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Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction

Abstract

Although netrins are an important family of neuronal guidance proteins, intracellular mechanisms that mediate netrin function are not well understood. Here we show that netrin-1 induces tyrosine phosphorylation of proteins including focal adhesion kinase (FAK) and the Src family kinase Fyn. Blockers of Src family kinases inhibited FAK phosphorylation and axon outgrowth and attraction by netrin. Dominant-negative FAK and Fyn mutants inhibited the attractive turning response to netrin. Axon outgrowth and attraction induced by netrin-1 were significantly reduced in neurons lacking the FAK gene. Our results show the biochemical and functional links between netrin, a prototypical neuronal guidance cue, and FAK, a central player in intracellular signaling that is crucial for cell migration.

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Figure 1: Regulation of tyrosine phosphorylation by netrin-1.
Figure 2: Activation of the Fyn tyrosine kinase by netrin-1.
Figure 3: Formation of protein-protein interaction complexes of DCC with FAK and Fyn.
Figure 4: Inhibition of netrin-induced axon outgrowth by Src family kinase inhibitors.
Figure 8: Inhibition of netrin-induced turning of spinal cord axons by a dominant-negative FAK mutant and a kinase-dead Fyn mutant.
Figure 5: Reduced axonal responses to netrin-1 in FAK mutants.
Figure 6: Axonal outgrowth from dissociated neurons of heterozygous and FAK-null embryos.
Figure 7: Loss of netrin attraction of cortical axons in FAK mutants.

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References

  1. Hedgecock, E.M., Culotti, J.G. & Hall, D.H. The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 4, 61–85 (1990).

    Article  CAS  PubMed  Google Scholar 

  2. Kennedy, T.E., Serafini, T., de la Torre, J.R. & Tessier-Lavigne, M. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78, 425–435 (1994).

    Article  CAS  PubMed  Google Scholar 

  3. Serafini, T. et al. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 78, 409–424 (1994).

    Article  CAS  PubMed  Google Scholar 

  4. Merz, D.C. & Culotti, J.G. Genetic analysis of growth cone migration in Caenorhabditis elegans. J. Neurobiol. 44, 281–288 (2000).

    Article  CAS  PubMed  Google Scholar 

  5. Ishii, N., Wadsworth W.G., Stern, B.D., Culotti, J.G. & Hedgecock, E.M. UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans. Neuron 9, 873–881 (1992).

    Article  CAS  PubMed  Google Scholar 

  6. Tessier-Lavigne, M., Placzek, M., Lumsden, A.G., Dodd, J. & Jessell, T.M. Chemotropic guidance of developing axons in the mammalian central nervous system. Nature 336, 775–778 (1988).

    Article  CAS  PubMed  Google Scholar 

  7. Placzek, M., Tessier-Lavigne, M., Jessell, T. & Dodd, J. Orientation of commissural axons in vitro in response to a floor plate-derived chemoattractant. Development 110, 19–30 (1990).

    CAS  PubMed  Google Scholar 

  8. Bovolenta, P. & Dodd, J. Guidance of commissural growth cones at the floor plate in embryonic rat spinal cord. Development 109, 435–447 (1990).

    CAS  PubMed  Google Scholar 

  9. Bovolenta, P. & Dodd, J. Perturbation of neuronal differentiation and axon guidance in the spinal cord of mouse embryos lacking a floor plate: analysis of Danforth's short-tail mutation. Development 113, 625–639 (1991).

    CAS  PubMed  Google Scholar 

  10. Bernhardt, R.R., Nguyen, N. & Kuwada, J.Y. Growth cone guidance by floor plate cells in the spinal cord of zebrafish embryos. Neuron 8, 869–882 (1992).

    Article  CAS  PubMed  Google Scholar 

  11. Harris, R., Sabatelli, L.M. & Seeger, M.A. Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila netrin/UNC-6 homologs. Neuron 17, 217–228 (1996).

    Article  CAS  PubMed  Google Scholar 

  12. Kolodziej, P.A. et al. Frazzled encodes a Drosophila member of the DCC immunoglobin subfamily and is required for CNS and motor axon guidance. Cell 87, 197–204 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. Mitchell, K.J. et al. Genetic analysis of Netrin genes in Drosophila: netrins guide CNS commissural axons and peripheral motor axons. Neuron 17, 203–215 (1996).

    Article  CAS  PubMed  Google Scholar 

  14. Wang, H., Copeland, N.G., Gilbert, D.J., Jenkins, N.A. & Tessier-Lavigne, M. Netrin-3, a mouse homolog of human NTN2L, is highly expressed in sensory ganglia and shows differential binding to netrin receptors. J. Neurosci. 19, 4938–4947 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Koch, M. et al. A novel member of the netrin family, β-netrin, shares homology with the β chain of laminin: identification, expression, and functional characterization. J. Cell Biol. 151, 221–234 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hedgecock, E.M., Culotti, J.G., Hall, D.H. & Stern, B.D. Genetics of cell and axon migrations in C. elegans. Development 100, 365–382 (1987).

    CAS  PubMed  Google Scholar 

  17. Chan, S.S.Y. et al. UNC-40, a C. elegans homolog of DCC (Deleted in Colorectal Cancer), is required in motile cells responding to UNC-6 Netrin cues. Cell 87, 187–195 (1996).

    Article  CAS  PubMed  Google Scholar 

  18. Leung-Hagesteijn, C. et al. UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans. Cell 71, 289–299 (1992).

    Article  CAS  PubMed  Google Scholar 

  19. Keino-Masu, K. et al. Deleted in colorectal cancer (DCC) encodes a netrin receptor. Cell 87, 175–185 (1996).

    Article  CAS  PubMed  Google Scholar 

  20. Fazeli, A. et al. Phenotype of mice lacking functional Deleted in colorectal cancer (DCC) gene. Nature 386, 796–804 (1997).

    Article  CAS  PubMed  Google Scholar 

  21. Ackerman, S.L. et al. The mouse rostral cerebellar malformation gene encodes an UNC-5-like protein. Nature 386, 838–842 (1997).

    Article  CAS  PubMed  Google Scholar 

  22. Leonardo, E.D. et al. Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors. Nature 386, 833–838 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Przyborski, S.A., Knowles, B.B. & Ackerman, S.L. Embryonic phenotype of Unc5h3 mutant mice suggests chemorepulsion during the formation of the rostral cerebellar boundary. Development 125, 41–50 (1998).

    CAS  PubMed  Google Scholar 

  24. Engelkamp, D. Cloning of three mouse Unc5 genes and their expression patterns at mid-gestation. Mech. Dev. 118, 191–197 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Hamelin, M., Zhou, Y., Su, M.-W., Scott, I.M. & Culotti, J.G. Expression of the UNC-5 guidance receptor in the touch neurons of C. elegans steers their axons dorsally. Nature 364, 327–330 (1993).

    Article  CAS  PubMed  Google Scholar 

  26. de la Torre, J.R. et al. Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC. Neuron 19, 1211–1224 (1997).

    Article  CAS  PubMed  Google Scholar 

  27. Hong, K. et al. A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell 97, 927–941 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Merz, D.C., Zheng, H., Killeen, M.T., Krizus, A. & Culotti, J.G. Multiple signaling mechanisms of the unc-6/netrin receptors unc-5 and unc-40/dcc in vivo. Genetics 158, 1071–1080 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Colavita, A. & Culotti, J.G. Suppressors of ectopic UNC-5 growth cone steering identify eight genes involved in axon guidance in Caenorhabditis elegans. Dev. Biol. 194, 72–85 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Huang, X., Cheng, H.-J., Tessier-Lavigne, M. & Jin, Y. MAX-1, a novel PH/MyTH4/FERM domain cytoplasmic protein implicated in netrin-mediated axon repulsion. Neuron 34, 563–576 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Ming, G.L. et al. Adaptation in the chemotactic guidance of nerve growth cones. Nature 417, 411–418 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Forcet, C. et al. Netrin-1-mediated axon outgrowth requires deleted in colorectal cancer-dependent MAPK activation. Nature 417, 443–447 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Campbell, D.S. & Holt, C.E. Apoptotic pathway and MAPKs differentially regulate chemotropic responses of retinal growth cones. Neuron 37, 939–952 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Shekarabi, M. & Kennedy, T.E. The netrin-1 receptor DCC promotes filopodia formation and cell spreading by activating Cdc42 and Rac1. Mol. Cell. Neurosci. 19, 1–17 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Li, X. Saint-Cyr-Proulx, E., Aktories, K. & Lamarche-Vane, N. Rac1 and Cdc42 but not RhoA or Rho kinase activities are required for neurite outgrowth induced by the Netrin-1 receptor DCC (deleted in colorectal cancer) in N1E-115 neuroblastoma cells. J. Biol. Chem. 277, 15207–15214 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Guan, J.L, Trevithick, J.E. & Hynes, R.O. Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. Cell Regul. 2, 951–964 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kornberg, L., Earp, S.E., Turner, C.E., Procktop, C. & Juliano, R.L. Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of β1 integrins. Proc. Natl. Acad. Sci. USA 88, 8392–8396 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Guan, J.L. & Shalloway, D. Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Nature 358, 690–692 (1992).

    Article  CAS  PubMed  Google Scholar 

  39. Hanks, S.K., Calalb, M.B., Harper, M.C. & Patel, S.K. Focal adhesion protein tyrosine kinase on fibronectin. Proc. Natl. Acad. Sci. USA 89, 8487–8491 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Schaller, M.D., Borgman, C.A., Cobb, B.S., Reynolds, A.B. & Parsons, J.T. pp125FAK, a structurally distinctive protein tyrosine kinase associated with focal adhesions. Proc. Natl. Acad. Sci. USA 89, 5192–5196 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Parsons, J.T. Focal adhesion kinase: the first ten years. J. Cell Sci. 116, 1409–1416 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Wong, K. et al. Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 107, 209–221 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Colamarino, S.A. & Tessier-Lavigne, M. The axonal chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons. Cell 81, 621–629 (1995).

    Article  CAS  PubMed  Google Scholar 

  44. Richards, L.J., Koester, S.E., Tuttle, R. & O'Leary, D.D. Directed growth of early cortical axons is influenced by a chemoattractant released from an intermediate target. J. Neurosci. 17, 2445–2458 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Beggs, H.E. et al. FAK deficiency in cells contributing to the basal lamina results in cortical abnormalities resembling congenital muscular dystrophies. Neuron 40, 501–514 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Li, W. et al. Activation of FAK and Src are receptor-proximal events required for netrin signaling. Nat. Neurosci. 7, 1213–1221 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Ren, X.-R. et al. Focal adhesion kinase in netrin-1 signaling. Nat. Neurosci. 7, 1204–1212 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Ming, G.L. et al. Phospholipase C-γ and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance. Neuron 23, 139–148 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Tong, J. et al. Netrin stimulates tyrosine phosphorylation of the UNC-5 family of netrin receptors and induces Shp2 binding to the RCM cytodomain. J. Biol. Chem. 276, 40917–40925 (2001).

    Article  CAS  PubMed  Google Scholar 

  50. Killeen, M. et al. UNC-5 function requires phosphorylation of cytoplasmic tyrosine 482, but its UNC-40-independent functions also require a region between the ZU-5 and death domains. Dev. Biol. 251, 348–366 (2002).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to S. Hanks for FAK phosphorylation site mutants; to J. Cooper, T. Tezuka and T. Yamamoto for Fyn mutants; to W.-C. Xiong and K.-L. Guan for communications prior to publication; and to the National Institutes of Health (CA107193 to Y.R. and NS19090 to L.F.R.), the Klingenstein Foundation and the National Brain Tumor Foundation for support. L.F.R. is an investigator of the Howard Hughes Medical Institute. H.B. was supported by a National Research Service Award fellowship from the NIH.

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Correspondence to Yi Rao.

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Supplementary information

Supplementary Fig. 1

Control experiment for Fig. 1b. Cortical neurons were treated with the control HEK conditioned medium for 5 min. Tyrosine phosphorylation was examined and was not found to be affected. (JPG 21 kb)

Supplementary Fig. 2

Control experiment for that in Fig. 1h. Control depletion was carried out with six anti-phospho-specific antibodies (against phospho-Pak1/Pak2, phospho-p38 MapK, phospho-ATF-2, phospho-CREB, phospho-GSK 3a/b and phospho-p38) could not reduce the induction of p130 phosphorylation by netrin-1. (JPG 21 kb)

Supplementary Fig. 3

Specificity of each phospho-specific anti-FAK antibody used in Fig. 1j. Wild type FAK and mutants lacking Y397, Y407, Y576/Y577 and Y861 were tagged with the myc epitope and the mutant lacking Y925 was tagged with the HA epitope. They were expressed in HEK cells and pulled down by anti-myc or anti-HA antibodies (bottom row). Each of the precipitated mutant protein was run with the wild type FAK and probed for phosphorylation at specific sites by individual antibodies and shown in separate panels. Of the two panels with 576/577 mutants, the first panel (in the top row) was probed with anti-phospho-576 antibody while the panel in the middle row was probed with anti-phospho-577 antibody. (JPG 44 kb)

Supplementary Fig. 4

Effects of Src kinase inhibitors on attraction of cortical axons by netrin. (a-f) Cortical explants from E15 embryos were co-cultured with HEK aggregates for 16 to 18 hours in the presence of pharmacological reagents before immunostaining with the TuJ1 antibody. The number of axon bundles extended from cortical explants proximally towards the control HEK aggregate is similar to that extended distally (a). The number of proximally projected axon bundles is larger than that of distally projected when cortical explants were co-cultured with HEK aggregates secreting netrin-1 (b). Asymmetric axon outgrowth was inhibited by PP2 (0.2 µM) (c) or SU6656 (f) but not by PP3 (0.2 µM) (d) or DMSO (e). (g) Quantification of axon bundles. Proximal/distal ratios were calculated from the numbers of axon bundles in the proximal part divided by those in the distal part. The P/D ratio are: HEK, 1.1 ± 0.2; Net, 2.1 ± 0.5; Net+PP2, 1.0 ± 0.2; Net+PP3, 2.0 ± 0.3; Net+DMSO, 2.0 ± 0.3; Net+SU6656, 1.1 ± 0.1. The numbers on the top (n=) indicate the numbers of explants tested. The P values for the differences (by Student's t test) are: < 0.001 between control and Net, < 0.001 between Net and Net+PP2, < 0.001 between Net+PP2 and Net+PP3, > 0.1 between control and Net+PP2, and > 0.1 between Net and Net+PP3, < 0.001 between Net+DMSO and Net+SU6656. (h) Quantification of the length of axon bundles. The proximal/distal ratios were calculated from the length of axon bundles in the proximal part divided by those in the distal part. The P/D ratio are: HEK, 1.1 ± 0.7; Net, 4.1 ± 0.7; Net ± PP2, 1.1 ± 0.2; Net ± PP3, 3.9 ± 0.5; Net+DMSO, 4.0 ± 0.9; Net+SU6656, 1.2 ± 0.3. The numbers on the top (n=) indicate the numbers of explants tested. The P values for the differences (by Student's t test) are: < 0.001 between control and Net, < 0.001 between Net and Net+PP2, < 0.001 between Net+PP2 and Net+PP3, > 0.1 between control and Net+PP2, and > 0.1 between Net and Net+PP3, < 0.001 between Net+DMSO and Net+SU6656. HEK, control HEK cells; NET, Netrin-1 secreting HEK cells. (JPG 74 kb)

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Liu, G., Beggs, H., Jürgensen, C. et al. Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction. Nat Neurosci 7, 1222–1232 (2004). https://doi.org/10.1038/nn1331

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