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Extracellular phosphorylation drives the formation of neuronal circuitry

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.

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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.

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.

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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.

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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.

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Correspondence to Philippe P. Monnier.

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Supplementary Note 1 and Supplementary Figures 1–21

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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

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