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Activation of FAK and Src are receptor-proximal events required for netrin signaling

Nature Neuroscience volume 7, pages 1213–1221 (2004)Cite this article

Abstract

The axon guidance cue netrin is importantly involved in neuronal development. DCC (deleted in colorectal cancer) is a functional receptor for netrin and mediates axon outgrowth and the steering response. Here we show that different regions of the intracellular domain of DCC directly interacted with the tyrosine kinases Src and focal adhesion kinase (FAK). Netrin activated both FAK and Src and stimulated tyrosine phosphorylation of DCC. Inhibition of Src family kinases reduced DCC tyrosine phosphorylation and blocked both axon attraction and outgrowth of neurons in response to netrin. Mutation of the tyrosine phosphorylation residue in DCC abolished its function of mediating netrin-induced axon attraction. On the basis of our observations, we suggest a model in which DCC functions as a kinase-coupled receptor, and FAK and Src act immediately downstream of DCC in netrin signaling.

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Figure 1: Netrin-stimulated tyrosine phosphorylation of DCC and FAK.
Figure 2: Src interacts with DCC and is activated by netrin.
Figure 3: Tyr1420 in DCC is phosphorylated and the P3 domain in DCC interacts with FAK.
Figure 4: The 1400PXXP motif in DCC and the SH3 domain in Src are required for DCC and Src interaction.
Figure 5: Src activity is required for netrin-induced axon growth.
Figure 6: Src function or DCC tyrosine phosphorylation is required for DCC-mediated axon attraction.

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Acknowledgements

We thank B. Kruger for critical reading of the manuscript, T. Zhu for technical assistance, and W. Xiong for communicating unpublished information. This work is supported by grants from the National Institutes of Health (K.-L.G.), a Rackham Graduate Fellowship (H.V.) and the Pharmocological Sciences Training Program (GMO7767 to J.A.).

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Authors and Affiliations

  1. Life Sciences Institute, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Weiquan Li, Jeeyong Lee, Jennifer Aurandt & Kun-Liang Guan

  2. Department of Biological Chemistry, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Jeeyong Lee, Haris G Vikis, Jennifer Aurandt & Kun-Liang Guan

  3. Department of Biochemistry, New York University School of Medicine, New York, 10016, New York, USA

    Seung-Hee Lee & Kyonsoo Hong

  4. Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, 63110, Missouri, USA

    Guofa Liu & Yi Rao

  5. Department of Molecular Medicine, Cornell University, Ithaca, 14853, New York, USA

    Tang-Long Shen & Jun-Lin Guan

  6. Departments of Human Genetics and Internal Medicine and Pathology, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Eric R Fearon

  7. Department of Molecular Cellular Developmental Biology, Howard Hughes Medical Institute, University of Colorado, Boulder, 80309, Colorado, USA

    Min Han

  8. Institute of Gerontology, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Kun-Liang Guan

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Supplementary information

Supplementary Fig. 1

DCC tyrosine phosphorylation and interaction with FAK and Src. (a) Netrin induces time- and dose-dependent tyrosine phosphorylation of DCC. HEK 293 cells were transfected with the indicated plasmids, stimulated with netrin and tyrosine phosphorylation of immunoprecipitated DCC was observed as a function of time (0-30 min) and concentration (0-2 μg/ml). (b) In vitro pulldown of FAK by GST-DCC. HEK293 cells were transfected with HA-FAK and lysates were incubated with GST and GST-DCC intracellular domain produced from HEK293 cells (lanes 1-2) or E.coli (lanes 3-4). The presence of FAK was determined by α-HA. Shown also is a Coomassie blue stained membrane of the GST-DCC protein. (c) Dose dependent inhibition of DCC tyrosine phosphorylation by PP2. PP2 was used at various concentrations for 60 min and the phosphorylation state of immunoprecipitated DCC was determined by phosphotyrosine IB. (d) Netrin-DCC induction of FAK site specific phosphorylation and inhibition by PP2. FAK was isolated by IP and probed with the indicated FAK phospho-specific antibodies. The presence of PP2 was indicated. (e) In vitro pulldown of Src and FAK by GST-DCC. Src and HA-FAK proteins immunoprecipitated from transfected 293 cell under high stringency conditions were incubated with GST-DCC purified from E.coli. The complex was precipitated with anti-Src or anti-HA (for HA-FAK) and the presence of DCC was detected by IB. (f) Netrin does not affect the interaction of DCC with FAK. The experiment was performed as described in Fig. 2f. (g) Fyn interacts with DCC through the same mechanism as Src. HEK 293 cells were transfected with indicated plasmids. Src or Fyn was immunoprecipitated and the presence of DCC in IP was determined by IB. (h) PP2 has no effects on the interaction of DCC and FAK. HEK 293 cells were transfected with the indicated plasmids and where treated with PP2. HA-FAK was immunoprecipitated and the presence of DCC in the IP was determined by IB. (JPG 92 kb)

Supplementary Fig. 2

A model for FAK and Src in netrin signaling. FAK interacts via its CTD with the P3 domain of DCC. Src interacts in part via its SH3 domain to the P1400XXP motif in DCC and in part via its SH2 domain to phosphorylated Y397 on FAK. Src phosphorylates Y1420 in DCC. Note that molecules except the intracellular domain of DCC are not drawn to scale. CTD, C-terminal domain; NTD, N-terminal domain of FAK. (GIF 40 kb)

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Li, W., Lee, J., Vikis, H. et al. Activation of FAK and Src are receptor-proximal events required for netrin signaling. Nat Neurosci 7, 1213–1221 (2004). https://doi.org/10.1038/nn1329

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