Abstract
Extracellular netrin-1 and its receptor deleted in colorectal cancer (DCC) promote axon branching in developing cortical neurons. Netrin-dependent morphogenesis is preceded by multimerization of DCC, activation of FAK and Src family kinases, and increases in exocytic vesicle fusion, yet how these occurrences are linked is unknown. Here we demonstrate that tripartite motif protein 9 (TRIM9)-dependent ubiquitination of DCC blocks the interaction with and phosphorylation of FAK. Upon netrin-1 stimulation TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination of DCC is reduced, which promotes an interaction with FAK and subsequent FAK activation. We found that inhibition of FAK activity blocks elevated frequencies of exocytosis in vitro and elevated axon branching in vitro and in vivo. Although FAK inhibition decreased soluble N-ethylmaleimide attachment protein receptor (SNARE)-mediated exocytosis, assembled SNARE complexes and vesicles adjacent to the plasma membrane increased, suggesting a novel role for FAK in the progression from assembled SNARE complexes to vesicle fusion in developing murine neurons.
© 2017 Plooster, Menon, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
MeSH terms
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Animals
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Axons / enzymology
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Axons / metabolism*
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Membrane / metabolism
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DCC Receptor / metabolism*
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Exocytosis / physiology
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Female
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Focal Adhesion Kinase 1 / genetics
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Focal Adhesion Kinase 1 / metabolism
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HEK293 Cells
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Humans
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Male
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Membrane Fusion
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Mice
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism*
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Netrin-1 / genetics
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Netrin-1 / metabolism
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Neurogenesis / physiology
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Neurons / cytology
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Neurons / enzymology
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Neurons / metabolism
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Phosphorylation
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Pregnancy
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Signal Transduction
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Tripartite Motif Proteins / genetics
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Tripartite Motif Proteins / metabolism*
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism*
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Ubiquitination
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src-Family Kinases / metabolism
Substances
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Carrier Proteins
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DCC Receptor
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DCC protein, human
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Dcc protein, mouse
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Nerve Tissue Proteins
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Ntn1 protein, mouse
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Tripartite Motif Proteins
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Netrin-1
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TRIM9 protein, human
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Trim9 protein, mouse
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Ubiquitin-Protein Ligases
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Focal Adhesion Kinase 1
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Ptk2 protein, mouse
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src-Family Kinases