) Developmental neurobiology: a destructive switch for neurons. Nature 442:365–366PubMedCrossRefGoogle Scholar
  11. 11.
    Petrucelli L, Dawson TM (2004) Mechanism of neurodegenerative disease: role of the ubiquitin proteasome system. Ann Med 36:315–320PubMedCrossRefGoogle Scholar
  12. 12.
    Sakurai M, Ayukawa K, Setsuie R, Nishikawa K, Hara Y, Ohashi H, Nishimono M, Abe T, Kudo Y, Sekiguchi M, Sato Y, Aoki S, Noda M, Wada K (2006) Ubiquitin C-terminal hydrolase L1 regulates the morphology of neural progenitor cells and modulates their differentiation. J Cell Sci 119:162–171PubMedCrossRefGoogle Scholar
  13. 13.
    Staropoli JF, McDermott C, Martinat C, Schulman B, Demireva E, Abeliovich A (2003) Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitotic neurons from kainate excitotoxicity. Neuron 37:735–749PubMedCrossRefGoogle Scholar
  14. 14.
    Klimaschewski L, Hausott B, Ingorokva S, Pfaller K (2006) Constitutively expressed catalytic proteasomal subunits are up-regulated during neuronal differentiation and required for axon initiation, elongation and maintenance. J Neurochem 96:1708–1717PubMedCrossRefGoogle Scholar
  15. 15.
    Sakata E, Yamaguchi Y, Kurimoto E, Kikuchi J, Yokoyama S, Yamada S, Kawahara H, Yokosawa H, Hattori N, Mizuno Y, Tanaka K, Kato K (2003) Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain. EMBO Rep 4:301–306PubMedCrossRefGoogle Scholar
  16. 16.
    Shimura H, Hattori N, Kubo S, Mizuno Y, Asakawa S, Minoshima S, Shimizu N, Iwai K, Chiba T, Tanaka K, Suzuki T (2000) Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase. Nat Genet 25:302–305PubMedCrossRefGoogle Scholar
  17. 17.
    Choi J, Levey AI, Weintraub ST, Rees HD, Gearing M, Chin LS, Li L (2004) Oxidative modifications and down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated with idiopathic Parkinson’s and Alzheimer’s diseases. J Biol Chem 279:13256–13264PubMedCrossRefGoogle Scholar
  18. 18.
    Gong B, Cao Z, Zheng P, Vitolo OV, Lie S, Staniszewski A, Moolman D, Zhang H, Shelanski M, Arancio O (2006) Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory. Cell 126:775–788PubMedCrossRefGoogle Scholar
  19. 19.
    Deveraux Q, Ustrell V, Pickart C, Rechsteiner M (1994) A 26S protease subunit that binds ubiquitin conjugates. J Biol Chem 269:7059–7061PubMedGoogle Scholar
  20. 20.
    Ferrell K, Deveraux Q, van Nocker S, Rechsteiner M (1996) Molecular cloning and expression of a multiubiquitin chain binding subunit of the human 26S protease. FEBS Lett 381:143–148PubMedCrossRefGoogle Scholar
  21. 21.
    Kawahara H, Kasahara M, Nishiyama A, Ohsumi K, Goto T, Kishimoto T, Saeki Y, Yokosawa H, Shimbara N, Murata S, Chiba T, Suzuki K, Tanaka K (2000) Developmentally regulated, alternative splicing of the Rpn10 gene generates multiple forms of 26S proteasomes. EMBO J 19:4144–4153PubMedCrossRefGoogle Scholar
  22. 22.
    van Nocker S, Sadis S, Rubin DM, Glickman M, Fu H, Coux O, Wefes I, Finley D, Vierstra RD (1996) The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover. Mol Cell Biol 16:6020–6028PubMedGoogle Scholar
  23. 23.
    Glickman MH, Rubin DM, Coux O, Wefes I, Pfeifer G, Cjeka Z, Baumeister W, Fried VA, Finley D (1998) A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94:615–623PubMedCrossRefGoogle Scholar
  24. 24.
    Verma R, Oania R, Graumann J, Deshaies RJ (2004) Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system. Cell 118:99–110PubMedCrossRefGoogle Scholar
  25. 25.
    Haracska L, Udvardy A (1997) Mapping the ubiquitin-binding domains in the p54 regulatory complex subunit of the Drosophila 26S protease. FEBS Lett 412:331–336PubMedCrossRefGoogle Scholar
  26. 26.
    Hofmann K, Falquet LA (2001) Ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems. Trends Biochem Sci 26:347–350PubMedCrossRefGoogle Scholar
  27. 27.
    Young P, Deveraux Q, Beal RE, Pickart CM, Rechsteiner M (1998) Characterization of two polyubiquitin-binding sites in the 26S protease subunit 5a. J Biol Chem 273:5461–5467PubMedCrossRefGoogle Scholar
  28. 28.
    Hiyama H, Yokoi M, Masutani C, Sugasawa K, Maekawa T, Tanaka K, Hoeijmakers JH, Hanaoka F (1999) Interaction of hHR23 with S5a: the ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of the 26S proteasome. J Biol Chem 274:28019–28025PubMedCrossRefGoogle Scholar
  29. 29.
    Kikukawa Y, Minami R, Shimada M, Kobayashi M, Tanaka K, Yokosawa H, Kawahara H (2005) Unique proteasome subunit Xrpn10c is a specific receptor for the antiapoptotic ubiquitin-like protein Scythe. FEBS J 272:6373–6386PubMedCrossRefGoogle Scholar
  30. 30.
    Walters KJ, Kleijnen MF, Goh AM, Wagner G, Howley PM (2002) Structural studies of the interaction between ubiquitin family proteins and proteasome subunit S5a. Biochemistry 41:1767–1777PubMedCrossRefGoogle Scholar
  31. 31.
    Elsasser S, Chandler-Militello D, Muller B, Hanna J, Finley D (2004) Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome. J Biol Chem 279:26817–26822PubMedCrossRefGoogle Scholar
  32. 32.
    Fu H, Sadis S, Rubin DM, Glickman M, van Nocker S, Finley D, Vierstra RD (1998) Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1. J Biol Chem 273:1970–1981PubMedCrossRefGoogle Scholar
  33. 33.
    Wilkinson CR, Seeger M, Hartmann-Petersen R, Stone M, Wallace M, Semple C, Gordon C (2001) Proteins containing the UBA domain are able to bind to multi-ubiquitin chains. Nature Cell Biol 3:939–943PubMedCrossRefGoogle Scholar
  34. 34.
    Shimada M, Kanematsu K, Tanaka K, Yokosawa H, Kawahara H (2006) Proteasomal ubiquitin receptor RPN-10 controls sex determination in Caenorhabditis elegans. Mol Biol Cell 17:5356–5371PubMedCrossRefGoogle Scholar
  35. 35.
    Kawahara H, Yokosawa H (1994) Intracellular calcium mobilization regulates the activity of 26S proteasome during metaphase-anaphase transition in the ascidian meiotic cell cycle. Dev Biol 166:623–633PubMedCrossRefGoogle Scholar
  36. 36.
    Clagett-Dame M, McNeill EM, Muley PD (2006) Role of all-trans retinoic acid in neurite outgrowth and axonal elongation. J Neurobiol 66:739–756PubMedCrossRefGoogle Scholar
  37. 37.
    Sato C, Matsuda T, Kitajima K (2002) Neuronal differentiation-dependent expression of the disialic acid epitope on CD166 and its involvement in neurite formation in Neuro2A cells. J Biol Chem 277:45299–45305PubMedCrossRefGoogle Scholar
  38. 38.
    Seeger M, Peterson RH, Wilkinson CRM, Wallace M, Samejima I, Taylor MS, Gordon C (2003) Interaction of APC/Cyclosome and proteasome protein complexes with multiubiquitin chain binding proteins. J Biol Chem 278:16791–16796PubMedCrossRefGoogle Scholar
  39. 39.
    Szlanka T, Haracska L, Kiss I, Deak P, Kurucz E, Ando I, Viragh E, Udvardy A (2003) Deletion of proteasomal subunit S5a/Rpn10/p54 causes lethality, multiple mitotic defects and overexpression of proteasomal genes in Drosophila melanogaster. J Cell Sci 116:1023–1033PubMedCrossRefGoogle Scholar
  40. 40.
    Smalle J, Kurepa J, Yang P, Emborg TJ, Babiychuk E, Kushnir S, Vierstra RD (2003) The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-specific function in abscisic acid signaling. Plant Cell 15:965–980PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Yoko Tayama
    • 1
  • Hiroyuki Kawahara
    • 1
  • Ryosuke Minami
    • 1
  • Masumi Shimada
    • 1
  • Hideyoshi Yokosawa
    • 1
  1. 1.Department of Biochemistry, Graduate School of Pharmaceutical SciencesHokkaido UniversitySapporoJapan

Personalised recommendations