Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Pregnenolone activates CLIP-170 to promote microtubule growth and cell migration

Nature Chemical Biology volume 9, pages 636–642 (2013)Cite this article

Subjects

Abstract

Pregnenolone (P5) is a neurosteroid that improves memory and neurological recovery. It is also required for zebrafish embryonic development. However, its mode of action is unclear. Here we show that P5 promotes cell migration and microtubule polymerization by binding a microtubule plus end–tracking protein, cytoplasmic linker protein 1 (CLIP-170). We captured CLIP-170 from zebrafish embryonic extract using a P5 photoaffinity probe conjugated to diaminobenzophenone. P5 interacted with CLIP-170 at its coiled-coil domain and changed it into an extended conformation. This increased CLIP-170 interaction with microtubules, dynactin subunit p150Glued and LIS1; it also promoted CLIP-170–dependent microtubule polymerization. CLIP-170 was essential for P5 to promote microtubule abundance and zebrafish epiboly cell migration during embryogenesis, and overexpression of the P5-binding region of CLIP-170 delayed this migration. P5 also sustained migration directionality of cultured mammalian cells. Our results show that P5 activates CLIP-170 to promote microtubule polymerization and cell migration.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: P5 regulates cell motility and promotes microtubule growth.
Figure 2: P5 interacts with CLIP-170.
Figure 3: P5 changes the conformation of CLIP-170.
Figure 4: P5 activates CLIP-170 and regulates its location on microtubules.
Figure 5: P5 and CLIP-170 coordinately regulate microtubule assembly.
Figure 6: P5 and CLIP-170 coordinately regulate microtubule assembly and epiboly movement during zebrafish development.

Similar content being viewed by others

Accession codes

Primary accessions

Ensembl

References

  1. Hu, M.C. et al. Steroid deficiency syndromes in mice with targeted disruption of Cyp11a1. Mol. Endocrinol. 16, 1943–1950 (2002).

    Article  CAS  Google Scholar 

  2. Hsu, H.J., Liang, M.R., Chen, C.T. & Chung, B.C. Pregnenolone stabilizes microtubules and promotes zebrafish embryonic cell movement. Nature 439, 480–483 (2006).

    Article  CAS  Google Scholar 

  3. Grigoryev, D.N., Long, B.J., Njar, V.C. & Brodie, A.H. Pregnenolone stimulates LNCaP prostate cancer cell growth via the mutated androgen receptor. J. Steroid Biochem. Mol. Biol. 75, 1–10 (2000).

    Article  CAS  Google Scholar 

  4. Iqbal Choudhary, M. et al. Pregnenolone derivatives as potential anticancer agents. Steroids 76, 1554–1559 (2011).

    Article  CAS  Google Scholar 

  5. Flood, J.F., Morley, J.E. & Roberts, E. Memory-enhancing effects in male mice of pregnenolone and steroids metabolically derived from it. Proc. Natl. Acad. Sci. USA 89, 1567–1571 (1992).

    Article  CAS  Google Scholar 

  6. Guth, L., Zhang, Z. & Roberts, E. Key role for pregnenolone in combination therapy that promotes recovery after spinal cord injury. Proc. Natl. Acad. Sci. USA 91, 12308–12312 (1994).

    Article  CAS  Google Scholar 

  7. Bianchi, M. & Baulieu, E.E. 3β-Methoxy-pregnenolone (MAP4343) as an innovative therapeutic approach for depressive disorders. Proc. Natl. Acad. Sci. USA 109, 1713–1718 (2012).

    Article  CAS  Google Scholar 

  8. Marx, C.E. et al. Proof-of-concept trial with the neurosteroid pregnenolone targeting cognitive and negative symptoms in schizophrenia. Neuropsychopharmacology 34, 1885–1903 (2009).

    Article  CAS  Google Scholar 

  9. Ritsner, M.S. et al. Pregnenolone and dehydroepiandrosterone as an adjunctive treatment in schizophrenia and schizoaffective disorder: an 8-week, double-blind, randomized, controlled, 2-center, parallel-group trial. J. Clin. Psychiatry 71, 1351–1362 (2010).

    Article  CAS  Google Scholar 

  10. Irwin, R.P. et al. Pregnenolone sulfate augments NMDA receptor mediated increases in intracellular Ca2+ in cultured rat hippocampal neurons. Neurosci. Lett. 141, 30–34 (1992).

    Article  CAS  Google Scholar 

  11. Sabeti, J., Nelson, T.E., Purdy, R.H. & Gruol, D.L. Steroid pregnenolone sulfate enhances NMDA-receptor–independent long-term potentiation at hippocampal CA1 synapses: role for L-type calcium channels and σ-receptors. Hippocampus 17, 349–369 (2007).

    Article  CAS  Google Scholar 

  12. Kavaliers, M. & Wiebe, J.P. Analgesic effects of the progesterone metabolite, 3 α-hydroxy-5 α-pregnan-20-one, and possible modes of action in mice. Brain Res. 415, 393–398 (1987).

    Article  CAS  Google Scholar 

  13. Matsunaga, M., Ukena, K., Baulieu, E.E. & Tsutsui, K. 7α-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc. Natl. Acad. Sci. USA 101, 17282–17287 (2004).

    Article  CAS  Google Scholar 

  14. Fontaine-Lenoir, V. et al. Microtubule-associated protein 2 (MAP2) is a neurosteroid receptor. Proc. Natl. Acad. Sci. USA 103, 4711–4716 (2006).

    Article  CAS  Google Scholar 

  15. Galjart, N. CLIPs and CLASPs and cellular dynamics. Nat. Rev. Mol. Cell Biol. 6, 487–498 (2005).

    Article  CAS  Google Scholar 

  16. Nakano, A. et al. AMPK controls the speed of microtubule polymerization and directional cell migration through CLIP-170 phosphorylation. Nat. Cell Biol. 12, 583–590 (2010).

    Article  CAS  Google Scholar 

  17. Li, H. et al. Phosphorylation of CLIP-170 by Plk1 and CK2 promotes timely formation of kinetochore-microtubule attachments. EMBO J. 29, 2953–2965 (2010).

    Article  CAS  Google Scholar 

  18. Arnal, I., Heichette, C., Diamantopoulos, G.S. & Chretien, D. CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues. Curr. Biol. 14, 2086–2095 (2004).

    Article  CAS  Google Scholar 

  19. Fukata, M. et al. Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170. Cell 109, 873–885 (2002).

    Article  CAS  Google Scholar 

  20. Coquelle, F.M. et al. LIS1, CLIP-170's key to the dynein/dynactin pathway. Mol. Cell Biol. 22, 3089–3102 (2002).

    Article  CAS  Google Scholar 

  21. Watson, P. & Stephens, D.J. Microtubule plus-end loading of p150Glued is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells. J. Cell Sci. 119, 2758–2767 (2006).

    Article  CAS  Google Scholar 

  22. Lomakin, A.J. et al. CLIP-170–dependent capture of membrane organelles by microtubules initiates minus-end directed transport. Dev. Cell 17, 323–333 (2009).

    Article  CAS  Google Scholar 

  23. Kholmanskikh, S.S. et al. Calcium-dependent interaction of Lis1 with IQGAP1 and Cdc42 promotes neuronal motility. Nat. Neurosci. 9, 50–57 (2006).

    Article  CAS  Google Scholar 

  24. Swiech, L. et al. CLIP-170 and IQGAP1 cooperatively regulate dendrite morphology. J. Neurosci. 31, 4555–4568 (2011).

    Article  CAS  Google Scholar 

  25. Neukirchen, D. & Bradke, F. Cytoplasmic linker proteins regulate neuronal polarization through microtubule and growth cone dynamics. J. Neurosci. 31, 1528–1538 (2011).

    Article  CAS  Google Scholar 

  26. Sun, X. et al. Microtubule-binding protein CLIP-170 is a mediator of paclitaxel sensitivity. J. Pathol. 226, 666–673 (2012).

    Article  CAS  Google Scholar 

  27. Lansbergen, G. et al. Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization. J. Cell Biol. 166, 1003–1014 (2004).

    Article  CAS  Google Scholar 

  28. Lee, H.S. et al. Phosphorylation controls autoinhibition of cytoplasmic linker protein-170. Mol. Biol. Cell 21, 2661–2673 (2010).

    Article  CAS  Google Scholar 

  29. Small, J.V., Geiger, B., Kaverina, I. & Bershadsky, A. How do microtubules guide migrating cells? Nat. Rev. Mol. Cell Biol. 3, 957–964 (2002).

    Article  CAS  Google Scholar 

  30. Mimori-Kiyosue, Y., Shiina, N. & Tsukita, S. The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules. Curr. Biol. 10, 865–868 (2000).

    Article  CAS  Google Scholar 

  31. Clark, M.A. & Shay, J.W. The role of tubulin in the steroidogenic response of murine adrenal and rat Leydig cells. Endocrinology 109, 2261–2263 (1981).

    Article  CAS  Google Scholar 

  32. Heal, W.P., Dang, T.H. & Tate, E.W. Activity-based probes: discovering new biology and new drug targets. Chem. Soc. Rev. 40, 246–257 (2011).

    Article  CAS  Google Scholar 

  33. Romeo, E. et al. Effects of antidepressant treatment on neuroactive steroids in major depression. Am. J. Psychiatry 155, 910–913 (1998).

    Article  CAS  Google Scholar 

  34. Grigoryev, D.N. et al. Cytochrome P450c17-expressing Escherichia coli as a first-step screening system for 17α-hydroxylase-C17,20-lyase inhibitors. Anal. Biochem. 267, 319–330 (1999).

    Article  CAS  Google Scholar 

  35. Rhéaume, E. et al. Structure and expression of a new complementary DNA encoding the almost exclusive 3 β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase in human adrenals and gonads. Mol. Endocrinol. 5, 1147–1157 (1991).

    Article  Google Scholar 

  36. Schumacher, M. et al. Steroid hormones and neurosteroids in normal and pathological aging of the nervous system. Prog. Neurobiol. 71, 3–29 (2003).

    Article  CAS  Google Scholar 

  37. Komarova, Y. et al. EB1 and EB3 control CLIP dissociation from the ends of growing microtubules. Mol. Biol. Cell 16, 5334–5345 (2005).

    Article  CAS  Google Scholar 

  38. Peris, L. et al. Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends. J. Cell Biol. 174, 839–849 (2006).

    Article  CAS  Google Scholar 

  39. Dragestein, K.A. et al. Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends. J. Cell Biol. 180, 729–737 (2008).

    Article  CAS  Google Scholar 

  40. Bieling, P. et al. CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites. J. Cell Biol. 183, 1223–1233 (2008).

    Article  CAS  Google Scholar 

  41. Burkhard, P., Stetefeld, J. & Strelkov, S.V. Coiled coils: a highly versatile protein folding motif. Trends Cell Biol. 11, 82–88 (2001).

    Article  CAS  Google Scholar 

  42. Hamdi, M., Ferreira, A., Sharma, G. & Mavroidis, C. Prototyping bio-nanorobots using molecular dynamics simulation and virtual reality. Microelectron. J. 39, 190–201 (2008).

    Article  CAS  Google Scholar 

  43. Carr, C.M. & Kim, P.S. A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell 73, 823–832 (1993).

    Article  CAS  Google Scholar 

  44. Moore, J.R., Li, X., Nirody, J., Fischer, S. & Lehman, W. Structural implications of conserved aspartate residues located in tropomyosin's coiled-coil core. BioArchitecture 1, 250–255 (2011).

    Article  Google Scholar 

  45. Hu, M.C., Guo, I.C., Lin, J.H. & Chung, B.C. Regulated expression of cytochrome P-450scc (cholesterol-side-chain cleavage enzyme) in cultured cell lines detected by antibody against bacterially expressed human protein. Biochem. J. 274, 813–817 (1991).

    Article  CAS  Google Scholar 

  46. Hoogenraad, C.C., Akhmanova, A., Grosveld, F., De Zeeuw, C.I. & Galjart, N. Functional analysis of CLIP-115 and its binding to microtubules. J. Cell Sci. 113, 2285–2297 (2000).

    CAS  PubMed  Google Scholar 

  47. Ozer, R.S. & Halpain, S. Phosphorylation-dependent localization of microtubule-associated protein MAP2c to the actin cytoskeleton. Mol. Biol. Cell 11, 3573–3587 (2000).

    Article  CAS  Google Scholar 

  48. Lai, P.Y. et al. Steroidogenic Factor 1 (NR5A1) resides in centrosomes and maintains genomic stability by controlling centrosome homeostasis. Cell Death Differ. 18, 1836–1844 (2011).

    Article  CAS  Google Scholar 

  49. Chang, W.H. et al. UniQua: a universal signal processor for MS-based qualitative and quantitative proteomics applications. Anal. Chem. 85, 890–897 (2013).

    Article  CAS  Google Scholar 

  50. Tamura, K. et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Takashima (Osaka University) and N. Galjart (Erasmus Medical Center) for CLIP-170 plasmids, S. Tsukita (Kyoto University) for the EB1-GFP plasmid, S. Halpain (University of California–San Diego) for the MAP2c plasmid and Y. Mimori-Kiyosue (RIKEN) for the GFP-CLIP-115 plasmid, generated by A. Akhmanova (Utrecht University). We thank W.-y. Ku and C.-w. Hsu for help in drawing the graphical abstract. RNAi reagents were obtained from the National RNAi Core Facility Platform at Academia Sinica (NSC 97-3112-B-001-016). The wild-type zebrafish were from Taiwan Zebrafish Core Facility at Academia Sinica (NSC 100-2321-B-001-030). C.-T.C. was supported by the National Science Council (NSC 97-2113-M-002-002-MY3 and NSC99-3112-B-001-002) and the National Taiwan University, and B.-c.C. was supported by the National Science Council (NSC101-2321-B-001-001, NSC 102-2311-B-001-013-MY3), the National Health Research Institute (NHRI-EX102-10210SI) and Academia Sinica.

Author information

Authors and Affiliations

  1. Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan

    Jui-Hsia Weng & Bon-chu Chung

  2. Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

    Jui-Hsia Weng, Sok-Keng Tong, Sue-Ping Lee & Bon-chu Chung

  3. Department of Chemistry, National Taiwan University, Taipei, Taiwan

    Ming-Ren Liang, Chien-Han Chen, Tzu-Chiao Huang & Chao-Tsen Chen

  4. Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan

    Yet-Ran Chen

Authors
  1. Jui-Hsia Weng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ming-Ren Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chien-Han Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sok-Keng Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tzu-Chiao Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sue-Ping Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yet-Ran Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Chao-Tsen Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Bon-chu Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.-H.W. designed the study, performed most of the experiments and wrote the manuscript; M.-R.L. synthesized P5s-NBPN, OH-NBPN and Cho-NBPN; C.-H.C. synthesized P5-NBPN and 7-OH-P5; S.-K.T. examined zebrafish clip1 expression patterns; T.-C.H. synthesized P5β-NBPN; S.-P.L. stained CLIP-170 for EM analysis; Y.-R.C. performed LC/MS/MS identification; C.-T.C. devised P5-photoaffinity probes and supervised syntheses of P5 derivatives; B.-c.C. designed the study, oversaw its execution and wrote the manuscript.

Corresponding authors

Correspondence to Chao-Tsen Chen or Bon-chu Chung.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–18 and Supplementary Notes 1–7 (PDF 5125 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Weng, JH., Liang, MR., Chen, CH. et al. Pregnenolone activates CLIP-170 to promote microtubule growth and cell migration. Nat Chem Biol 9, 636–642 (2013). https://doi.org/10.1038/nchembio.1321

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1321

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing