Molecular and Cellular Biochemistry

, Volume 381, Issue 1–2, pp 221–231 | Cite as

Nuclear import of aristaless-related homeobox protein via its NLS1 regulates its transcriptional function

  • Wenduo Ye
  • Wenbo Lin
  • Alan M. Tartakoff
  • Qilin Ma
  • Tao Tao


Nucleocytoplasmic transport of transcription factors is essential in eukaryotes. We previously reported the presence of two functional NLSs in the homeodomain protein, aristaless-related homeobox (Arx) protein, which is a key transcriptional repressor of LMO1, SHOX2, and PAX4 during development. NLS2, that overlaps the homeodomain, is recognized directly by multiple importin βs, but not by importin αs. In this study, we found that the N-terminal NLS1 of Arx is targeted by multiple importin α proteins, including importin α3 and α5. Both in vivo and in vitro assays demonstrated that nuclear import of Arx via NLS1 is mediated by the importin α/β pathway. Mutagenesis analysis indicated that two basic amino acids, 84K and 87R, are essential to the function of NLS1, and that their mutation prevents interactions of Arx with importin αs. Interestingly, inhibition of nuclear import of Arx via NLS1 clearly attenuates its ability of transcriptional repression, suggesting that nuclear import of Arx via NLS1 contributes to its transcriptional function.


Arx Importin α/β Nuclear localization signal Transcriptional repression 



This study was supported by Grants from National Science Foundation of China (#30971669 to Dr. Tao Tao; #31100599 to Dr. Wenbo Lin), and a Grant from Xiamen Center for Brain Research (#NK5888 to Dr. Qilin Ma). Dr. Alan Tartakoff is supported by NIH Grant R01-GM089872. Plasmids expressing importin αs were kindly provided by Dr. Nancy C. Reich of Stony Brook University. The (TAATTA)4 luciferase reporter plasmid was a gift from Dr. Jeffrey A Golden of Harvard University.


  1. 1.
    Tartakoff AM, Tao T (2010) Comparative and evolutionary aspects of macromolecular translocation across membranes. Int J Biochem Cell Biol 42:214–229PubMedCrossRefGoogle Scholar
  2. 2.
    Ploski JE, Shamsher MK, Radu A (2004) Paired-type homeodomain transcription factors are imported into the nucleus by karyopherin 13. Mol Cell Biol 24:4824–4834PubMedCrossRefGoogle Scholar
  3. 3.
    Ye W, Lin W, Tartakoff AM, Tao T (2011) Karyopherins in nuclear transport of homeodomain proteins during development. Biochim Biophys Acta 1813:1654–1662PubMedCrossRefGoogle Scholar
  4. 4.
    Collombat P, Mansouri A, Hecksher-Sorensen J, Serup P, Krull J, Gradwohl G, Gruss P (2003) Opposing actions of Arx and Pax4 in endocrine pancreas development. Genes Dev 17:2591–2603PubMedCrossRefGoogle Scholar
  5. 5.
    Kitamura K, Yanazawa M, Sugiyama N, Miura H, Iizuka-Kogo A, Kusaka M, Omichi K, Suzuki R, Kato-Fukui Y, Kamiirisa K, Matsuo M, Kamijo S, Kasahara M, Yoshioka H, Ogata T, Fukuda T, Kondo I, Kato M, Dobyns WB, Yokoyama M, Morohashi K (2002) Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans. Nat Genet 32:359–369PubMedCrossRefGoogle Scholar
  6. 6.
    Gecz J, Cloosterman D, Partington M (2006) ARX: a gene for all seasons. Curr Opin Genet Dev 16:308–316PubMedCrossRefGoogle Scholar
  7. 7.
    Friocourt G, Parnavelas JG (2010) Mutations in ARX result in several defects involving GABAergic neurons. Front Cell Neurosci 4:4PubMedGoogle Scholar
  8. 8.
    Shoubridge C, Fullston T, Gecz J (2010) ARX spectrum disorders: making inroads into the molecular pathology. Hum Mutat 31:889–900PubMedCrossRefGoogle Scholar
  9. 9.
    Colombo E, Collombat P, Colasante G, Bianchi M, Long J, Mansouri A, Rubenstein JL, Broccoli V (2007) Inactivation of Arx, the murine ortholog of the X-linked lissencephaly with ambiguous genitalia gene, leads to severe disorganization of the ventral telencephalon with impaired neuronal migration and differentiation. J Neurosci 27:4786–4798PubMedCrossRefGoogle Scholar
  10. 10.
    Shoubridge C, Cloosterman D, Parkinson-Lawerence E, Brooks D, Gecz J (2007) Molecular pathology of expanded polyalanine tract mutations in the Aristaless-related homeobox gene. Genomics 90:59–71PubMedCrossRefGoogle Scholar
  11. 11.
    Lin W, Ye W, Cai L, Meng X, Ke G, Huang C, Peng Z, Yu Y, Golden JA, Tartakoff AM, Tao T (2009) The roles of multiple importins for nuclear import of murine aristaless-related homeobox protein. J Biol Chem 284:20428–20439PubMedCrossRefGoogle Scholar
  12. 12.
    Goldfarb DS, Corbett AH, Mason DA, Harreman MT, Adam SA (2004) Importin alpha: a multipurpose nuclear-transport receptor. Trends Cell Biol 14:505–514PubMedCrossRefGoogle Scholar
  13. 13.
    Mason DA, Stage DE, Goldfarb DS (2009) Evolution of the metazoan-specific importin alpha gene family. J Mol Evol 68:351–365PubMedCrossRefGoogle Scholar
  14. 14.
    Harreman MT, Hodel MR, Fanara P, Hodel AE, Corbett AH (2003) The auto-inhibitory function of importin alpha is essential in vivo. J Biol Chem 278:5854–5863PubMedCrossRefGoogle Scholar
  15. 15.
    Stewart M (2003) Structural biology. Nuclear trafficking. Science 302:1513–1514PubMedCrossRefGoogle Scholar
  16. 16.
    Fagerlund R, Kinnunen L, Kohler M, Julkunen I, Melen K (2005) NF-{kappa}B is transported into the nucleus by importin {alpha}3 and importin {alpha}4. J Biol Chem 280:15942–15951PubMedCrossRefGoogle Scholar
  17. 17.
    Welch K, Franke J, Kohler M, Macara IG (1999) RanBP3 contains an unusual nuclear localization signal that is imported preferentially by importin-alpha3. Mol Cell Biol 19:8400–8411PubMedGoogle Scholar
  18. 18.
    Quensel C, Friedrich B, Sommer T, Hartmann E, Kohler M (2004) In vivo analysis of importin alpha proteins reveals cellular proliferation inhibition and substrate specificity. Mol Cell Biol 24:10246–10255PubMedCrossRefGoogle Scholar
  19. 19.
    Fulp CT, Cho G, Marsh ED, Nasrallah IM, Labosky PA, Golden JA (2008) Identification of Arx transcriptional targets in the developing basal forebrain. Hum Mol Genet 17:3740–3760PubMedCrossRefGoogle Scholar
  20. 20.
    Kosugi S, Hasebe M, Entani T, Takayama S, Tomita M, Yanagawa H (2008) Design of peptide inhibitors for the importin alpha/beta nuclear import pathway by activity-based profiling. Chem Biol 15:940–949PubMedCrossRefGoogle Scholar
  21. 21.
    Liu L, McBride KM, Reich NC (2005) STAT3 nuclear import is independent of tyrosine phosphorylation and mediated by importin-alpha3. Proc Natl Acad Sci U S A 102:8150–8155PubMedCrossRefGoogle Scholar
  22. 22.
    Harreman MT, Cohen PE, Hodel MR, Truscott GJ, Corbett AH, Hodel AE (2003) Characterization of the auto-inhibitory sequence within the N-terminal domain of importin alpha. J Biol Chem 278:21361–21369PubMedCrossRefGoogle Scholar
  23. 23.
    Fanara P, Hodel MR, Corbett AH, Hodel AE (2000) Quantitative analysis of nuclear localization signal (NLS)-importin alpha interaction through fluorescence depolarization. Evidence for auto-inhibitory regulation of NLS binding. J Biol Chem 275:21218–21223PubMedCrossRefGoogle Scholar
  24. 24.
    Shoubridge C, Tan MH, Seiboth G, Gecz J (2012) ARX homeodomain mutations abolish DNA binding and lead to a loss of transcriptional repression. Hum Mol Genet 21:1639–1647PubMedCrossRefGoogle Scholar
  25. 25.
    Kordowich S, Collombat P, Mansouri A, Serup P (2011) Arx and Nkx2.2 compound deficiency redirects pancreatic alpha- and beta-cell differentiation to a somatostatin/ghrelin co-expressing cell lineage. BMC Dev Biol 11:52PubMedCrossRefGoogle Scholar
  26. 26.
    Colasante G, Collombat P, Raimondi V, Bonanomi D, Ferrai C, Maira M, Yoshikawa K, Mansouri A, Valtorta F, Rubenstein JL, Broccoli V (2008) Arx is a direct target of Dlx2 and thereby contributes to the tangential migration of GABAergic interneurons. J Neurosci 28:10674–10686PubMedCrossRefGoogle Scholar
  27. 27.
    Friocourt G, Poirier K, Rakic S, Parnavelas JG, Chelly J (2006) The role of ARX in cortical development. Eur J Neurosci 23:869–876PubMedCrossRefGoogle Scholar
  28. 28.
    Yoshihara S, Omichi K, Yanazawa M, Kitamura K, Yoshihara Y (2005) Arx homeobox gene is essential for development of mouse olfactory system. Development 132:751–762PubMedCrossRefGoogle Scholar
  29. 29.
    Picard D, Yamamoto KR (1987) Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J 6:3333–3340PubMedGoogle Scholar
  30. 30.
    Theodore M, Kawai Y, Yang J, Kleshchenko Y, Reddy SP, Villalta F, Arinze IJ (2008) Multiple nuclear localization signals function in the nuclear import of the transcription factor Nrf2. J Biol Chem 283:8984–8994PubMedCrossRefGoogle Scholar
  31. 31.
    Jakel S, Gorlich D (1998) Importin beta, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells. EMBO J 17:4491–4502PubMedCrossRefGoogle Scholar
  32. 32.
    Waldmann I, Walde S, Kehlenbach RH (2007) Nuclear import of c-Jun is mediated by multiple transport receptors. J Biol Chem 282:27685–27692PubMedCrossRefGoogle Scholar
  33. 33.
    Quan Y, Ji ZL, Wang X, Tartakoff AM, Tao T (2008) Evolutionary and transcriptional analysis of karyopherin beta superfamily proteins. Mol Cell Proteomics 7:1254–1269PubMedCrossRefGoogle Scholar
  34. 34.
    Shoubridge C, Tan MH, Fullston T, Cloosterman D, Coman D, McGillivray G, Mancini GM, Kleefstra T, Gecz J (2010) Mutations in the nuclear localization sequence of the Aristaless related homeobox; sequestration of mutant ARX with IPO13 disrupts normal subcellular distribution of the transcription factor and retards cell division. Pathogenetics 3:1–15PubMedCrossRefGoogle Scholar
  35. 35.
    Cho G, Nasrallah MP, Lim Y, Golden JA (2012) Distinct DNA binding and transcriptional repression characteristics related to different ARX mutations. Neurogenetics 13:23–29PubMedCrossRefGoogle Scholar
  36. 36.
    Aratani S, Oishi T, Fujita H, Nakazawa M, Fujii R, Imamoto N, Yoneda Y, Fukamizu A, Nakajima T (2006) The nuclear import of RNA helicase A is mediated by importin-alpha3. Biochem Biophys Res Commun 340:125–133PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Xiamen University School of Life SciencesXiamenChina
  2. 2.Department of Pathology & Cell Biology ProgramCase Western Reserve University School of MedicineClevelandUSA
  3. 3.Department of NeurologyNo. 1 Hospital Affiliated to Xiamen University School of MedicineXiamenChina

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