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:

Extracellular phosphorylation drives the formation of neuronal circuitry

Nature Chemical Biology volume 15, pages 1035–1042 (2019)Cite this article

Subjects

Abstract

Until recently, the existence of extracellular kinase activity was questioned. Many proteins of the central nervous system are targeted, but it remains unknown whether, or how, extracellular phosphorylation influences brain development. Here we show that the tyrosine kinase vertebrate lonesome kinase (VLK), which is secreted by projecting retinal ganglion cells, phosphorylates the extracellular protein repulsive guidance molecule b (RGMb) in a dorsal–ventral descending gradient. Silencing of VLK or RGMb causes aberrant axonal branching and severe axon misguidance in the chick optic tectum. Mice harboring RGMb with a point mutation in the phosphorylation site also display aberrant axonal pathfinding. Mechanistic analyses show that VLK-mediated RGMb phosphorylation modulates Wnt3a activity by regulating LRP5 protein gradients. Thus, the secretion of VLK by projecting neurons provides crucial signals for the accurate formation of nervous system circuitry. The dramatic effect of VLK on RGMb and Wnt3a signaling implies that extracellular phosphorylation likely has broad and profound effects on brain development, function and disease.

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

Fig. 1: RGMb regulates retinal axon mapping.
Fig. 2: Divergences between RGMa and RGMb.
Fig. 3: RGMb phosphorylation regulates cell-surface presence and axonal growth.
Fig. 4: VLK phosphorylates RGMb to modulate axonal growth.
Fig. 5: p-RGMb regulates LRP5–Wnt3a-mediated axonal growth.
Fig. 6: RGMb regulates retino-tectal mapping.

Similar content being viewed by others

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its supplementary information or from the authors upon reasonable request.

References

  1. Hornbeck, P. V. et al. PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res. 40, D261–D270 (2012).

    Article  CAS  Google Scholar 

  2. Bordoli, M. R. et al. A secreted tyrosine kinase acts in the extracellular environment. Cell 158, 1033–1044 (2014).

    Article  CAS  Google Scholar 

  3. Samad, T. A. et al. DRAGON: a member of the repulsive guidance molecule-related family of neuronal- and muscle-expressed membrane proteins is regulated by DRG11 and has neuronal adhesive properties. J. Neurosci. 24, 2027–2036 (2004).

    Article  CAS  Google Scholar 

  4. Samad, T. A. et al. DRAGON, a bone morphogenetic protein co-receptor. J. Biol. Chem. 280, 14122–14129 (2005).

    Article  CAS  Google Scholar 

  5. Ma, C. H. et al. The BMP coreceptor RGMb promotes while the endogenous BMP antagonist noggin reduces neurite outgrowth and peripheral nerve regeneration by modulating BMP signaling. J. Neurosci. 31, 18391–18400 (2011).

    Article  CAS  Google Scholar 

  6. Kam, J. W. et al. RGMB and neogenin control cell differentiation in the developing olfactory epithelium. Development 143, 1534–1546 (2016).

    Article  CAS  Google Scholar 

  7. Cang, J. & Feldheim, D. A. Developmental mechanisms of topographic map formation and alignment. Annu. Rev. Neurosci. 36, 51–77 (2013).

    Article  CAS  Google Scholar 

  8. Sperry, R. W. Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl Acad. Sci. USA 50, 703–710 (1963).

    Article  CAS  Google Scholar 

  9. Drescher, U. et al. In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases. Cell 82, 359–370 (1995).

    Article  CAS  Google Scholar 

  10. Schmitt, A. M. et al. Wnt–Ryk signalling mediates medial-lateral retinotectal topographic mapping. Nature 439, 31–37 (2006).

    Article  CAS  Google Scholar 

  11. Hindges, R., McLaughlin, T., Genoud, N., Henkemeyer, M. & O’Leary, D. EphB forward signaling controls directional branch extension and arborization required for dorsal–ventral retinotopic mapping. Neuron 35, 475–487 (2002).

    Article  CAS  Google Scholar 

  12. Flanagan, J. G. Neural map specification by gradients. Curr. Opin. Neurobiol. 16, 59–66 (2006).

    Article  CAS  Google Scholar 

  13. Cheng, H. J., Nakamoto, M., Bergemann, A. D. & Flanagan, J. G. Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cell 82, 371–381 (1995).

    Article  CAS  Google Scholar 

  14. Monnier, P. P. et al. RGM is a repulsive guidance molecule for retinal axons. Nature 419, 392–395 (2002).

    Article  CAS  Google Scholar 

  15. Tessier-Lavigne, M. & Goodman, C. S. The molecular biology of axon guidance. Science 274, 1123–1133 (1996).

    Article  CAS  Google Scholar 

  16. Tamagnone, L. et al. Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell 99, 71–80 (1999).

    Article  CAS  Google Scholar 

  17. Bonhoeffer, F. & Huf, J. Position-dependent properties of retinal axons and their growth cones. Nature 315, 409–410 (1985).

    Article  CAS  Google Scholar 

  18. Hornberger, M. R. et al. Modulation of EphA receptor function by coexpressed ephrinA ligands on retinal ganglion cell axons. Neuron 22, 731–742 (1999).

    Article  CAS  Google Scholar 

  19. Tassew, N. G. et al. Modifying lipid rafts promotes regeneration and functional recovery. Cell Rep. 8, 1146–1159 (2014).

    Article  CAS  Google Scholar 

  20. Kalia, M. et al. Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3′-kinase-dependent machinery. Mol. Biol. Cell 17, 3689–3704 (2006).

    Article  CAS  Google Scholar 

  21. Prevostel, C., Alice, V., Joubert, D. & Parker, P. J. Protein kinase Cα actively downregulates through caveolae-dependent traffic to an endosomal compartment. J. Cell Sci. 113, 2575–2584 (2000).

    CAS  PubMed  Google Scholar 

  22. Shyng, S. L., Heuser, J. E. & Harris, D. A. A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits. J. Cell Biol. 125, 1239–1250 (1994).

    Article  CAS  Google Scholar 

  23. Cooper, A. & Shaul, Y. Clathrin-mediated endocytosis and lysosomal cleavage of hepatitis B virus capsid-like core particles. J. Biol. Chem. 281, 16563–16569 (2006).

    Article  CAS  Google Scholar 

  24. Ding, Y. et al. Genome-wide analysis of dorsal and ventral transcriptomes of the Xenopus laevis gastrula. Dev. Biol. 426, 176–187 (2017).

    Article  CAS  Google Scholar 

  25. Thakar, S., Chenaux, G. & Henkemeyer, M. Critical roles for EphB and ephrin-B bidirectional signalling in retinocollicular mapping. Nat. Commun. 2, 431 (2011).

    Article  Google Scholar 

  26. Mao, B. et al. Kremen proteins are Dickkopf receptors that regulate Wnt/β-catenin signalling. Nature 417, 664–667 (2002).

    Article  CAS  Google Scholar 

  27. Mao, J. et al. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol. Cell 7, 801–809 (2001).

    Article  CAS  Google Scholar 

  28. Tamai, K. et al. LDL-receptor-related proteins in Wnt signal transduction. Nature 407, 530–535 (2000).

    Article  CAS  Google Scholar 

  29. MacDonald, B. T. & He, X. Frizzled and LRP5/6 receptors for Wnt/β-catenin signaling. Cold Spring Harb. Perspect. Biol. 4, a007880 (2012).

    Article  Google Scholar 

  30. Yamamoto, H., Sakane, H., Michiue, T. & Kikuchi, A. Wnt3a and Dkk1 regulate distinct internalization pathways of LRP6 to tune the activation of β-catenin signaling. Dev. Cell 15, 37–48 (2008).

    Article  CAS  Google Scholar 

  31. Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R. & Brock, R. A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8, 848–866 (2007).

    Article  CAS  Google Scholar 

  32. Nusse, R. & Clevers, H. Wnt/β-catenin signaling, disease, and emerging therapeutic modalities. Cell 169, 985–999 (2017).

    Article  CAS  Google Scholar 

  33. Zhu, W. et al. IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis. Nature 454, 345–349 (2008).

    Article  CAS  Google Scholar 

  34. Harada, H., Takahashi, Y., Kawakami, K., Ogura, T. & Nakamura, H. Tracing retinal fiber trajectory with a method of transposon-mediated genomic integration in chick embryo. Dev. Growth Differ. 50, 697–702 (2008).

    Article  Google Scholar 

  35. Funahashi, J. et al. Role of Pax-5 in the regulation of a mid-hindbrain organizer’s activity. Dev. Growth Differ. 41, 59–72 (1999).

    Article  CAS  Google Scholar 

  36. Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of the chick embryo. 1951. Dev. Dyn. 195, 231–272 (1992).

    Article  CAS  Google Scholar 

  37. Sato, Y. et al. Stable integration and conditional expression of electroporated transgenes in chicken embryos. Dev. Biol. 305, 616–624 (2007).

    Article  CAS  Google Scholar 

  38. Lê, S., Josse, J., & Husson, F. FactoMineR: an R package for multivariate snalysis. J. Stat. Softw. https://doi.org/10.18637/jss.v025.i01 (2008).

  39. Savier, E. et al. A molecular mechanism for the topographic alignment of convergent neural maps. eLife 6, e20470 (2017).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Krembil Foundation (P.P.M.), the Glaucoma Research Society of Canada (P.P.M.), the Heart and Stroke Foundation of Ontario (grant NA7067 to P.P.M.) and the Canadian Institutes for Health Research (grants MOP106666 and MOP-85014 to P.P.M.). We thank M. Whitman for the gift of the VLKKM mutant.

Author information

Author notes

  1. These authors contributed equally: Hidekiyo Harada, Nahal Farhani.

Authors and Affiliations

  1. Krembil Research Institute, Vision Division, Krembil Discovery Tower, Toronto, Ontario, Canada

    Hidekiyo Harada, Nahal Farhani, Xue-Fan Wang, Shuzo Sugita, Jason Charish, Michael Reber & Philippe P. Monnier

  2. Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada

    Jason Charish & Philippe P. Monnier

  3. Department of Biochemistry, Donnelly Center, University of Toronto, Toronto, Ontario, Canada

    Liliana Attisano

  4. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Michael Moran

  5. Montreal Neurological Institute, Montral, Quebec, Canada

    Jean-Francois Cloutier

  6. Department of Ophthalmology and Vision Sciences, Faculty of Medicine, University of Toronto, Ontario, Toronto, Canada

    Michael Reber & Philippe P. Monnier

  7. CNRS UPR3212, University of Strasbourg, Strasbourg, France

    Michael Reber

  8. Lunenfeld Tannenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    Rod Bremner

Authors
  1. Hidekiyo Harada
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nahal Farhani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xue-Fan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shuzo Sugita
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jason Charish
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Liliana Attisano
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michael Moran
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jean-Francois Cloutier
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michael Reber
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Rod Bremner
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Philippe P. Monnier
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.H. performed the chick in vivo studies and biochemical experiments. N.F. carried out outgrowth experiments on dissociated RGCs. X.-F.W. and M.R. performed analysis and imaging of pathfinding in mice. R.B. and S.S. designed some of the constructs to generate CRISPR mice and expression plasmids. L.A. co-developed the in vitro system that allows electroporation in RGCs and study of cell-surface localization. J.-F.C. established the RGMb mouse model. M.M. performed MS on RGMb. J.C. performed in situ hybridization for RGMb. P.P.M. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Philippe P. Monnier.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Note 1 and Supplementary Figures 1–21

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Harada, H., Farhani, N., Wang, XF. et al. Extracellular phosphorylation drives the formation of neuronal circuitry. Nat Chem Biol 15, 1035–1042 (2019). https://doi.org/10.1038/s41589-019-0345-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-019-0345-z

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